PAR2-Gatekeepers For Proteases in The Brain, Thesis, RJ Lohman 2008 Uq2

Protease-activated receptor 2; a gate-keeper for protease activity in the brain
Rink-Jan Lohman
Submitted in total fulfillment of the requirements of the degree of Doctor of Philosophy June 2008 Departments of Pharmacology and Medicine The University of Melbourne
Printed on archival quality paper
From little things, big things grow
Rink-Jan Lohman
PAR2 and trypsin in the brain
Abstract Many proteases, including tissue plasminogen activator (tPA), thrombin and motopsin are highly expressed in neurons and glia of the brain, in particular in highly plastic regions such as the hippocampus. Trypsin is also expressed in the brain, however, very little is known about its distribution or role. Digestive enzymes have important roles in neuronal plasticity related to learning and memory, both in cell responsiveness, such as long-term potentiation and long-term depression, and in cellmatrix reorganisation to enable new synaptic connections to be formed. During inflammatory insults to the brain such proteases are up-regulated in situ and can enter the matrix following disruption of the blood-brain barrier. This may then lead to excessive plastic changes, hyperactivity and cell damage and death, as is observed in acquired epilepsies such as temporal lobe epilepsy (TLE). A subset of serine proteases have receptors called protease-activated receptors (PARs), of which four exist. The protease trypsin and trypsin-like proteases (i.e. mast cell tryptase) activate PAR2. The synthetic hexamer peptide SLIGRL also selectively activates PAR2 without the need for proteolysis, making it a useful pharmacological tool to investigate possible functions of the receptor without any side-effects of endogenous proteolytic agonists. PAR2 is generally regarded to be involved in inflammation. All PARs are expressed in the brain yet their roles here are not well characterised, although PAR2 appears to be neuroprotective in certain neurological disease states that involve inflammation, such as stroke and chronic infection like HIV. Roles for PAR2 in other disorders that also involve proteases, such as epilepsy, have not been studied. Therefore, the purpose of this study was to investigate such roles of centrally-expressed PAR2 using SLIGRL as an agonist in conscious, instrumental rats. Using immunohistochemistry, both trypsin and PAR2 were found to be highly
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colocalised to neurons in all regions of the brain examined, particularly in the hippocampus. Also, trypsin colocalised with tPA in granular structures resembling large dense core vesicles and it was hypothesised that these proteases are released together from PAR2-positive neurons upon common stimuli to facilitate memory formation.
Activation of central PAR2 with SLIGRL injected directly (i.c.v.) into the cerebrospinal fluid (CSF) induced dose-dependent behavioural changes, the highest doses causing neurotoxicity that was manifest as behavioural catatonia and seizure. The maximum tolerated dose of SLIGRL for i.c.v. injection was 0.15mg/kg. Using mass-spectrometry, SLIGRL, injected subcutaneously (s.c), was found to cross from blood to CSF and decay from CSF suggestive of first order kinetics with a half-life of approximately 25 minutes. Thus, it was assumed from these experiments that SLIGRL is able to readily and quickly enter the brain following a peripheral administration, and, as such may induce central effects.
In both the electrical amygdala kindling and the post-kainic acid induced status epilepticus models of limbic epileptogenesis (i.e. TLE) in rats, central (i.c.v.) administration of SLIGRL (0.15mg/kg) caused pronounced anti-seizure and antiepileptogenic effects including a raised seizure threshold, an attenuated progression from lower order to higher order convulsive seizures and a reduction in the number of animals having seizures of higher order compared to rats treated with an inactive control peptide, LRGILS (0.15mg/kg i.c.v.). This same pattern of outcomes occurred in the kindling model when SLIGRL was administered subcutaneously (s.c.), supporting the previous data (above) that SLIGRL can enter the brain as an active
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molecule. These data show that animals treated with SLIGRL required a greater insult to induce seizure and epileptogenesis and suggest that, like in other neurological disease models, SLIGRL is neuroprotective for epileptogenic insults.
Histological examination of the kindled rat brains using immunofluorescence revealed that animals treated with the LRGILS, which were epileptic, had significantly elevated levels of trypsin in neurons of the hippocampus much like that shown for tPA in similar models of epileptogenesis. In comparison, rats treated with SLIGRL that were protected from epilepsy, showed no elevations of trypsin expression in any region analysed over those of sham animals. Therefore, PAR2 activation appears to activate a negative feedback mechanism to regulate trypsin expression in neurons of the brain, much like that previously described by others in the airways and pancreas.
Since proteases are involved in learning and memory, this thesis also examined behavioural effects of peripherally administered SLIGRL using common ethological assays such as the elevated plus maze (EPM), open field test, sucrose consumption test and the Morris water maze (MWM) in rats. SLIGRL (1.5mg/kg, s.c.) had no effects on the anxiety states or levels of depression in rats, nor did it induce any sedation. The same treatment, however, caused significant deficits in experiencedependent motivational learning in both the MWM and the test-retest paradigm of the EPM. These data suggest, for the first time, that PAR2 and possibly trypsin play important roles in the regulation of memory formation. The memory deficit observed here may be related to the SLIGRL-induced trypsin reduction in the hippocampus described above. Thus, SLIGRL may cause a decrease in trypsin (and possibly tPA) that is otherwise required for memory formation. Perhaps PAR2 acts to stop
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production of these normally important enzymes in the brain and thus it may function as a gatekeeper to inhibit further trypsin production and release and thereby minimise damage to the brain.
In conclusion, the results of this thesis indicate that trypsin and PAR2 are involved not only in normal hippocampal functioning such as learning and memory, but also are protective against epileptogenesis following brain insults. It is hypothesised that non-proteolytic agonists of PAR2 may be useful as anti-epileptogenic or neuroprotective therapies.
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Declaration
This thesis contains no material which has been accepted for the award of any other degree or diploma in any University. To the best of my knowledge and belief, this thesis comprises only my original work towards the PhD except where otherwise cited. Apart from the intellecualisation from my superiors and others, the majority of the work presented here is that of my own. This thesis is less than 100,000 words in length, exclusive of tables, bibliographies and appendices.
Rink-Jan Lohman
Rink-Jan Lohman
Publications
1. Lohman, R.J., T.J. O'Brien, and T.M. Cocks, Protease-activated receptor-2 regulates trypsin expression in the brain and protects against seizures and epileptogenesis. Neurobiol Dis, 2008. 30(1): p. 84-93. 2. Lohman, R.J., Liu, L., Morris, M,. OBrien T.J., Validation of a method for localised microinjection of drugs into thalamic subregions in rats for epilepsy pharmacological studies. J Neurosci Methods, 2005. 146(2): p. 191-7.
Seminars
1. Lohman R-J. Trypsin and protease-activated receptor 2 in the rat brain; roles in epileptogenesis, learning and memory. Institute for Molecular Biosciences, The University of Queensland. Brisbane, May 2008. 2. Lohman R-J. Trypsin and protease-activated receptor 2 in the rat brain; roles in epileptogenesis, learning and memory. 1/ Dept. Pharmacology, The University of Melbourne, February, 2008. 2/ Dept. Medicine, The Royal Melbourne Hospital, April 2008. 3. Lohman R-J. PAR2 in the kindling model of seizure and epileptogenesis. Inflammation Symposium, The Royal Melbourne Hospital, August 2006. 4. Lohman R-J. Extinguishing the fire within; PAR2 in the kindling model of seizure and epileptogenesis. Research in Progress. Department of Pharmacology, The University of Melbourne 2006.
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Posters and abstracts

1. Lohman R-J, OBrien TJ, Cocks, TM. (2008). The involvement of proteaseactivated receptor 2 in learning and memory. Australian Neuroscience Society Conference, Hobart, January 2008. 2. Lohman R-J, OBrien TJ, Cocks, TM. (2007). Trypsin colocalises with tissue plasminogen activator in PAR2 positive neurons in the brain; a protective role in epilepsy. International Brain Research Organisation Conference, Melbourne, July, 2007. 3. Lohman R-J, OBrien TJ, Cocks, TM. (2006). Anti-convulsant and antiepileptogenic effects of protease-activated receptor 2 (PAR2) in the electrical kindling model of epilepsy. 1/ Epilepsy Society of Australia, 21st Annual Scientific Meeting, Melbourne, October 2006. 2/ Australian Medical Research Congress, Melbourne, November 2006. 3/ Medical Research Week, The Royal Melbourne Hospital, 2006. 4. Lohman R-J, OBrien TJ, Cocks, TM. (2006). Protease-activated receptor 2 and trypsin in the rat brain; and immunohistochemistry study. Medical Research Week, The Royal Melbourne Hospital, 2005.
Chapter
1. Myers D, OBrien T, Berkovic S, Lohman R-J, Reid C, Williams D. Exvivo electrophysiology and optical imaging of brain slices in the GAERS epilepsy model Chapter XI, epilepsy program, The University of Melbourne epilepsy retreat 2003.
Awards
1. Best poster prize; Epilepsy Society of Australia, 21st Annual Scientific Meeting, Melbourne, October 2006.Lohman R-J, OBrien TJ, Cocks, TM. (2006). Anti-convulsant and anti-epileptogenice effects of protease-activated receptor 2 (PAR2) in the electrical kindling model of epilepsy.
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Acknowledgements
To my primary supervisor and mentor, Assoc. Professor Tom Cocks, who not only taught me how to be a true scientist, but also that not all things in life are as they seem. Tom, I could not have achieved what I have without your support, be it philosophical, psychological or financial, I cannot thank you enough. As tough a nut you are to crack, it has only made me a better researcher and philosopher. I now have a fantastic career which I could have only imagined, thanks to you, and in the future I can envisage a very prosperous collaboration.
To my co-supervisor, Assoc. Professor Terry OBrien, Terry, your knowledge is immense, and I can only bewilder at how you can function so highly. You are truly a great role model. I could not have had more enthusiastic guiding lights than you and Tom. I thank you for the relentless patience, wisdom and the intellectual input.
Thank you to my good friends and colleagues, Dr Bianca Jupp and Valantina (Aunty) Jovanovska, your support and friendship has always been undying, both in and out of the laboratory, and it has been invaluable. I wish you all the best for a fantastic and prosperous career..
To all past and present members of the OBrien Lab, thank you for the constantly changing and dynamic research environment. It is always inspirational to see such fantastic young minds at work.
To my close friends, Nathan and Aaron Hall, I cannot thank you two enough for your rock solid support and mateship. You were, and probably always will be, willing to
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hear my excitements and concerns throughout my studies and life. The intellecualisation from the devils advocates made me not only re-asses the meaning of my results, but also to realise that there are still always more than one point of view. I truly know who my friends are, and will never forget.
Last but not least, I would like to thank my family, whose love and support will always continue. The underlying and effortless support me realise what the most important things in life really are. I could not have done this with out any of you.
Finally, I dedicate this thesis to my late great grandfather, Louis Jacob Joseph Muskens (1872-1937), who pioneered the epilepsy research field, establishing both the International League Against Epilepsy and the journal Epilepsia. It truly feels like I am walking on the shoulders of giants.
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Table of Contents 1 Epilepsy and inflammation of the brain; Roles for proteases and their receptors in learning, memory and epileptogenesis.....................1
Epilepsy................................................................................................................................................1 1.1.1 The burden of epilepsy..........................................................................................................1 1.1.2 Types of Epilepsy...................................................................................................................3 Seizures.................................................................................................................................................5 1.1.3 Treatment of seizures in epileptic patients..........................................................................7 Temporal Lobe Epilepsy (TLE).........................................................................................................8 1.1.4 Temporal lobe epileptogenesis.............................................................................................9 The hippocampus..............................................................................................................................10 Plasticity, learning and memory in the hippocampus ...................................................................14 1.1.5 A cellular basis for memory; long-term potentiation.......................................................15 Neuronal plasticity and serine proteases in the hippocampus......................................................17 1.1.6 Tissue plasminogen activator (tPA)...................................................................................19 1.1.7 Thrombin.............................................................................................................................19 1.1.8 Motopsin/neurotrypsin......................................................................................................20 1.1.9 Trypsin.................................................................................................................................20 1.1.10 Protease inhibitors.............................................................................................................21 Roles of serine proteases in learning and memory.........................................................................21 The hippocampus, seizures and TLE..............................................................................................23 1.1.11 Histopathologies of TLE...................................................................................................24 1.1.11.1 Hippocampal sclerosis.................................................................................................24 1.1.11.2 Granule cell dispersion of the dentate gyrus ..............................................................26 1.1.11.3 Mossy fibre sprouting..................................................................................................27 Inflammation of the hippocampus and seizure..............................................................................28 1.1.12 Cytokines and seizure.......................................................................................................28 1.1.13 Proteases and seizure........................................................................................................29 Protease activated receptors (PARs)...............................................................................................32 1.1.14 PAR2; pro- or anti-inflammatory?..................................................................................37 1.1.15 PARs in the brain .............................................................................................................40 Animal models of epilepsy................................................................................................................42 1.1.16 The electrical amygdala kindling model of TLE............................................................42 1.1.17 The kainic acid model of TLE..........................................................................................44 1.1.18 The Genetic Absence Epilepsy Rats of Strasbourg (GAERS) ......................................45 Aims and Hypothesis.........................................................................................................................47
2 General Materials and Methods.........................................................49

Animals and postoperative care.......................................................................................................49 Drugs and peptides............................................................................................................................49 Injection protocols.............................................................................................................................50 2.1.1 i.c.v. peptide administration...............................................................................................50 2.1.2 Peripheral peptide injection...............................................................................................51 Surgeries.............................................................................................................................................51 2.1.3 Implantable devices (see Figure 2.1) .................................................................................51 2.1.4 General surgical technique.................................................................................................53 2.1.5 Post-operative care..............................................................................................................54
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Confirmation of surgical accuracy..................................................................................................56 2.1.6 i.c.v. cannula placement......................................................................................................56 2.1.7 Intra-amygdala bipolar electrode placement....................................................................57 Cerebrospinal fluid (CSF) extraction..............................................................................................57 2.1.8 Preparation of CSF samples...............................................................................................58 Models of epilepsy.............................................................................................................................59 2.1.9 The kindling apparatus.......................................................................................................59 2.1.10 Electrical amygdala kindling ...........................................................................................59 2.1.11 Kainic acid induced status epilepticus.............................................................................61 2.1.12 Twenty-four hour recording protocol.............................................................................63 2.1.13 Seizure classification- The scale of Racine......................................................................63 Behavioural monitoring, assessment and scoring..........................................................................66 2.1.14 Elevated plus maze............................................................................................................68 2.1.15 Open field test ...................................................................................................................69 2.1.16 Morris water maze ...........................................................................................................69 2.1.17 Light-dark box ..................................................................................................................71 2.1.18 Sucrose consumption test .................................................................................................71 Tissue collection and processing......................................................................................................72 2.1.19 Transcardial perfusion and rat brain fixation................................................................72 2.1.20 Cryoprotection and tissue storage...................................................................................73 2.1.21 Cryostat sectioning............................................................................................................74 Histology and immunohistochemistry.............................................................................................75 2.1.22 Antibodies and antigens....................................................................................................75 2.1.23 Immunohistochemistry protocol......................................................................................76 2.1.24 Positive and negative controls..........................................................................................78 2.1.25 Pre-absorption experiments.............................................................................................78 2.1.26 Thionin staining protocol..................................................................................................79 2.1.27 Timms staining protocol..................................................................................................79 Microscopy and image acquisition..................................................................................................80 2.1.28 Bright field and fluorescence microscopy.......................................................................80 2.1.29 Confocal microscopy.........................................................................................................81 Quantitation of data..........................................................................................................................81 2.1.30 Quantitation of kindling EEG data.................................................................................81 2.1.31 Quantitation of 24h video-EEG data from kainic acid rats..........................................82 2.1.32 Quantitation of immunofluorescence and mossy fibre sprouting.................................82 2.1.33 Statistical analysis..............................................................................................................84
3 Immunohistochemical localisation of PAR2 and trypsin in the rat brain.........................................................................................................87

Introduction.......................................................................................................................................87 Methods..............................................................................................................................................88 3.1.1 Tissue preparation...............................................................................................................88 3.1.2 Immunohistochemistry.......................................................................................................89 Results................................................................................................................................................89 3.1.3 PAR2 in the rat hippocampus............................................................................................89 3.1.4 PAR2 expression in the cortex, thalamus, striatum, amygdala and white matter tracts ........................................................................................................................................................93 3.1.5 Trypsin expression in the rat hippocampus......................................................................94 3.1.6 Trypsin expression in the cortex, thalamus, striatum, amygdala and white matter tracts..............................................................................................................................................96 3.1.7 Trypsin and tPA colocalise in PAR2-positive neurons....................................................99 3.1.8 PAR2 and trypsin in the pancreas...................................................................................100 3.1.9 Primary antibody pre-absorption....................................................................................100
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Discussion.........................................................................................................................................104
4 The pharmacokinetics of SLIGRL in cerebrospinal fluid.............111

Introduction.....................................................................................................................................111 Methods............................................................................................................................................114 4.1.1 Animals, surgery and confirmation of cannula placement............................................115 4.1.2 i.c.v. peptide injection .......................................................................................................115 4.1.3 CSF extraction...................................................................................................................115 4.1.4 Preparation of CSF samples.............................................................................................115 4.1.5 LCMS-MS..........................................................................................................................116 4.1.6 Confirmation of SLIGRL detection using LCMS .........................................................116 4.1.7 Calculation of half-life (t1/2) of SLIGRL........................................................................117 Results..............................................................................................................................................118 4.1.8 Reliability of CSF extraction from lateral ventricles ....................................................118 4.1.9 Confirmation of SLIGRL detection using LCMS .........................................................118 4.1.10 SLIGRL in CSF following i.c.v. administration...........................................................121 4.1.11 Detection of SLIGRL in CSF following peripheral (i.p.) injection.............................123 Discussion.........................................................................................................................................125
5 The behavioural neurotoxicity of centrally administered SLIGRL 130

Introduction.....................................................................................................................................130 Methods ...........................................................................................................................................131 5.1.1 Animals, surgery and cannula position confirmation....................................................131 5.1.2 Drugs, peptides and i.c.v. injection..................................................................................131 5.1.3 Experimental design and EEG monitoring.....................................................................132 5.1.4 Behavioural monitoring, assessment and scoring..........................................................133 Results..............................................................................................................................................133 5.1.5 Confirmation of the scoring system in NEC rats; ketamine causes neurotoxicity......133 5.1.6 SLIGRL (i.c.v.) induces dose-dependant neurotoxic behaviours.................................134 5.1.6.1 NEC rats......................................................................................................................134 5.1.6.2 GAERS........................................................................................................................137 Discussion.........................................................................................................................................140
6 Anticonvulsant and anti-epileptogenic effects of PAR2 and SLIGRL in the electrical kindling and kainic acid models of TLE. 144
Introduction.....................................................................................................................................144 Materials and Methods...................................................................................................................146 6.1.1 Animals and post-operative care......................................................................................146 6.1.2 Drugs and peptides............................................................................................................146 6.1.3 Surgeries.............................................................................................................................146 6.1.4 Afterdischarge threshold (ADT) determination in the electrical amygdala kindling model............................................................................................................................................147 6.1.5 Electrical amygdala kindling ...........................................................................................147 6.1.6 Kainic acid-induced status epilepticus............................................................................148 6.1.7 Daily peptide administrations..........................................................................................148 6.1.8 Twenty-four hour recording protocol.............................................................................148 6.1.9 Immunohistochemistry and histology.............................................................................148 Results..............................................................................................................................................149 6.1.10 i.c.v. SLIGRL elevates ADT during kindling...............................................................149 6.1.11 i.c.v. SLIGRL administration prevents kindling-induced seizure.............................150 6.1.12 s.c. SLIGRL delivery elevates afterdischarge threshold..............................................152
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6.1.13 s.c. SLIGRL attenuates kindling-induced seizure........................................................154 6.1.14 i.c.v. SLIGRL administration attenuates kainic acid induced status epilepticus ....155 6.1.15 i.c.v. SLIGRL increases latency of spontaneous seizure and reduces seizure duration following status epilepticus........................................................................................................156 6.1.16 PAR2 and trypsin colocalise in neurons in the brain ..................................................159 6.1.17 PAR2 and trypsin increase expression during kindling..............................................160 6.1.18 SLIGRL treatment does not affect mossy fibre sprouting..........................................161 Discussion ........................................................................................................................................161
7 The involvement of PAR2 in fear-aversive learning......................174

Introduction.....................................................................................................................................174 Materials and methods...................................................................................................................176 7.1.1 Animals...............................................................................................................................176 7.1.2 Peptides...............................................................................................................................176 7.1.3 Behavioural testing protocols...........................................................................................176 Results..............................................................................................................................................177 7.1.4 SLIGRL does not affect rat behaviour on the open field test.......................................177 7.1.5 SLIGRL has no effect on light-dark box behaviours.....................................................178 7.1.6 SLIGRL has no effect on sucrose consumption .............................................................180 7.1.7 SLIGRL prevents fear avoidance learning on the elevated plus maze .......................180 7.1.8 Effect of SLIGRL administration on Morris water maze behaviours.........................188 Discussion.........................................................................................................................................191
8 General Discussion............................................................................199
Future perspectives.........................................................................................................................209
9 Final Conclusion................................................................................213 10 References........................................................................................214 We are the music makers, and we are the dreamers of dreams..238
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List of Figures
Figure 1.1 The human brain 28 Figure 1.2 The human hippocampus 29
Figure 1.3 Long-term potentiation........................................................18 Figure 1.5 Inflammation-driven epileptogenesis.................................34 Figure 1.6 Protease-activated receptor 2 .............................................35
Table 1.1 Protease-activated receptor proteolytic and peptide agonists...............................................................................................................................................36
Figure 2.2 Representation of surgical locations of implantable devices 55 Figure 2.3 Representation of final internal locations of implantable devices......................................................................................................55 Figure 2.4 kindling apparatus...............................................................60 Figure 2.5 Schema of kindling appratus...............................................60 Figure 2.7 Typical spontaneous seizure following status epilepticus in the rat.......................................................................................................65
Table 2.1 neurotoxic behavioural scoring legend...........................................................................67 Table 2.2 Neurotoxic behaviour duration scoring legend.............................................................67
Table 2.3 Primary and secondary antibodies used..............................76 Figure 2.8 Regions of interest (ROI) used in the immunofluorescence quantitation experiments.......................................................................85 Figure 2.9 Timms-stained hippocampus and region of interest schema.....................................................................................................86 Figure 3.5 PAR2 and trypsin colocalise in all regions of the rat brain 101 Figure 3.6 Trypsin and tPA colocalise to LDCVs in PAR2 positive neurons..................................................................................................102
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Figure 3.7 PAR2 and trypsin expression in the pancreas.................103 Figure 4.1 CSF can be extracted from the lateral ventricles............119 Figure 4.2 Original LCMS curve of SLIGRL in CSF.......................120 Figure 4.3 SLIGRL can be detected in CSF Figure 4.4 SLIGRL decays with first-order kinetics.........................122 Figure 4.5 SLIGRL crosses from blood to CSF.................................124 Figure 4.6 SLGRIL decays from CSF with first order kinetics.......124 Figure 4.7 Passage of peptides across the blood-brain barrier........129 Figure 5.1 Schematic of neurotoxicity experiment............................132 Figure 5.2 Dose-response curve to ketamine......................................138 Figure 5.3 Dose-response curve to SLIGRL......................................138 Figure 5.4 Examples of EEG patterns caused by SLIGRL..............139 Figure 6.6 Timms staining in the hippocampus g-induced trypsin expression..............................................................................................167 Figure 7.1 SLIGRL has no effect on the behaviours of rats on the open field test. ......................................................................................179 igure 7.2 SLIGRL has no effect on light-dark box behaviours........181 Figure 7.3 SLIGRL has no effect on sucrose consumption in rats. .182 Figure 7.5 SLIGRL impairs fear-avoidance on the elevated plus maze 186 Figure 8.1 Hypothesised feedback inhibition of trypsin by PAR2...208
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Chapter 1 General Introduction
Chapter 1 Epilepsy and inflammation of the brain; Roles for proteases and their receptors in learning, memory and epileptogenesis
Epilepsy Epilepsy describes a broad category of brain disease that involve spontaneous recurrent seizures. It affects people of all ages regardless of sex, race or social status, making it one of the worlds most prevalent neurological conditions. In 2007, the World Health Organisation (WHO) estimated that more than 50 million people worldwide have some form of epileptic syndrome, a figure that is not only growing with the earths population at 2.4 million new cases globally per annum, but also highlights the fact that there is no true preventative cure for this disease (WHO, 2008).
1.1.1
The burden of epilepsy
The word epilepsy comes from the Greek epilavainem, meaning to be taken hold of, and the term seizure means to grasp forcibly. These descriptive terms reflect early beliefs that epilepsy was a sacred disease in that patients suffering from seizures were thought to be possessed by demons as a punishment for previous acts [1] and were described as moonstruck or lunatic. Early treatment of epilepsy generally involved patients being locked up in a sanatorium or mental institute, as epilepsy was considered a psychiatric disorder. This remained the case until Hippocrates in around 400BC suggested that epilepsy was a disease of the brain and as such should be treated with drugs or diet and not with exorcism or religious incantations [1]. However, even today, a stigma still exists for epileptic patients. Epileptics are often ostracised, stemming from a lack of understanding of epilepsy as a syndrome and a fear of epileptic people having a seizure [2]. Thus, patients are often labelled as
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different or ill and excluded from many social activities due to the potential risk of seizure. There are various types of epileptic syndromes, which will be discussed in depth later. Some, however, have such mild physical manifestations (i.e. absence seizures) that they may go unrecognised by the general public. In reality, in the developed world, up to 80% of patients with epilepsy are well managed by their medication and can thus lead relatively normal lives (WHO, 2008).
Epilepsy can be associated with a considerable burden of impaired quality of life and disability, as it is often associated with a range of co-morbidities such as physical injuries and psychiatric disorders. It can often lead to premature mortality. When onset of epilepsy occurs during childhood or adolescence, there can be serious ongoing effects on psychological development, educational accomplishment and the transition to working life. Furthermore, patients may be restricted in performing certain basic yet dangerous tasks such as driving a car. In Australia, if a patient has an isolated seizure, they are liable to loose their drivers license for a minimum of three months and a patient diagnosed with chronic epilepsy must be seizure-free, that is, not had a seizure, for a minimum period of two years before being able to drive again (www.epinet.org.au). About 20% of patients loose their license permanently, potentially costing them their jobs as well as their independence and further contributing to their stigmatisation.
There is always a risk of physical injury for epileptics, mainly as a result of falls during seizures, with head injuries, such as cerebral haemorrhage, possibly adding to the risk of further seizure. In addition, the unpredictable nature of seizures can lead to psychiatric conditions such as depression and anxiety, both of which can add to the
stigma of having a mental illness.
1.1.2
Types of Epilepsy
Epilepsy does not describe a single disease, rather it broadly defines a group of neurological diseases and syndrome which have in common the occurrence of spontaneous, recurrent seizures. Diagnosis of a particular syndrome is based upon the patients symptoms, such as seizure type, pathophysiology, cause, onset and management of seizure. Certainly, the greatest diagnosing factor is the prevalence of seizure, however, due to there very nature (i.e. losses of consciousness), the patient may themselves not be fully aware of the symptoms, or that they had a seizure at all, in some cases. For this reason, an eyewitness account of the seizure manifestations is usually required for a physician to fully understand the type of seizure and potentially the form of epilepsy encountered.
The origins of epilepsy are genetic, acquired or idiopathic [1]. Genetically-based forms of epilepsy are usually generalised conditions, such as absence epilepsy, generalised epilepsy with febrile seizures (GEFS) or certain focal epilepsies [3]. These can affect entire families. These are more often than not a result of a defect in a gene encoding a voltage- or ligand-gated ion channel [4, 5] such as T-types calcium channels (i.e.CaV.3.2/CACNA1H) [3] (Kyi et al, unpublished observations), sodium channels (i.e. SCN1A and SCN1B)[3], the GABAA receptor (GABRA1) and chloride channels (i.e. CLCN2)[6]. Furthermore, some families may be genetically susceptible to acquiring epileptic syndromes due to a genetic background which renders them more at risk of seizure following a brain insult.
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Patients with acquired epilepsy have known precipitating causes or pathologies of the condition, such as cerebral infections (meningitis or encephalitis), sustained head injury, haemorrhagic or ischaemic stroke, brain tumour or other forms of neurological insults or neurodegenerative diseases like Huntingtons [7] and Alzheimers disease [8]. In developing countries, many cases of acquired epilepsy are caused by preventable parasitic infections, such as neurocysticercosis, schistosomiasis and malaria (WHO).
A third type of onset is idiopathic epilepsy, which defines patients with no known physical brain lesion or identified genetic mutation that underlies the cause of their seizures. Many such cases, however, will presumably fall into the genetic category as more seizure-causing gene mutations are discovered [4].
The most common causes of epilepsy in children is either genetic or cerebral infection, as often indicated by febrile seizures [9]. In young adults the greatest cause of epilepsy is traumatic brain injury, often the result of falls or motor vehicle accidents (www.braininjury.com.au), whilst in older people the most common causes are tumour, stroke and degenerative conditions such as Alzheimers disease [8]. These forms of epilepsy are generally associated with an acquired defect or lesion (scar) in the brain around where the seizure pattern originates (i.e. the seizure focus) [10]. Even though these epileptic conditions are acquired, they are secondary to the primary condition (i.e. Alzheimers) and are thus considered co-morbidities. Epileptic seizures are often co-morbid to other neurological conditions, for instance, people with developmental mental retardation or other developmental brain defects often have epileptic seizures. This thesis will primarily concentrate on temporal lobe epilepsy,
the most common form of acquired epilepsy.
Seizures A seizure is an unprovoked, sudden burst of highly synchronised excitatory electrical activity in the brain that is usually accompanied by physical and psychological manifestations, ranging from mild altered states of awareness and sensory or emotional hallucinations, known as auras, to physical manifestations such as mild facial clonus, tonic-clonic limb jerking and severe losses in balance, consciousness and posture. Seizures can be partial or focal, occurring and remaining in a specific region of the brain, or generalised, involving the entire brain simultaneously. The latter types of seizure are usually associated with losses of consciousness. Partial seizures can also spread to become secondarily generalised seizures, progressively involving more brain regions. Seizures can also be classed as simple or complex, the latter referring to when losses of consciousness are experienced.
A seizure is believed to be caused by a disruption in the balance between neuronal excitation and inhibition within the brain [1, 6, 11], a phenomenon referred to as a paroxysmal depolarisation shift [1, 12], where an entire group of neurons simultaneously depolarise making them more susceptible to hyperactive burst firing and seizure-like discharges. In a complex partial seizure, for instance, neuronal excitation can spread from the focus to incorporate other regions of the brain, such as the sensory and motor cortices, causing secondary generalised and more severe seizures described above. As the seizure spreads over the motor and sensory cortices, the region of excitation of the homunculus is reflected by the physical manifestations. For instance, a seizure may begin with a simple finger twitch evolving to include the
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whole arm, shoulder and eventually the entire body, such as experienced during a tonic-clonic seizure. This is termed a Jacksonian March after British Neurologist John Hughlings-Jackson who first described this progression [13].
The stimulus required to induce such burst firing in any one region is called the seizure threshold. People with epilepsy have a lower seizure threshold, so a physiological input stimulus can induce aberrant hyperexcitability and seizures [1, 6, 11]. Most anti-convulsant medications work by raising the seizure threshold, thereby reducing the relative excitation, dampening the paroxysmal depolarisation shift and as such, reduce the risk of seizure [1, 6, 11, 14-19].
It has long been postulated that seizures beget seizures [20-23], i.e. if a seizure has occurred the chance of having another is increased. This may be the crux of the so called vicious cycle of epileptogenesis, where one seizure leads to another and so forth. Such heightened seizure risk may be due to the described long-lasting paroxysmal depolarisation shift within groups of neurons in the brain, which may never return to a normal polarisation state. Therefore, epileptic brains appear to experience long-lasting and excessive potentials for neuronal synchronisation and hyperexcitability and therefore may respond to normal physiological signalling with excessive burst firing, courtesy of the depolarisation shift described.
1.1.3
Treatment of seizures in epileptic patients
To date the most effective treatment for epileptic patients are anticonvulsants, such as carbamazepine, phenytoin and valproate, and more recently developed drugs such as lamotrogine and levetiracetam. These medications merely prevent onset and spread of seizures, but the underlying epileptic condition still exists and seizures will recur if the medication is ceased. Furthermore, up to 30% of patients become refractory to their medications and as such, constant re-evaluation of therapeutic regimes and changes in medications administered are generally required.
The anti-seizure mechanism of action of most anticonvulsants is not fully understood, although it is recognised that they generally function to increase the seizure threshold by reducing cationic channel function, increasing anionic currents and/or interfering with neurotransmission [15, 16, 18, 19, 24-26]. However, the nature of action of these medications means they can often lead to neurological side effects such as speech and motor disturbances, sedation, confusion and deficits in concentration and memory [17, 27-30]. Furthermore, in a subset of patients, certain medications can result in seizure aggravation, particularly for carbamazepine in absence epilepsies [31-34] and in patients prescribed lamotrigine [30, 35], vigabatrin and gabapentin [30] with symptomatic generalised epilepsies. Other common side effects of these drugs include rashes [30], bone mineral density loss [30, 36], weight gain [30, 37, 38] alterations in balance (Dr Sandra Petty, verbal communication) and cognitive impairment [17, 28, 30]. Finally, some of these medications have been reported to cause life-threatening, adverse reactions such as multiple organ failure for lamotrigine [30].
For the most severe cases of temporal lobe epilepsy, where patients become resistant
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to anti-convulsant treatment, the epileptic hippocampus is surgically removed unilaterally, in a process known as hippocampal resection. The outcomes are generally very good, with the majority of patients becoming seizure free following resection, as well as having a substantially better neuropsycology [39]. However, only a minority of patients with medically refractory epilepsy are suitable for epilepsy surgery.
One of the great challenges for improving epilepsy therapies is to develop neuroprotective treatments that interfere with the process of epileptogenesis, i.e. drugs that are truly disease modifying. Although this is primarily due to the lack of knowledge of the intricacies of the epileptogenic period and the specific underlying causes of the epileptic pathophysiologies, recent evidence suggests that inflammatory processes may be involved in the ontogenesis of epilepsy [40-44]. Thus, perhaps such anti-epileptogenic treatments may best target inflammation, rather than neuronal excitability. Temporal Lobe Epilepsy (TLE) TLE is the most common form of acquired epilepsy and it is usually caused by brain insults such as head trauma, tumour, stroke or cerebral infection such as meningitis or encephalitis. TLE patients, whether young or old, usually experience complex partial seizures and rarely only simple partial seizures. As the name suggests, TLE has the seizure focus in the temporal lobe, however, due to the high inter-connectivity of neurons in this region to others, the seizure usually spreads to higher brain structures such as the motor and sensory cortices. Thus, most patients experience complex partial seizures followed by secondary-generalised and more severe tonic-clonic seizure after the seizure has spread from the focus to the motor cortex.
The temporal lobe is found bilaterally in the human brain (Figure 1.1) lying beneath the sylvian fissure of the parietal and frontal lobes and in front of the occipital lobes. It is part of the telencephalon (or cerebrum) and houses the greater part of the limbic system; the hippocampus, fornix and the amygdala.
1.1.4
Temporal lobe epileptogenesis
TLE is often regarded to be a disease of the hippocampus, because most histopathologies associated with the syndrome are most obvious in this region of the brain [1]. Its ontogenesis, however, is poorly understood, although after an initial precipitating insult such as head trauma, vascular trauma (i.e. stroke or haemorrhage), cerebral infection or tumour in the more fragile temporal lobe, a patient can live for months to years without a physical seizure, yet at some point may experience one spontaneously. If such seizures become recurrent, epilepsy is diagnosed. The latent, seizure-free period following the initial brain insult is referred to as the epileptogenic period during which pro-epileptic, histopathological changes occur, mainly in the hippocampus [45-49] but also in the amygdala [1, 50].
Inflammatory events in the brain following an initial insult may play a key role in the ontogenesis of epilepsy [41, 43, 51-58]. Also, each seizure is an insult to the brain and adds to the initial inflammatory response. This occurs not only because of glutamatergic toxicity [1, 59, 60], but also the release of inflammatory factors (i.e. proteases and cytokines) from both cells of the brain and vascular sources, such as mast cells entering the brain matrix and cerebrospinal fluid during seizure [40, 41, 43, 44, 54-56, 61-76].
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Since the hippocampus is a critical structure in TLE to the anatomy and neurophysiology of this important limbic structure will be described in some detail.
The hippocampus The hippocampal formation, an integral part of the mammalian limbic system, encompasses the entorhinal cortex, dentate gyrus, hippocampus proper and subiculum (Figure 1.2). It has extensive connections to the amygdala, the parahippocampal gyrus and the neocortex. The structure of the hippocampus is well conserved between the rat and human, making the rat a good model for studying human hippocampal functioning in health and disease.
The hippocampus is a highly organised neuronal network, divided into subregions with interconnecting lamina and synaptic pathways. The cornu ammonis (CA) or Ammons horn, (referring to the rams horn-like shape similar to that on the head of the Egyptian god Amun), can be divided into distinct layers called the stratum oriens, stratum pyramidale, stratum radiatum and stratum lacunosum-moleculare. The stratum pyramidale consists primarily of excitatory pyramidal cells divided into four regions termed the CA1, CA2, CA3 and CA3c (also known as the hilar region (dentate hilus) or CA4). The stratum oriens contains the basal dendrites of these neurons that project from the CA3c to the CA1 via the associational-commissural fibres of the CA2 region.
The major gateway from the entorhinal cortex into the hippocampus is the dentate gyrus. This consists of a layer of granule cells that send neuronal signals along
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granule cell axons, called mossy fibres (by Ramon y Cajal), towards the CA3 pyramidal neurons. In turn, these project via the Schaffer collaterals to join with association-commissural fibres that arise from the fornix and the contralateral CA3 region. The dentate gyrus can also be divided into layers: the stratum moleculare, stratum granulosum (granule cell layer) and dentate hilus. Apical dendrites of both granule and pyramidal cells extend into the stratum moleculare and stratum lacunosum-moleculare, respectively. The dentate hilus (CA4/CA3c) is difficult to characterise, as it known as a polymorphic region where, besides pyramidal neurons, cell types with as yet undefined properties exist [77-79].
Ultimately, signals enter the dentate gyrus from the entorhinal cortex via the perforant path and exit via the mossy fibre pathway, projecting to the CA3 and CA2. They then project to the CA1 pyramidal cells via the association-commissural fibres and via the entorhinal cortex to the neocortex. This forms what is known as the tri-synaptic pathway (Figure 1.2).
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Figure 1.1Regions of the human brain
Figure 1.1 Regions of the human brain. Picture obtained from www.science.ca/images/Brain_Witelson.jpg
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Figure 1.2 The human hippocampus
DG
Figure 1.2 The human hippocampus. A; diagram of the relative position of the hippocampus in the human brain (from;http://discovermagazine.com/2007/). B; cross section of a human hippocampus stained with thionin showing pyramidal cells of the cornu ammoni (CA1-3) and granule cells of the dentate gyrus (DG) (sub; subiculum) (image courtesy of Bianca Jupp). C; Shows simplified circuit diagram of the hippocampus with respect to the granule cells of the DG (filled circles) and the pyramidal cells of the CA1-3 (filled triangles), incorporating the mossy fibre pathway (mf); distinctly shows is the trisynaptic pathway from the enterorhinal cortex (EC ) via the perforant path (PP) to the DG, from the DG to CA3 and from the CA3 via Shaffer collaterals (sc) to the CA1 (from; www.behavioraland brainfunctions.com ). D; a more complex diagram of hippocampal circuitry showing directions of neural signals in and out of the hippocampus (from; www.bris.ac.uk) (ff; fornix, AC; associational commissural path).
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Plasticity, learning and memory in the hippocampus The hippocampus is a highly plastic structure, meaning it is susceptible to changes in cell responsiveness to certain stimuli, as demonstrated in long-term potentiation and long-term depression experiments [6, 80-83] (see below), as well as to changes in synaptic organisation and cell-to-cell connections [84-86]. Such plasticity of structure and function highlights the importance of the hippocampus in the formation of, in particular, short-term and declarative memories (e.g. where you left your car keys) [87-89]. These short term memories will eventually be stored as long-term memories in the neocortex (e.g. family names and birth dates) if the same input stimuli are frequently repeated as part of the learning process. Therefore, the hippocampus can be thought of as a short-term memory storage device, whereas the higher memory centres of the cortex store more permanent long-term memories. The highly plastic nature of the hippocampus, however, also makes it vulnerable to pathological changes, which can lead to various disease states such as Alzheimers disease [90-92] and epilepsy [29, 49, 93-97].
Some of the best evidence for hippocampal functioning in both learning, memory and disease comes from a monumental blunder in the 1950s on a TLE patient referred to as H.M. [98]. H.M. suffered from severe generalised seizures which were insufficiently controlled by anti-convulsant therapy. Therefore, H.M. was selected for radical temporal lobe surgery during which the greater part of the temporal lobe was removed bilaterally, including both hippocampi and the majority of both amygdalae. Following surgery, H.M. no longer suffered from seizures but now suffered from severe amnesia of all post-surgical events, although he still was able to recall events
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prior to hippocampal removal [98]. It was quickly concluded that the hippocampus is a crucial structure not only in learning and memory formation, but also in seizure generation and epilepsy. Consequently, in order to avoid such disastrous memory outcomes, only unilateral hippocampal resections are now performed on only the worst of patients with medically refractory TLE.
1.1.5
A cellular basis for memory; long-term potentiation
In the hippocampal pyramidal and granular neurons, new cellular memories are thought to be created by lasting changes in cell responsiveness likely due to changes in synapse number as well as the types and levels of receptor, neurotransmitter and protein expression within these synapses [80, 83, 99]. This is achieved through a process known as long-term potentiation (LTP). LTP describes a heightened response in the magnitude and persistence of the synaptic excitatory post-synaptic potential (EPSP) of post-synaptic neurons in response to a tetanic pre-synaptic input [81, 83, 100]. This is caused by an increase in responsiveness to the post-synaptic NMDA receptor-dependent signal transduction mechanisms and/or an increase in the population of NMDA receptors on the post-synaptic membrane [81]. Thus, a given input can cause lasting changes in cell excitability (Figure 1.3). By contrast, long-term depression (LTD) describes lasting decreases in synaptic responsiveness that follow certain types of electrical stimulation in the hippocampus, that may be caused by opposite effects on neurotransmitter and receptor properties as described above for LTP [101], resulting in a dampened EPSP. LTP and LTD are thought to be the cellular basis for learning and memory [82, 83].
Three distinct types of LTP have been identified (LTP1, LTP2 and LTP3) which are
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characterised by the persistence of the signal potentiation and the degree of recruitment of protein synthesis and gene transcription [81, 83]. All three forms of LTP are NMDA- and calcium-dependent [81, 83].
LTP1 (or early LTP (eLTP)) is induced by the weakest stimuli and is the shortest lived of the three types of LTP, decaying back to baseline within minutes. LTP1 is independent of protein synthesis and most likely involves post-translational protein modifications and ryanodine (Ry) receptor-induced calcium release from intracellular stores [81].
LTP2 (intermediate LTP (iLTP)) is induced by moderate stimuli and is more robust than LTP1. It is dependent on protein synthesis but not gene transcription. Unlike LTP1, LTP2 is dependant on intracellular calcium liberated by inositol (1,4,5) triphosphate (IP3), and not Ry receptors [81].
LTP3 (late LTP (lLTP)) is the most robust of all the LTPs and is induced by the greatest stimulus strentgh. It can last for months in vivo and weeks in vitro [81, 83]. LTP3 is dependent on gene translation and transcription, as well as L-type voltagegated calcium channels, with no requirement for intracellular calcium stores [81].
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Neuronal plasticity and serine proteases in the hippocampus As well as changes in neurotransmitter and receptor sensitivities as described in LTP, structural changes in pre- and post-synaptic cell-to-cell connections also contribute to plasticity in the hippocampus. By cleavage of the cell matrix and the pruning of existing synapses, proteases can create a physical environment for the formation of new synaptic connections to facilitate the formation of memories [84, 85, 88, 99, 102, 103].
Many proteases are expressed in the brain, including cathepsins, caspases and calpains (calcium-dependent cysteine proteases), matrix metalloproteases (MMPs) and serine proteases. This thesis concentrates on the latter. Low levels of serine proteases including tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) [40, 42, 73, 104-107], neuropsin [63, 87, 103, 108-110], motopsin/neurotrypsin [103, 111-113], thrombin [65, 87, 103, 114-120], trypsin [73, 121] and various trypsin-like enzymes such as mesotrypsin [122-124], P22 [122, 125] and myencephalon-specific protease [126, 127], are normally present in limbic regions of the brain including the hippocampus. Some of these are discussed in detail below.
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Figure 1.3 Long-term potentiation

Stimulating electrode Recording electrode
Figure 1.3 Long-term potentiation (LTP). A; shows a basic experimental set-up to record LTP with a stimulating electrode in the Schaffer collaterals and a recording electrode in the pyramidal cell layer of the hippocampus. B; shows an example of both early LTP (LTP1) and late LTP (LTP3) where the slope of the EPSP stays elevated from baseline for a long duration following trains of stimulation to the Schaffer collateral pathway. Note the greater the trains, the greater and more robust the response (from; http://www.unmc.edu/Physiology/Mann/pix_19/ltp6.gif).
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1.1.6
Tissue plasminogen activator (tPA)
tPA is the most studied of the serine proteases expressed in the brain and it is involved in a wide range of protease-dependent processes generally regarded as neuromodulatory [42, 64, 87, 107, 128, 129]. In the brain tPA converts plasminogen to the active protease plasmin which then degrades extracellular matrix adhesion proteins such as laminin [85, 86] with a resultant disruption of cell connections and synaptic integrity. In addition, tPA has been shown to potentiate NMDA receptor signalling, possibly via NR1-subunit cleavage [130] that is seemingly independent of plasminogen activation [131]. In hippocampal pyramidal neurons, tPA is stored in large dense core vesicles (LDCVs) in the soma and axons [132, 133]. tPA is considered a marker for LDCVs [132]. During neuronal stimulation LDCVs contents such as tPA are presumably exocytosed onto the matrix [132-134].
1.1.7
Thrombin
Prothrombin mRNA and protein have been extracted from primary neuronal cultures and from both human and rat brains at various stages of development [65, 87, 114, 116-120]. Astrocytes and microglia also express thrombin [135-137] and perhaps interestingly, the principle activator of the zymogen prothrombin, Factor Xa, is present and functional in the rat brain [138, 139]. Much like tPA, thrombin can potentiate glutamatergic signalling via NMDA receptors in hippocampal CA1 neurons [66, 87].
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1.1.8
Motopsin/neurotrypsin
The trypsin-like protease motopsin (or neurotrypsin), has been detected at the mRNA and protein levels in the brain [111-113, 122]. This serine protease appears to have important roles in brain development since deletion or mutation of the motopsin gene causes non-syndromic mental retardation in humans [111, 112]. Also, a species specific homologue in Drosophila (Tequila) has been implicated in long-term memory formation [140], although this has been challenged [141]. Motopsin is stored in vesicles within neurons, although distinctly separate from those containing tPA [111].
1.1.9
Trypsin
Trypsin has been shown to be present in pyramidal cells in human hippocampi using immunohistochemistry [121] and as the zymogen trypsinogen at the mRNA and protein levels [103]. The endogenous activator of trypsinogen in the intestine, enterokinase, is also expressed in the brain at the mRNA level [142, 143], thus, in a similar manner to thrombin, regulation of trypsin activity may be intrinsic in the brain.
The subcellular trypsin localisation in the brain has, however, not been well characterised, yet this knowledge is vital to fully understand its regulation and function. Perhaps like tPA [132, 133] and motopsin [111], trypsin is stored in specific vesicles, much like it is in acinar cells of the pancreas [144].
Trypsin has been found to induce epileptiform discharges in rat brain slices, responses
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that were blocked by the NMDA receptor antagonist MK-801 [145]. Therefore, much like tPA and thrombin, trypsin may potentiate glutamatergic signalling at the level of NMDA receptor.
Most available evidence indicates that the isoform of trypsin expressed in the brain is the extra-pancreatic type called trypsin IV or mesotrypsin (PRSS3) [122-124]. According to a Blast search (http://www.ncbi.nlm.nih.gov/blast/bl2seq/ wblast2.cgi? 2), trypsin IV shares approximately 93% homology with the pancreatic trypsin (trypsin I (PRSS1)), and thus similarities in both structure and function of these two proteases may be expected.
1.1.10 Protease inhibitors As well as many serine proteases, an equally impressive number of serine protease inhibitors (serpins) are found in the brain. These include protease-nexin-1 (PN1) [146, 147], plasminogen activator inhibitor (PA-I) [148], anti-thrombin III [117, 149] and neuroserpin [150-155]. Neuroserpin is exclusively expressed by neurons [151, 153], and PN1 by both glia and neurons [91, 147, 156, 157]. Perhaps relevant to the outcomes of this thesis, neuroserpin is stored in LDCVs together with tPA [152] indicating that this protease and its major endogenous inhibitor are released together, perhaps to tightly regulate matrix and synaptic reorganisation.
Roles of serine proteases in learning and memory The concept of protease-mediated memory formation has arisen from in vivo behavioural studies that suggest serine proteases like tPA [158-160], neuropsin [109] and thrombin [161, 162] are important in both emotional reward and stress-induced,
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anxiety-like behaviours [102, 131, 159]. Specifically, the inhibition of certain serine proteases [150] or deletion of the gene encoding tPA, for instance, prevents the development of both anxiety-like behaviours following acute-restraint stress [102, 159] and neophobic or fear aversive behaviours [150]. Furthermore, over-expression of tPA results in enhanced learning performances in vivo [163]. These behaviours are thought to be related to protease-dependent synaptic remodelling and neuronal structural changes, such as dendritic spine-loss [164-166].
Electrophysiological experiments have also described effects of proteases on learning phenomena [64, 67, 84, 87, 99, 108, 128, 163, 167-170]. LTP and LTD are dependant on NMDA receptor signalling [81] and proteolytic synaptic reorganisation [84, 85, 170]. tPA is an early gene activated in LTP [64] and it has been shown to be essential in the enhancement of post-tetanic changes in late-phase LTP (LTP3) induction [87, 128, 163, 171, 172]. Likewise, inhibition of LTP3 has been shown using tPA inhibitors [173] and LTP3 cannot be induced in tPA gene knock-out (k/o) mice [172]. Other brain proteases, such as MMP-9 [168, 169, 174] and neuropsin [108, 109] also appear to be essential in both early and late phase LTP (LTP1 and LTP3) [87, 108, 109]. Finally, as discussed above, tPA [130], thrombin [66] and trypsin [145] appear to potentiate NMDA receptor signalling possibly by proteolytic cleavage of the NR subunits of NMDA receptors [130, 175]. Thus, serine proteases may facilitate cellular learning (i.e. LTP) by enhancement of NMDA receptor signalling.
Overall, these studies suggest that many serine proteases expressed in the brain, in
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particular tPA, play important mechanistic roles in memory formation and learning in the hippocampus. In a whole animal such protease-dependent learning may relate to, in particular, the development of fear-motivated learning and memory following stressful situations [159].
As briefly alluded to earlier, the co-storage of tPA (and most likely other proteases) and neuroserpin in LDCVs of pyramidal neurons indicates that these are likely to be released together from the cell as a neuromodulatory package to mediate neuronal plasticity in the hippocampus. Thus, by cleavage of the adhesion molecules forming the cell matrix, like laminin, [83, 85, 125, 170], and together with many other factors (such as the cytokines IL-1 [52, 83, 176] and TNF [177]), proteases have the potential to promote fibre outgrowth [178-180], synaptogenesis [181], as well as facilitate LTP by NMDA-mediated mechanisms. Therefore, such proteases and inflammatory factors may provide a multi-faceted approach to synaptic plasticity and memory formation following their release from neurons and other cells of the hippocampus [89].
The hippocampus, seizures and TLE The hippocampus is believed to be critically important in the generation of seizures in patients with TLE. As mentioned earlier, seizures are initiated by a hypersynchronous, paroxysmal depolarisation shift that begins in a group of neurons located at the seizure focus (i.e. in the temporal lobe in TLE) [182]. This hyperexcitability can then spread to surrounding neurons, progressively affecting the entire brain as seen in a generalised seizure. The hippocampus may be central to the synchronisation of this spread [6, 10]. Whilst excessive neuronal excitation, or
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impaired inhibition, in such a neuronal group is intuitively the cause of such depolarisation shifts, the onset of hyperexcitability in the seizure focus is not well understood. In epileptic hippocampi plastic changes similar, but excessive, to LTP have frequently been reported [6, 12, 29, 64, 94-97, 183]. Perhaps these proepileptic changes in LTP allow hippocampal neurons to depolarise aberrantly in response to physiological stimuli, such as that provided by glutamatergic signalling, thus potentially inducing a paroxysmal depolarisation shift, a reduction in seizure threshold and an increased potential for seizure [6, 29, 59, 94, 184].
The specific mechanisms underlying excessive plasticity associated with epilepsy are not well understood, although inflammatory events within the brain following insult is most likely a major contributing factor [41, 43, 51, 53, 54, 56, 62], as will be discussed later.
1.1.11 Histopathologies of TLE 1.1.11.1 Hippocampal sclerosis The most common histopathology found at autopsy or from surgically-excised human temporal lobes taken from TLE patients is hippocampal sclerosis, which consists of a loss of pyramidal cells in the hippocampus [1] (Figure 1.4) predominantly in the CA1, CA3 and hilar (CA3c/CA4) regions of the hippocampus [1]. It is controversial, however, whether this cell loss in hippocampal sclerosis contributes to seizure generation, or is rather is a consequence or epiphenomenon of the recurrent seizures [78, 95, 185-187]. Animals in the electrical amygdala kindling model of epilepsy generally not only fail to show any significant cell loss in the hippocampus [185] and also fail to exhibit spontaneously occurring seizures (see Chapter 2). By contrast,
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however, studies using status epilepticus models of epileptogenesis have shown progressive cell losses in the CA1 and CA3 regions of the hippocampus that correlate with the appearance of spontaneous seizure [86, 188, 189], similar to that found in humans. Cell loss in the hippocampus may, therefore, be related to spontaneouslyoccurring seizures rather than those induced by an electrical stimulation, such as in kindling. Such cell loss can also contribute to progressive alterations in potential for memory formation in epileptic patients [17, 28, 30].
Hippocampal sclerosis is also associated with an invasion and proliferation of reactive astrocytes into the hippocampus, a process known as astrogliosis, or scarring [1, 190-195]. Astrocytes have been classically thought of as the glue between neurons that holds neuronal networks together. However, modern ideas suggests these cells 'talk and listen' to neurons and thereby maintain a normal extracellular environment. As such, astrocytes are regarded as vital support cells to neurons [44, 190, 193-198], influencing neuronal survival by providing glucose, regulating neurotransmitter (glutamate and GABA) uptake and release, scavenging free radicals, transporting water [198] and producing pro-inflammatory cytokines such as TNFa [41, 62, 183, 199, 200], IL-1 [41, 44, 176, 201, 202] and IL-6 [62], as well as other inflammatory mediators like prostaglandins (PGE2) and nitric oxide (NO) [58, 198]. An example of the importance of these cells may be observed in models of brain injury where they have been shown to be important in regulating neurite outgrowth, synaptic plasticity and neuronal regeneration. Thus astrocytes may be important in epileptogenesis [191, 193-195] but also in recovery after brain injury [198, 203]. In the brain, astrocytes become reactive during ischaemia, Parkinsons disease, Alzheimers disease, hypoxia, trauma and seizure [58, 115, 190, 191, 195, 197, 204,
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205]. When reactive, their morphology changes from a polygonal to a stellate shape, with large processes extending up to hundreds of microns in between nerve cells [193, 195]. When this occurs, the end-feet of astrocytes supporting the vascular integrity of the blood-brain barrier, swell and retract [206], making it permeable and leaky. In epileptic brains, hippocampal astrocytes become reactive and stellate [195] and these have a deficit in glutamate uptake that may contribute to the paroxysmal depolarisation shifts of the surrounding neurons [195]. The ensuing high levels of extra-synaptic glutamate may therefore contribute to hyper-excitability, seizure-like electrical activity and neuronal damage and cell death [191, 195, 197].
Also, the stellation of reactive astrocytes in epileptic brains, especially in the hippocampus, may contribute to the synchronisation of seizure [195], where many nerve cells have the potential to react to input information from only a few astrocytes.
1.1.11.2 Granule cell dispersion of the dentate gyrus Epileptic brains frequently demonstrate dispersion of granule cells in the dentate gyrus [49, 93, 207-209] (Figure 1.4). This dispersion is not only associated with cell loss, but also with excessive ectopic granule cell neurogenesis [49], with neurogenesis taking prevalence over loss (Dr Bianca Jupp, personal communication). This granule cell dispersion observed in epileptic brains is also associated with an inappropriate degree of cell migration, causing a broadening of the dentate gyrus [1, 10]. Why this occurs during epileptogenesis is unknown, although some evidence supports the involvement of mutations or deficiencies in a gene encoding a protein, reelin, essential for neuronal migration [210-213]. Altered reelin expression is induced by seizure [212] that in turn may alter the structural and migratory properties of new
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granule cells within the dentate gyrus [212]. How this phenomenon contributes to epilepsy and seizure is unknown, although it has been suggested to contribute to hyperexcitability, particularly in the ectopic cells of the hilar region [1, 10, 212, 214] which can lead to seizure generation.
1.1.11.3 Mossy fibre sprouting Another histopathology of TLE originating in the dentate gyrus is aberrant dendrite growth of the mossy fibre pathway, known as mossy fibre sprouting. It is prevalent in the human epileptics and has also been reported in both electrical kindling [215-224] and status epilepticus animal models of epilepsy [215, 225-227]. Mossy fibre sprouting is most commonly found in the outer molecular layer of the dentate gyrus, where the axons of the granule cells sprout new collaterals which connect with the proximal dendrites of mossy fibres in the inner molecular layer [10, 48, 218]. This forms recurrent circuits within the dentate gyrus that may extend into the hilar region and may enhance the susceptibility for seizure [216, 226, 228-230] by inducing selfperpetuating, recurrent excitation and aberrant and persistent electrical signaling in the hippocampus [12, 48, 215]. Like for hippocampal sclerosis (see above), however, whether mossy fibre sprouting actually contributes to seizure is still controversial [219, 221, 222, 231, 232]. It has been proposed that rather than contributing to hyperexcitability and seizure, mossy fibre sprouting may function as a compensatory mechanism against aberrant hyperexcitability during seizure. This is because some of the newly-synthesised recurrent axonal sprouts can innervate a sparse network of inhibitory interneurons in the hilus that have extensively divergent axons connecting with excitatory granular neurons [1]. Therefore, it is plausible these few inhibitory neurons may inhibit a large group of excitatory dentate neurons, thereby dampening
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seizure risk. Lastly, mossy fibre sprouting may be incidental to epileptogenesis since the same phenomenon has been demonstrated in pathologies that do not usually involve seizures, such as bipolar affective disorder [233] and Alzheimers disease [234-236].
Inflammation of the hippocampus and seizure The role of inflammation of the brain and seizure initiation has, until recently, received little attention. High levels of many cytokines and proteases have been found in the brain during and after seizure in both humans and in animal models [41, 43, 51, 53, 54, 56, 62]. Thus presumably, a balance between cytokines, proteases and protease inhibitors exists for normal brain functioning [129, 151]. However, if large amounts of these factors are released after brain injury as an inflammatory response, it follows that damage may occur due to the disruption of this balance.
1.1.12 Cytokines and seizure Pro-inflammatory cytokines such as IL-1, IL-6 and TNF are rapidly induced after traumatic and excitotoxic brain insults. Recent clinical and experimental evidence suggests that such inflammatory events in the brain may substantially contribute to epileptogenesis. For example, TLE patients express high levels of the proinflammatory cytokines IL-6 [70, 71, 237], IL-1 , TNF [54, 238], as well as the endogenous IL-1 receptor antagonist [71] in cerebrospinal fluid (CSF) compared to non-epileptic patients. Various animal models of seizure and epilepsy show increased pro-inflammatory cytokines (IL-1, IL-6 and TNF ) at the mRNA and protein levels in the hippocampus following seizure [41, 43, 44, 51, 55, 154, 202, 239, 240], as well as increased cytokine levels in CSF [61, 62, 241, 242], much like observed in the
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human. Furthermore, bacterial lipopolysaccharide [51] and intracerebroventricular (i.c.v.) treatment with high doses of IL-1 and TNF- induced seizure in mice [43, 44, 239]. In contrast, administration of IL-1 in rats in the kindling-model of epileptogenesis showed a reduced acquisition of behavioural seizures [243]. Although it is known that seizures affects the permeability of the blood-brain barrier by astrocyte end-foot retraction [43, 75], it appears that microglia and astrocytes are also the first cells to produce cytokines after seizure [41, 202]. High levels of these cytokines may also contribute to post-status epileptogenesis, such as cell loss [41, 202]. Therefore, cytokines may be critical in the inflammatory cascade that can lead to epileptogenesis. 1.1.13 Proteases and seizure Many studies suggest that serine proteases such as tPA and thrombin up-regulate and have pathogenic properties in Alzhiemers disease [90, 91, 122, 126, 205, 244-246], experimental autoimmune encephalitis/multiple sclerosis [114, 116, 126, 139, 204, 244, 247-250], stroke [65, 105, 130, 139, 149, 244, 245, 251-255], seizure and epilepsy [40, 43, 44, 63-69, 72, 84, 129, 154, 155]. A range of genes encoding proteases like cathepsins, caspases, calpains, matrix metalloproteases and
plasminogen activators (both tPA and urokinase-PA) have recently been shown to be prominently activated in the hippocampus during both the early and latent phases of epileptogenesis following electrically-induced status epilepticus, with some remaining activated for as long as three months following this insult [73].
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Figure 1.4 Hippocampal histopathologies
Figure 1.4 Examples of human hippocampal pathophysiologies. A and B; Hippocampal sclerosis. A; shows a healthy human hippocampus with pyramidal cells of the CA regions intact. B; Shows an epileptic hippocampus following surgical resection, clearly demonstrating severe pyramidal cell losses in the CA regions (arrows) (images courtesy of Bianca Jupp). C and D; Granule cell dispersion. C; shows a healthy dentate gyrus D; shows a significant widening of the dentate gyrus (arrows) in an epileptic brain. Note also the thinning of the pyramidal cell layer (from; http://www.nature.com/jcbfm/journal/v20/n1/thumbs / 9590866f1th.jpg).
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Furthermore, in pyramidal neurons of the hippocampus, increased levels of proteases such as tPA, thrombin, myelencephalon-specific protease and trypsin (IV) correlate with pro-epileptogenic changes such as neuronal loss [67, 255, 256], LTP [63-69, 84], mossy-fibre sprouting [67] and generation of seizure [40, 43, 44, 63-69, 72, 73, 84, 129, 154, 155]. Whilst the origins of these proteases are most likely neurons and glia, they may also enter the brain from vascular sources following increased permeability of the blood brain barrier during haemorrhagic stroke or seizure [54, 71, 74-76]. Thus, elevated levels of proteases in the hippocampus following brain insult may facilitate pro-epileptogenic plastic changes such as excessive matrix degradation, potentially leading to cell loss (i.e. hippocampal sclerosis) [63, 69, 257] and excessive neuronal sprouting (i.e. mossy fibre sprouting) [67]. Furthermore, by excessive NMDA receptor potentiation [64, 66, 84, 87, 145], proteases may also contribute to the paroxysmal depolarisation shift, leading to changes in neuronal hyperexcitability and the risk of seizure. It is therefore possible that such excessive protease activity could lead to neuronal damage and death. An epileptic seizure is in itself an insult to the brain, and as such fuels the inflammatory fire of proteases and cytokines, as discussed above. Inflammatory events in the brain may thus not only lead to seizures and neuronal damage, but also further inflammatory factor release, thereby potentially leading to further seizure. This suggests that recurrent seizures and epileptogenesis may be the result of a dysregulated or excessive inflammatory response in the brain that appears to be maintained by recurrent seizure-induced inflammatory responses. Thus, a vicious cycle of inflammation-driven seizure and seizure driven-inflammation is instigated (Figure 1.5). This concept may provide a neurobiological explanation for the observation that seizures beget seizures, with inflammatory factors central to such a
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vicious cycle. Ultimately, inhibition of proteases with neuroserpin, or a loss of tPA protein in particular (i.e. tPA gene k/o) significantly attenuates experimental seizure induction and epileptogenesis [42, 43, 129, 154, 155, 258], suggesting that such proteases may be central to epileptogenesis. Perhaps protease inhibition during inflammatory events in the brain following insult may be a good target for future antiepileptogenic therapies. This will be further investigated in the body of this thesis. Protease activated receptors (PARs) PARs are a unique four member sub-family (PAR1, PAR2, PAR3 and PAR4) of Gprotein coupled receptors (GPCRs) that are activated by proteolytic cleavage of their N-terminal extracellular domain by specific serine proteases. Following such cleavage, the newly exposed N-terminal domain of each PAR acts as a tethered ligand that binds intramolecularly to an extracellular binding domain of the receptor to initiate intracellular signalling (Figure 1.6). A myriad of intracellular events are then initiated including IP3-mediated calcium release, protein phosphorylation and MAPK, nitric oxide synthase (NOS-1) and cyclo-oxygenase (COX-1) activation, ultimately leading to a range of cell-specific biological events. PARs are thought to have evolved as a mechanism for cells to detect and respond to the presence of certain extracellular catalytically-active serine proteases [259].
Thrombin principally activates PAR1, PAR3 and PAR4 and trypsin and tryptic-like proteases (e.g. mast cell tryptase) principally activate PAR2. There is, however, a growing list of candidate protease activators for all PARs (see Table 1.1). Possibly related to the outcomes of the studies outlined in this thesis, it needs to be stressed that trypsin can activate PAR1 and PAR4 [139], but not PAR3, and tPA has also been suggested as a possible activator of PAR1 [160], yet thrombin does not activate PAR2
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[139, 260]. Therefore, many proteases may have the potential to activate multiple PARs.
The cloning of PARs in the 1990s led to the synthesis of small peptide agonists (predominantly hexamers/6mers) that mimicked the last six amino acids of the tethered ligand sequence for each receptor [260-262], except for PAR3 where there is no functional peptide ligand available [139] (see Table 1.1). Such peptides have been extensively used as effective pharmacological tools to study the function of PARs in various biological systems without any proteolytic side-effects of their endogenous protease agonists. Little research, however, has been directed towards the pharmacokinetics of these peptides. As such, in vivo studies using them are all but limited to presumed acute and localised effects of PAR peptides, given they are expected to be rapidly metabolised in a whole animal by systemic and hepatic means.
Whilst the use of PAR-activating peptides is common in both in vitro and in vivo studies, concern as to their specificity may be warranted. For example, the most commonly used PAR2 peptide, SLIGRL (Ser-Leu-Ile-Gly-Arg-Leu), has been claimed to exert a range of PAR2-independent effects [263]. These include activation of mast cells [264], intestinal transport induction [265], contraction of mouse trachea [266] and umbilical vein [267] smooth muscle, and activation of chloride ion secretion across mouse airway epithelium [266]. These responses perhaps represent an interaction of SLIGRL with other receptors, such as neurokinin receptors (NK-1Rlike, NK2) [266, 268].
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Figure 1.5 Inflammation-driven epileptogenesis
Brain insult
Epileptogenesis
Figure 1.5 Inflammation-driven epileptogenesis. Following an initial brain insult (head trauma, stroke, cerebral infection etc.), cytokines and proteases are released from neurons and glia as an inflammatory response as well as possibly entering the brain matrix from usually blood bound source such as mast-cells following blood-brain barrier breakdown. Such inflammatory factors can induce seizure. The seizure itself is an insult to the brain and the inflammatory response is amplified, setting up a vicious cycle reminiscent of the theory in epilepsy that seizures beget seizures. Thus, inflammation may drive recurrent seizure generation and epileptogenesis.
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Trypsin
H3N+
Tethered ligand
Tethered ligand binding site
SLIGRL
extracellular
cell membrane
intracellular
COO-
Figure G-protein 1.6 Protease-activated receptor 2
MAPK
IP3
Figure 1.6 Protease-activated receptor 2 (PAR2). PAR2 is a seven transmembrane spanning G-protein coupled receptor activated by proteolytic cleavage of the N-terminus domain by trypsin. This exposes a new N-terminal sequence termed the tethered ligand sequence, which folds back to the active site located on the second extracellular loop and activates the receptor. The Gprotein-mediated responses include IP3 induced Ca++ release and MAPK pathway activation leading to gene transcription. The synthetic hexamer peptide SLIGRL (single amino acid code) mimics the tethered ligand sequence and can activate the receptor independently of proteolytic cleavage, making this and similar peptide a valuable pharmacological tools for functional studies on PAR2.
Furthermore, the PAR1 peptide SFLLRN (Ser-Phe-Leu-Leu-Arg-Asn) can activate

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PAR2 [269], but the PAR2 peptide SLIGRL does not activate PAR1. Novel PARactivating peptides have of late become available which are claimed to be more specific and potent than the original hexamer peptides. For instance, substitution of the serine molecule of SLIGRL with a furoyl ring, (i.e. 2-furoyl-LIGRL) [266] or addition of a seventh amino acid to the N-terminus domain (i.e. SLIGRLI) [270] have been reported to increase the potency at PAR2 10 to 100-fold [270]. Novel nonpeptidic and nore druggable compounds which conform better to Lipinskis rule of five [271] are also being created in search for a specific agonist [272]. The development of a specific antagonist will also prove to be valuable, both for research but perhaps also clinical purposes.
Given the focus of this thesis on trypsin expression in the brain and its relationship to plasticity associated with learning, memory and epileptogenesis, the bulk of the remaining discussion will focus primarily on PAR2.
PAR1 Prote a se a gonists Throm bin Trypsin Mesotyrpsin/trypsinIV? FVIIa Fxa APC Granzyme A Arginine specific gingipains-R
PAR2 Trypsin Mast-cell tryptase Trypsin IV/mesorypsin P22 kallikrein-5,-6,-14 FVIIa Fxa MT-SP1 Proteinase-3 Acrosein Arginine specific gingipains-R SLIGRL-NH2 SLIGKV-NH2 SFLLR-NH2 LIGRLO-NH2 Trans -cinnamoyl-LIGRLO-NH2 2-furoyl-LIGRLO-NH2
PAR3 Throm bin
PAR4 Throm bin Trypsin Mesotyrpsin/trypsinIV? kallikrein-14 Cathepsin G FVIIa Fxa Arginine specific gingipains-R
Pe ptide a gonists
SFLLR-NH2 TFLLR-NH2 TRagB TFRIFD
none
Table 1.1 Protease-activated receptor proteolytic and
GYPGQV-NH2 GFPGKP-NH2 GYPGKF-NH2 AYPGKF-NH2
peptide agonists1.1 Protease-activated receptor proteolytic and peptide agonists. Table Principle activators are in bold (Derived from Lou et al. 2007[139])
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1.1.14 PAR2; pro- or anti-inflammatory? PAR2 is ubiquitously expressed on epithelial and endothelial cells throughout the body which has led to the belief that they may be involved in barrier defense [273]. The plasma membrane location of PAR2 on many cell types thus enable these receptors to detect active, extracellular trypsin-like serine-proteases [259] and, probably via PGE2 [274, 275] and NO release [275-278], induce a range of effects when activated during an inflammatory insult.
Whether the effects of PAR2 activation are pro- or anti-inflammatory remains controversial. Evidence for a pro-inflammatory role comes from reports that PAR2 is up-regulated in many inflammatory disease states [204, 246, 276-292]. In addition, PAR2 has been reported to promote the rolling, adhesion and extravasation of leukocytes [280], and induces secretion of a wide range of inflammatory mediators including PGE2, TNF, NO, IL-6, IL-12, IL-1, monocyte chemo-attractant protein1, and macrophage inflammatory protein-1 [139, 293], as well as stimulating eosinophil infiltration [57], granulocyte influx and sensory nerve-dependant oedema [260, 279, 280, 294, 295]. Furthermore, it is widely reported that SLIGRL injected into the periphery of a rat induces allergic hypersensitivity, cough and hyperalgesia via sensory neuron activation [278, 279, 281, 284, 290, 292, 296, 297]. These reactions are considered to be mechanisms that promote an inflammatory event, and therefore it is widely believed that PAR2 is pro-inflammatory, and can exacerbate chronic inflammatory disease states [204, 298-301].
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Others, however, have claimed that PAR2 activation is anti-inflammatory, reducing the disease symptoms in a number of animal models of chronic inflammation, including colitis [302], asthma [303] and arthritis (Devlin and Cocks, unpublished observations). Evidence for a defensive or anti-inflammatory role for PAR2 can be found in studies on the role of PAR2 in the pancreas and the upper intestine. PAR2 is highly expressed in acinar cells [304] and ducts [305] of the pancreas and the epithelium of the small intestine [306], tissue that is regularly exposed to high concentrations of trypsinogen and/or trypsin. In both these tissue types, PAR2 activates mechanisms considered cytoprotective [273], and are largely driven by PGE2 and NO release from epithelia [273]. These include an inhibition of mast cell activation [260], reduction of smooth muscle contraction [273-275, 307, 308], an inhibition of cytokine release [302, 309-311] and fibrinolysis [312], protease, fluid and mucous secretion [259, 273, 275, 293, 304, 313, 314], and induction of fibroblast [315] and smooth muscle proliferation [316]. Furthermore, intraperitoneal injections of SLIGRL have been shown to reduce inflammatory cell infiltration in the airways, but paradoxically, intraperitoneal trypsin showed an opposite effect [273]. This apparent discrepancy may be related to either the non-selective effects of SLIGRL on receptors other than PAR2 (i.e. NK1) or the proteolytic effects of trypsin.
As already briefly alluded to, some of the best evidence that PAR2 is cytoprotective or anti-inflammatory comes from studies on the exocrine function of the pancreas. Trypsin and other powerful digestive enzymes (i.e. chymotrypsin) are stored as proenzymes within zymogen granules of pancreatic acinar cells in order to avoid selfdigestion and damage to pancreatic tissue upon release for transport to the small intestine [144, 317]. Inappropriate enzyme pre-activation, however, presents the
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pancreas with a challenging stress that can become life-threatening, as in the case of pancreatitis, during which trypsin is prematurely activated within the acinar cells [317]. As such, acinar cells of the pancreas highly express PAR2 and respond to its activation through at least two defensive mechanisms [304, 314, 318]. The first involves a feed-forward response, whereby activation of PAR2 using SLIGRL elevates intracellular calcium [318] leading to increased secretion of both proenzymes and fluid, thus effectively washing out any offending pre-activated enzymes from the pancreas [304]. The second proposed protective mechanism is that acinar PAR2 acts as the sensory arm of a negative feedback mechanism that upon activation shuts down ERK1/2 nuclear trafficking [314], thereby effectively preventing further zymogen transcription and excessive trypsinogen production. Therefore, in the pancreas PAR2 may induce cellular protection against excessive pre-activated trypsin via both increased secretion of pre-activated trypsin and inhibition of further production of trypsinogen [304, 314, 318]. Thus, as previously described in the airways [259], PAR2 in the pancreas appears to act as a monitor for proteolytic activity in the extracellular milieu, activating feedback mechanisms to effectively control trypsin production and secretion.
Perhaps on a speculative note, it does not make intuitive sense that a receptor like PARs so widely expressed and activated by a host of proteases is pro-inflammatory. If this was the case, it would be hypothesised that PAR2 could become self-destructive as simple infections or inflammatory events would potentially have catastrophic results due to amplified inflammatory responses induced by PAR2. Perhaps a more intuitive position is that PAR2 is cytoprotective and necessary for normal healing processes [259]. For instance, perhaps the well-reported sensory nerve-dependent,
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PAR2-induced hyperalgesia [278, 279, 281, 284, 290, 296, 297] is a signal to the brain that inflammatory events exist somewhere in the body, which as such acts as a warning system to the animal. Certainly, pain is a very good signal of damage, alerting the individual of the inflammatory event and leading to behaviours that prevent further damage to the area, thus, facilitating proper healing. Therefore, it is still the opinion of our laboratory that PAR2 acts as a monitor of inflammation and, as such, acts to modulate the ensuing inflammatory response vital for survival [259].
1.1.15 PARs in the brain All four PARs have been located in the rat brain [285, 309, 310, 319, 320]. Of the thrombin receptors, PAR1 is most abundant in the CA2 and CA3 of the pyramidal layer of the hippocampus, but it is also expressed in neurons of thalamic and amygdaloid nuclei and the cortex [319]. PAR1 has been located on astrocytes and microglia [321-326]. In neurons, the expression of PAR1 is highest on the cytoplasmic side of the perikaryal membrane and in neuronal processes [319]. Similarly, PAR3 is highly expressed in the pyramidal cells of the CA1, CA2, CA3, dentate hilus and granule cells of the dentate gyrus of the hippocampus [319], also being located in cortical neurons, particularly in layer 1. PAR3 is located in microglia [322] and astrocytes [323-325, 327-329]. The expression of PAR3 in neurons is generally restricted to the soma [319]. PAR4 is expressed in neuronal somata, axons and dendrites in all regions of the hippocampus, and in all cortical layers, as well as the thalamus, hypothalamus and amygdala [319].
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PAR2 is expressed on neurons in the CA1, CA2, CA3 and hilar regions of the hippocampus [285, 295, 309, 319, 320] but also in the all cortical layers, the amygdala, thalamic nuclei, the hypothalamus and the striatum [204, 319]. PAR2 is expressed on cultured astrocytes [285, 328, 330] and microglia [135]. Thus much like for thrombin, the presence of trypsin, its major activator, inhibitors (e.g. serpins) and receptor (e.g. PAR2), suggests intrinsic regulation and receptor interaction of this powerful proteases in the brain.
Whilst PAR2 has been clearly shown to activate peripheral sensory neurons in vivo [278, 279, 281, 284, 290, 296, 297], little is known about the effects of PAR2 activation on central neurons. By far the greatest response in neurons (and other cells) in culture to both trypsin and PAR2 peptides like SLIGRL is an IP3-mediated increase in intracellular calcium [260, 285, 286, 320, 331], although, the ultimate neuronal response is not well characterised. Smith-Swintowsky et al (1997) showed that SLIGRL is neurotoxic to cultured pyramidal neurons in a concentration-dependant manner, which was found to be related to calcium transients induced by PAR2 activation [320]. In astrocytes, activation of PAR2 with trypsin or SLIGRL has been shown to induce reversal of stellation of cultured astrocytes [332].
PAR2 is known to up-regulate during inflammation of the brain [139, 204, 244, 291, 294, 295, 309, 319], specifically in response to inflammatory mediators such as TNF and IL-1 [309]. Such up-regulation may exert neuroprotective effects in pathologies such as stroke and HIV-associated dementia. For instance, PAR2 k/o mice have been found to display a larger infarct volume than their wild-type litter mates following transient middle cerebral artery occlusion model of stroke [333].
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Likewise, PAR2 activation with intrastriatal SLIGRL administration reduces neuronal death in HIV-associated dementia in vivo, whereas neuronal death was exacerbated in PAR2 gene k/o mice in the same disease model [309]. Furthermore, activation of PAR2 can reduce neuronal death in vitro by specifically mitigating the p53 apoptotic inducer [309]. In contrast, however, in an animal model of multiple sclerosis (experimental autoimmune encephalitis (EAE)), it was found that PAR2 activation with SLIGRL caused oligodendrocyte toxicity and injury in vitro which when extrapolated to the in vivo situation may exacerbate the symptoms of EAE [204], since deficiency of PAR2 (i.e. PAR2 gene k/o) reduced the experimental symptoms of EAE possibly via reductions in levels of neuroinflammation and T-cell proliferation [204].
As proteases are involved in normal and pathogenic hippocampal plasticity, and PARs are highly expressed in plastic regions of the brain, like the hippocampus, there is the intriguing possibility that both trypsin and PAR2 are intimately involved in normal and pathological processes in the brain. Thus, this thesis attempts to uncover such roles for trypsin and PAR2 in the brain. Not only in normal hippocampal plasticity related to learning and memory, but also in pathogenesis like those underlying seizure and epilepsy.
Animal models of epilepsy 1.1.16 The electrical amygdala kindling model of TLE Kindling refers to an experimental process whereby focal seizures are either chemically or electrically provoked in various parts of the brain. Both types of kindling are extensively used in epilepsy research and whilst most regions of the brain
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are susceptible to kindling [334], those most sensitive are limbic structures such as the hippocampus, amygdala and surrounding cortices. Of these, the amygdala is most often used due to the relatively rapid seizure induction [335, 336]. The electrical amygdala kindling model of TLE was used in this thesis.
The electrical amygdala kindling model of TLE involves stereotaxically inserting a twisted steel bipolar electrode into the amygdala to electrically stimulate this region. Rats receive twice daily electrical stimulations via the implanted electrodes that are of sufficient strength to produce neuronal afterdischarges (ADs) recorded on the EEG [337]. The AD is a synchronised burst of excitatory neuronal activity appearing as seizure-like activity on the EEG. ADs are elicited by intra-amygdala electrical stimulations and the AD threshold (ADT) is the specific stimulus strength required to induce such an AD train. The ADT serves as an indicator for seizure threshold and thus acts as a good index for the effect of drugs on seizure threshold in kindling experiments [15]. With successive stimuli, ADT progressively and permanently decreases so what were previously subconvulsive stimulations begin to elicit high frequency and longer lasting AD trains that begin to manifest as focal seizures [338]. This state represents a permanent change in the ADT [335]. As kindling continues, this seizure-like AD electrical activity in the amygdala begins to spread to incorporate other regions of the brain and thus begin to manifest as convulsive, generalised seizures. Repeated stimulation therefore invokes increasingly severe seizure classes until bilateral clonic seizures occur (i.e. Class IV-V, see Chapter 2) that represents a permanent epileptogenic change to the brain and permanent heightened seizure susceptibility [338].
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The behavioural changes of the seizures generated by electrical kindling have resemblance to those seen in human complex partial seizures with secondary generalisation. These include head bobbing, eye blinking, chewing, myoclonic and clonic limb jerking, rearing and losses of balance and posture. The seizures are scored in severity from Class I V according to the system of Racine [339]. A more detailed description of the kindling-induced seizure manifestations can be found in the General Methods chapter of this thesis (Chapter 2).
The electrical amygdala kindling model is not usually associated with generation of spontaneous seizure [6], unless a process known as over-kindling is performed, where up to 280-300 stimulations are required [185]. The electrical amygdala kindling model is associated with pro-epileptic changes such as mossy fibre sprouting [215224] but not cell loss [185]. It is thought that electrical amygdala kindling is a good model for seizures of idiopathic, or cryptogenic, TLE patients.
Whilst the electrical kindling model of epilepsy may be a good model for seizure and epileptogenesis without cell losses, it is not a good model for the ontogenesis of TLE, as seizures are initiated by an electrical stimulus and as such there is no characteristic latent period prior to the onset of spontaneous seizures as seen in human epileptogenesis. Thus other models of epilepsy such as the kainic acid model, as used in this thesis, are required to better understand the pathogenic mechanisms that occur during epileptogenesis that lead to spontaneous seizure.
1.1.17 The kainic acid model of TLE TLE can be induced in rats by injection of the powerful glutamatergic agonist, kainic
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acid. This model of epileptogenesis has been well characterised as a good model of status epilepticus and TLE [188, 340]. Kainic acid induces status epilepticus (an ongoing and unrelenting seizure) in rats by excessive glutamatergic-like
hyperexcitability which can last for hours [188] (classes I-V, Racine scale, Chapter 2). Following recovery from status epilepticus, a latent period lasting from several days to weeks ensues during which neurobiological changes occur that ultimately lead to the development of spontaneous seizures. At this point animals are considered epileptic. These neurobiological changes correlate well with human pathologies such as hippocampal sclerosis, with cell losses in both the CA1 and CA3 regions [95, 341344], but not the CA2 [188, 345], astrogliosis [41] as well as mossy fibre sprouting [47, 97, 224], granule cell dispersion [346] and progressive hippocampal shrinkage [342]. Thus, along with a latent period and the appearance of spontaneous seizures, the pathophysiological changes that occur in the rat brain following kainic acidinduced status epilepticus are well correlated with human TLE.
1.1.18 The Genetic Absence Epilepsy Rats of Strasbourg (GAERS) GAERs are an inbred strain of Wistar rat which develop spontaneous absence-like seizures, associated with spike and wave discharges on EEG very similar to those observed in human absence epilepsy, such as a genetic predisposition and the specific involvement of thalamocortical circuitry with-out the involvement of the hippocampus. The EEG of GAERS exhibit bursts of spike and wave discharges (7 11 Hz, 200 600 mV, 0.5-40 sec duration) that begin and end abruptly and are associated with behavioural arrest and clonic twitches of the whiskers, face and neck muscles. GAERS have also been shown to have a similar pharmacological response profile to human epilepsy, with seizures being effectively suppressed by certain
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anticonvusants (e.g. valproic acid, ethosuximide, benzodiazepines), but others such as carbamazepine being ineffective or even aggravating the seizures. GAERS have a higher level of anxiety than there background strain (non-epileptic control; NEC) in our cohort [347].
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Aims and Hypothesis Since both PAR2 and trypsin are expressed in the brain, and proteases appear to be heavily involved in hippocampal plasticity related to learning, memory and the pathogenesis of epilepsy, the aims of this thesis were to characterise the expression of PAR2 and trypsin in the rat brain and to determine whether both are involved in neuronal plasticity. The principle hypothesis underpinning these aims is that PAR2 and trypsin in neurons of the brain are functionally linked. Specifically, wherever trypsin is expressed, PAR2 will be co-expressed to monitor its extracellular activity. In this manner, cells that synthesise, package and release enzymes, one of which is trypsin, are able to regulate the production and release of such proteases, potentially acting to reduce proteolytic damage to the brain matrix. Therefore, it is hypothesed that trypsin and other proteolytic enzymes play important roles in normal functioning of highly plastic regions of the brain, such as the hippocampus, that are reliant on the need for rapid synaptic reorganisation related not only to learning and memory, but also to pathogenesis like epilepsy. If PAR2 functions in a cytoprotective manner in the brain as it does in other tissue types such as the pancreas, upper intestine and airways, then agonists of PAR2 may be neuroprotective during pathogenesis. Therefore the activation of central PAR2 will be investigated in the course of this thesis with particular reference to plasticity in the hippocampus related to epilepsy, but also learning and memory.
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Chapter 2 Materials and Methods
Chapter 2 General Materials and Methods
Animals and postoperative care. Non-Epileptic Control (NEC) Wistar rats and Genetic Absence Epileptic Rats of Strasbourg (GAERS) of both sexes, aged between 12-14 weeks (170-250g), were obtained from breeding colonies at the Royal Melbourne Hospital. All experiments were approved by the ethics committee at the Royal Melbourne Hospital/Ludwig Institute, Melbourne, Australia. After surgeries, all animals were individually housed with food and water supplied ad libitum under a 12-hour light/12-hour dark cycle. Rats were handled twice daily during the 7 day post-surgical recovery period in order to familiarise them with the handler and the experimental procedures. For rats with an embedded intracerebroventricular cannula, the cannula dummy was unscrewed, removed and replaced daily during this period in order to prevent the cannula becoming blocked with inflammatory tissue and to habituate the animal to the experimental procedures. Drugs and peptides Ketamine (Parnell Laboratories) and xylazine (Troy Laboratories; 75mg/kg and 10mg/kg respectively) diluted in 0.9% sterile saline for anaesthesia were injected intraperitonealy (i.p). Carprofen (Rimadyl, Pfizer, UK; 5mg/kg in saline (1ml)) for post-surgical analgesia was delivered subcutaneously (s.c.). Angiotesin II (AngII) (1 M/ l in 0.9% sterile saline, Auspep, Australia) was injected
intracerebroventricularly (i.c.v.) for cannula placement validation. Pentobarbitone sodium (Lethabarb, Virbac Animal Health; 300mg/kg, i.p) was used for euthanasia prior to transcardial perfusion of each animal after experimental completion. The PAR2-activating peptide (Ser-Leu-Iso-Gly-Arg-Leu-NH2 (SLIGRL) and the full
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reversed peptide sequence (Leu-Arg-Gly-Iso-Leu-Ser-NH2 (LRGILS) AusPep, Australia), which is not recognised by PAR2 [1-5], both C-terminally amidated, were diluted in 0.9% sterile saline at the specific concentrations described in each experimental chapter (Chapters 4, 5, 6 and 7). Kainic acid (Ocean Produce International, Canada) for status epilepticus induction and diazepam (5mg/ml, DBL, Mayne Pharma) for seizure inhibition were diluted in saline at the described doses.
Injection protocols 2.1.1 i.c.v. peptide administration
Peptides (SLIGRL, LRGILS, AngII or saline vehicle) were administered manually at an approximate rate of 2 l/min using a distilled water (dH2O) -filled, polyethylene injection line (i.d. 0.28mm, o.d. 0.61mm, length 20cm (Microtube Extrusions, Australia)) one end of which was connected to a 10 l glass Hamilton syringe (Syringe Perfection, SGE, Australia) and the other to an internal injection cannula (the size coinciding with the implanted guide cannula, see Section 2.4.1) to be inserted into the embedded guide cannula after the dummy cannula was unscrewed and removed. An air bubble of approximately 2l was drawn into the injection line in order to separate the dH20 in the line from the peptide to be drawn into the line. This also acted as a guide as to when all the peptide had been injected. Rats were manually restrained during the injection procedure. The dummy cannula was removed and placed in 70% ethanol (in dH2O) for 1min and then washed in sterile saline and allowed to air dry, during which the embedded guide cannula tip was cleaned with a 70% ethanol soaked cotton bud. Measured volumes of peptide were drawn into the injection needle, which was then gently inserted into the embedded guide cannula. A maximum of 4 l was injected. The injection needle with attached line remained in
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the embedded cannula for another minute following injection to allow the injected solution to circulate throughout the cerebrospinal fluid (CSF) and prevent it from flowing back up the guide cannula following removal of the injection needle. The needle was then removed, the tip of the guide cannula again cleaned with a 70% ethanol soaked cotton bud and the dummy cannula replaced and screwed tight. The rat was then returned to its experimental cage. The line was flushed with 1ml 70% ethanol followed by 3ml sterile dH2O. Separate lines were used for each cohort (i.e. SLIGRL, LRGILS, saline, AngII and CSF extraction).
2.1.2
Peripheral peptide injection
For intraperitoneal (i.p.) drug/peptide administration, rats were injected in the lower left quadrant of the abdomen and for subcutaneous (s.c.) injections inter-scapularly in the loose skin at the scruff of the neck using a 30G insulin syringe. Rats were manually restrained during these procedures.
Surgeries 2.1.3 Implantable devices (see Figure 2.1)
Stainless steel guide cannulae (9.0mm length, 22 Gauge, inner diameter (i.d.) 0.39mm, outer diameter (o.d.) 0.71mm with a nylon pedestal, dummy cannulae (also known as a Stylet or Obturator, 9.5mm length, 0.36mm diameter stainless steel wire with nylon screw cap) and internal cannula for injection (28 gauge, i.d. 0.18mm, o.d. 0.36mm with 0.5ml projection (i.e. 9.5mm length)) were made to order (Plastics One, Australia). Guide
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Figure 2.1 implantable devices
B C
Figure 2.1 Implantable devices. A; custom built extradural electrode. B; anchor screw (nickel-alloy). C; bipolar electrode. D; intraventricular (guide) cannula. E; dummy cannula (Stylet/obturator). F; injection (internal) cannula.
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cannulae and bipolar electrodes were stereotaxically introduced into the lateral ventricle (for i.c.v. injections and CSF extractions) and the basal lateral amygdala (for recording and stimulations) respectively. Bipolar electrodes consisted of two Tefloninsulated twisted stainless steel wires (length; 10mm, diameter; 0.5mm) with a nylon pedestal (3.5mm diameter) and were also made to order (Plastics One, Australia). Extradural electrodes were custom built whereby nickel-alloy jeweller screws (1.4mm x 3.5mm, Mr Specs, Australia) were silver-soldered to 1.5mm diameter male gold connectors (1.3cm x 1.4mm; Farnell Components, Australia). These acted as ground and reference electrodes during recording. The same jeweller screws without the soldered connector pin were used as anchor screws for cap security.
2.1.4
General surgical technique
Once anaesthesia had been achieved, as tested by plantar reflex, the crown of the rat was shaved with electrical clippers and cleaned with a swab soaked in an iodine-based disinfectant (Betadine). An incision (approx. 1.5cm in length) was made in the skin and periosteum overlying the skull down the midline from the bridge of the nose to the occiput. The skin was retracted and the periosteum scraped away from the bone using the scalpel blade. Any bleeding from the bone was cauterised using a lowtemperature diathermy (Aaron Medical). Two burr holes were drilled bilaterally (1.4mm diameter) in the frontal bone (approx. 2mm anterior to the coronal suture) and two in the parietal bone (approx. 2mm anterior lambdoid suture) using a low speed electrical drill (Arlec Engraver). Extreme care was taken not to pierce the dura when drilling. The extradural electrodes described above (Section 2.4.1) were gently screwed no more than 2.5 rotations into the anterior burr holes using lockable forceps, thus not to pierce the dura. Similarly the anchor screws were screwed into posterior
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burr holes (see Figure 2.2 for a schema of the locations of these implantable devices).
The rat was transferred to a dual-arm, digital stereotaxic frame (Stoelting, USA) and bregma located. The guide cannula was inserted through a burr hole drilled in the skull overlying the left lateral ventricle (coordinates from bregma; antero-posteriorly (A-P) -1.2, medio-laterally (M-L) -1.6, dorso-ventrally (D-V) -3 to 3.5 mm, see Figure 2.2). Similarly, a twisted steel bipolar electrode was also inserted through a burr hole (co-ordinates, A-P; bregma - 3.0, M-L; bregma+5.0, D-V; bregma - 6.5, see Figure 2.2) and lowered to the level of the basal lateral amygdala on the right (Figure 2.3). All co-ordinates were obtained from the Rat Brain Atlas of Paxinos and Watson [348]. A mound of dental cement (Vertex, The Netherlands) was built up around the guide cannula, bipolar electrode, anchor screws and frontal electrode and allowed to set. The retracted skin was closed around the dental cement cap using a purse-string type suture technique (3.0 surgical silk (Ethicon)). The rat was removed from the frame and the guide cannula was then closed by carefully inserting the dummy and gently screwed shut.
2.1.5
Post-operative care
All animals were given carprofen analgesic (Rimadyl, 5 mg/kg in saline, s.c.) and placed on their side on a bed of tissues in their home cage with fresh sawdust bedding on top of a heat mat and allowed to recover from anaesthesia. Rats were frequently checked during this recovery period. Once animals regained consciousness, a dish of high-energy mush
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Figure 2.2 Representation of surgical locations of implantable devices Figure 2.3 Representation of final internal locations of implantable devices
[383]
[345]
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(50% infant formula and 50% powdered rat pellet mixed with tap water) and a dish of fresh water were provided on the floor of the cage. Normal rat food-pellets and drinking water were also provided.
For 7 days following surgery, rats were weighed daily and examined for any potential neurological deficits such as ipsiversive movements, ataxia, and atonia. If any weight losses of greater than 15% (than prior to surgery) and/or obvious locomotor deficits were maintained throughout the seven days with no sign of recovery, rats were deemed unsuitable for further experimentation and euthanised (Lethabarb). The majority of rats fully recovered from surgery. The day following surgery, half a tissue box and shredded paper was placed in each rats cage to provide some shelter and environmental enrichment.
Confirmation of surgical accuracy 2.1.6 i.c.v. cannula placement
All rats implanted with an i.c.v. cannula were subjected to experiments to confirm the correct placement of the cannula into the lateral ventricle. When injected directly i.c.v., AngII induces excessive drinking behaviour in rats [349, 350]. The experimental design involved comparing the volume of water (by weight) consumed 30min prior to and 30min after AngII injection. Thus, rats were injected with AngII (2nM in 2l in saline, i.c.v. over 1min as described in Section 2.3) and returned to their boxes with the water bottles replaced. Rats passed the test if they consumed 5times more water after injection of AngII than before [349]. For rats that did not drink at least 5-fold of water post-AngII injection, the same test was repeated the following day. For rats that failed the test twice, or whose average water consumption after
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injection over the two experiments was less than 5-fold than before injection, the cannula was presumed to be incorrectly placed and the animal was omitted from further experimentation. Rats that passed the test continued onto electrical kindling, kainate-induced epileptogenesis, CSF extraction or neurotoxicity experiments.
2.1.7
Intra-amygdala bipolar electrode placement
Rats implanted for the electrical amygdala kindling experiments specifically were required to have a functional electrode in the basal lateral amygdala for recording and stimulation. Electrodes for the kindling experiments were functionally tested by the appearance of an afterdischarge (AD) of greater than 6s following stimulation (at threshold, see Section 2.7.2). If a stimulus required to elicit such an AD train was greater than 400 A the electrode was deemed incorrectly placed outside of the basal lateral amygdala and the rat was omitted from electrical kindling. For other experimental groups in which the bipolar electrode only acted as a recording electrode (i.e. kainate-induced epileptogenesis and neurotoxicity experiments) the precise location was of less importance. Correct placements of electrodes were confirmed in the basal lateral amygdala microscopically in all experimental cohorts after the brain was fixed and stained with thionin.
Cerebrospinal fluid (CSF) extraction Rats were manually restrained and the dummy cannula removed and placed in 70% ethanol and then in sterile saline for another minute and allowed to air-dry. The tip of the embedded guide cannula was cleaned with a 70% ethanol soaked cotton bud, and the internal (injection) needle (same line and syringe set-up as for i.c.v. injection), following a quick wash in sterile saline, was gently introduced into the lateral
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ventricle guide cannula. The plunger was then slowly drawn back, and CSF extracted, which generally appeared clear, straw-coloured and blood-free. The needle was removed from the cannula and the dummy replaced. A good extraction was often preempted by observing CSF to well at the tip of the guide cannula following removal of the dummy. Once the sample had been extracted, the volume of CSF as indicated by the gradations on the syringe was recorded and the collected CSF sample transferred a pre-labelled 500 l microfuge tube and snap frozen on dry ice. Following this, the Hamilton syringe was removed from the line and the line was flushed with 70% ethanol and dH2O prior to the next serial extraction. Between extractions the rat was returned to its home cage and the injection cannula was left in 70% ethanol. All serial CSF samples were collected prior to preparation for analysis.
2.1.8
Preparation of CSF samples
Once all CSF samples were collected, they were thawed and transferred to fresh prelabelled microfuge tubes placed on ice. A volume of acetonitrile twice that of each collected CSF sample was added to precipitate any blood products and large proteins in the sample and the caps of the tubes rapidly closed. Diluted samples were then centrifuged at 32,500 rpm for 5min at 4C and supernatants carefully removed and transferred to fresh pre-labelled microfuge tubes. The samples were then diluted further with an equivalent volume of milliQ filtered water, effectively increasing the volume of original CSF samples six-fold, then snap frozen on dry ice for later LCMS analysis (see below).
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Models of epilepsy 2.1.9 The kindling apparatus
The kindling equipment consisted of a stimulus generator (Accupulser Pulse Generator -stimulator, A310, World Instruments), connected to a battery-operated, constant-current stimulus isolator (A360, World Instruments). The stimulus isolator was linked to an in-house built three-way switch box, which was connected to the bipolar electrodes implanted in the amygdala of the rat and the EEG head box (see Figure 2.4 and 2.5). The implanted ground and reference electrodes were connected to custom made leads that fed into the EEG head box (SynAmps, 32 channel, 150 gain AC/DC, Neuroscan). This was connected to an EEG amplifier (SynAmps, model 5083, Neuroscan) that supplied a PC running EEG acquisition software (Neuroscan X.0.Compumedics) (Figure 2.4 and 2.5). The three-way switch box was used to separate EEG recording from stimulus delivery to the intra-amygdala bipolar electrode.
2.1.10 Electrical amygdala kindling Twenty-four hours after ADT determination, rats were again connected to the kindling apparatus, and a 30-60s baseline EEG recorded. Kindling stimuli (1s train of 1ms biphasic square wave pulses, 60Hz) were then applied at the stimulus strength (A) related to the specific ADT for each individual rat. Rats were monitored for behavioural seizures and the duration of induced ADs were measured from the EEG after each stimulus. If a seizure was elicited by a stimulus, it was allowed to end, both behaviourally and electrographically. Seizures were scored according to the scale of
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Figure 2.4 kindling apparatus Figure 2.5 Schema of kindling appratus
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Racine [9-12](see below). A post-ictal (after the seizure) trace of at least 60s was continued before recording ended once baseline EEG had returned. The rat was disconnected from the attached leads and returned to the home cage. Rats were subject to kindling stimuli twice daily (minimum 4h apart), 5 days per week at the determined ADT stimulus strength. The end point of experimentation was set at 5 class V seizures (see Section 2.12 and Figure 2.6), animals being considered fullykindled, or after a maximum of 80 stimulations. Twenty-four hours after the fifth class V seizure (or 80th stimulation) rats were transcardially perfused as described below (Section 2.9)
2.1.11 Kainic acid induced status epilepticus Seven days post-surgery and following confirmation of the cannula placement, a separate cohort of rats (n=30) was injected with the potent glutamatergic agonist kainic acid (15mg/kg in saline, i.p.), which has been well-characterised for its ability to induce complex partial seizures, status epilepticus and temporal lobe epilepsy [188, 340, 354]. Following this first injection, rats were monitored both visually and on EEG for any signs of seizure-like behaviours. EEG traces were monitored for epileptiform spiking and wave patterns. Behaviours were monitored for
manifestations such as automatisms (excessive eye blinking, scratching, grooming, phantom chewing), head-bobbing, hyper-locomotion, confusion, wet-dog shakes, loss of consciousness and any form of seizure-like kinesia, such as myoclonic and clonic limb jerking, convulsive rearing and a loss of balance. Seizures were again classed based on the scale of Racine [339] (see below). Consciousness was determined by startle responses displayed by the rat in response to loud noises such as finger clicking or hand clapping in close proximity to the rat. If any startle response was displayed
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the rat was not considered to be in status epilepticus, even though the seizure activities (behavioural and EEG) may have been apparent.
One injection of kainic acid rarely induced status epilepticus in the cohort, thus if status epilepticus was not attained, rats were injected again with kainic acid. Injections were repeated every 60min until rats were confirmed to be in status epilepticus. If rats were displaying intermittent seizure-like behaviours, but not in status epilepticus per se, then they were given an additional half-dose of kainic acid (i.e. 7.5mg/kg). No more than 4 doses (i.e. a total of 60mg/kg) were administered. Status epilepticus was confirmed by a prevailing and ongoing electrographic epileptic wave forms (Figure 2.7) and behavioural seizures of progressively higher order classes associated with synchronised epileptiform activity on the EEG, during which consciousness was absent. Status epilepticus was also confirmed by at least one independent expert researcher on each occasion.
Rats remained in status epilepticus for 4h from onset, before receiving a bolus dose of diazepam (4mg/kg in saline, i.p). If rats remained in status epilepticus 20min later, another half-dose of diazepam was administered. This was repeated until rats were no longer in seizure. As described for the surgery, recovery consisted of placing the rats on a bed of tissue paper in boxes on top of a heat mat. Fresh water and high-energy mush were also supplied. Rats were further monitored until confirmed they were truly no longer in status epilepticus and sedate. The following day all rats were weighed and tested for neurological deficits, as described in Section 2.3.5.
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2.1.12 Twenty-four hour recording protocol Seven days post- status epilepticus, rats began a once-weekly 24h video-EEG recording protocol which continued for 8 weeks to assess the frequency and nature of spontaneous seizures. Rats were connected to an EEG amplifier (Powerlab ML750/4SP running 4 x BioAmp ML136) via the bipolar electrode and continually monitored for 24h. An infrared digital video camera mounted on the ceiling above the cages was used to record the movements and behaviours of the rats during EEG recordings. EEG (Chart, 5.3, Powerlab, ADInstruments) and video recordings (Quicktime Pro,) were made on a computer (Apple Macintosh G5). The lids of the cages had been previously blackened to avoid any infrared light reflections during night-cycle recordings, food and water were supplied ad libitum. Up to 4 rats were recorded at any one time. Using this system, EEGs could be later correlated to the recorded video thus providing a demonstration of seizure, seizure class or movement artefact to which the EEG corresponds.
This 24h protocol was repeated every 7 days for 8 weeks, and was conducted on the same day of the week every week in order to monitor the appearance of spontaneous seizure activity and the progression of epileptogenesis following status epilepticus. Twenty-four hours after the final (week 8) recording, rats were transcardially perfused with fixative (4% PFA) (Section 2.9) for histological analysis of the brain. Brains were collected and frozen. Animals which had died prior to end point were not able to be used for these purposes
2.1.13 Seizure classification- The scale of Racine Seizures observed in both electrical amygdala kindling and kainic acid experiments
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were scored according to the scale of Racine [339]. Physical and behavioural manifestations generally observed for a designated class are as follows: Class I; facial clonus, vibrissae twitching, ipsilateral eyelid closure. Class II; facial clonus, ipsilateral eyelid closure automated chewing, head bobbing. Class III; contralateral forelimb clonus including class II behaviours and bilateral eyelid closure. Class IV; convulsive bilateral forelimb clonus, rearing, generalised tonic-clonic convulsions. Class V; convulsive bilateral forelimb clonus, generalised tonic-clonic convulsion associated with rearing and a loss of balance. Often alternating unilateral hind limb convulsions (referred to as Hokey-Pokey seizure) or wild jumping (popcorn seizure) were observed, particularly in during status epilepticus induced by kainic acid. Even though apparently of higher order, these seizures were scored as class V to adhere to scientific convention as defined by Racine [339].
Post-ictal EEGs were usually quiescent, associated with very little and slow cortical activity compared to baseline EEG traces, as is usually observed in humans [355]. Generally higher order seizure classes (class IV and V) were followed by secondary seizure activity on EEG, even though the initial seizure had apparently ended behaviourally. These usually did not involve behavioural seizures greater than class I, but were associated with epileptiform-spiking activity on the EEG. Much like observed in human epileptic patients, rats experienced post-ictal confusion,
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Figure 2.6 Typical EEGs obtained during kindling
A
1mV 1sec
Stimulus on
Figure 2.6 Normal EEG traces obtained during electrical kindling in the rat. A; shows a normal resting rat EEG rhythm. B; shows a typical 6s AD train (arrow heads) following stimulation (stimulus on denotes the stimulus artifact). C; typical seizure-like rhythmic activity of a class V seizure.
Figure 2.7 Typical spontaneous seizure following status epilepticus in the rat
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lethargy and irritability, particularly after higher order class IV and class V seizures. Electrical kindling was discontinued when either the rat had exhibited 5 class V seizures (animals being considered fully-kindled) or if the rats were not fullykindled after a maximum of 80 stimulations had been applied, this being considered the end point of experimentation. Sham animals used for immunohistochemistry and Timms histology did not receive any peptide or electrical kindling stimuli but were handled identically during the kindling protocol
Behavioural monitoring, assessment and scoring Neurotoxic behaviours (see Chapter 5) were scored according to a scale developed in our laboratory for post-surgical and general health assessment and as described previously [15, 16, 356-358], but adapted to suit the current behaviours observed (see Table 2.1). The time spent in any particular behaviour observed was scored (Table 2.2). During such behavioural experiments, rats were monitored by visually observing patterns in changes of normal (baseline) behaviours, for in particular, neurotoxicity. Loss of consciousness and hypersensitivity were assessed by displayed startle responses (see above). No response indicated unconsciousness and a heightened response assumed hypersensitivity. Analgaesia and hyperalgaesia was assessed using plantar reflex. Rats were visually monitored for up to 130min, during which the behaviours were scored for both neurotoxicity (see Table 2.1 and Chapter 5) as well as the duration (min) of that particular behaviour (see Table 2.2). The most severe of the neurotoxic behaviours observed was used to calculate the final neurotoxic coefficient, as shown in Equation 2.1.
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neurological scoreObserved behaviour Table 2.1 neurotoxic behavioural scoring legend 0 1 2 3 4
normal behaviour shivering/hyperactivity/myoclonic jerks loss of balance/atonia/sedation loss of conciousness/catatonia barrel rolling/generalised seizure/anaesthesia
Table 2.2 Neurotoxic behaviour duration behavioural scoring legend. Observed Table 2.1 Neurotoxic scoring legend. behaviours ranging from no changes (i.e. normal behaviour) to severe neurotoxicity (barrell-rolling and seizure) were scored from 0-4.
duration score 1 2 3 4
Duration of behaviour 0-20min 21-40min 41-60min 61min+
Table 2.2 Neurotoxic behaviour duration scoring legend. Durations of the behaviours encountered (see Table 2.1) were scored from 1-4 depending, as shown.
Equation 2.1;
Toxicity coefficien t = Behavioura l score duration score
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2.1.14 Elevated plus maze The elevated plus maze is a well-established ethological tool for measuring the levels of fear and anxiety in rodents, although it has also been shown to be useful in studying fear aversive memory in rats [359]. The elevated plus maze consists of a black Perspex platform in the form of a plus, elevated 60 cm from the floor, with four arms (length 43cm; width 14cm) at right angles surrounding a centre platform (14cmx14cm). Two opposing arms enclosed by walls (30cm height) form the closed arms, the other two adjacent arms without walls form the open arms.
Experiments using the elevated plus maze are described in Chapter 7 of this thesis. Lighting conditions were set at ~100 lux at the tips of the open arms, ~70 lux in the centre of the maze and ~30 lux in the closed arms using strategically positioned spotlights.
Rats were placed in the centre arena of the elevated plus maze facing an open arm, after which the room was vacated. Tests lasted for 10min. Rats were tracked using Ethovision software (version 3.1.16, Noldus Information Technology, The Netherlands) and a camera mounted over head. Data was analysed by Ethovision for time spent in the open and closed arms, number of entries into each arm and distance travelled (open and closed arenas, and total). Risk assessments, operationally defined as two paws in an open arm, were determined manually by a blinded reviewer. Following each test, the maze was thoroughly cleaned with 70% ethanol prior to the next rat. Each test was repeated three times with 48h between runs.
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2.1.15 Open field test The open field test is a well-established technique used to measure anxiety and locomotion in rodents. The apparatus consists of a circular arena with a diameter of 100cm surrounded by white plastic walls 30cm high. An inner central circle of 66cm diameter in the middle of the arena forms the centre arena.
For experiments using the open field test refer to Chapter 7. The lighting levels in the centre arena were set at ~90 lux using strategically positioned spotlights.
Rats were placed in the centre of the open field and the room was vacated. Tests lasted for 10min. Ethovision software and an over-head camera (see above) were again used to track the rats for time spent in the centre arena of the field, number of entries into the centre field and distance travelled, both in the centre field and in the total arena. Faecal boli produced by the rats were counted after each test. Tests were repeated three times over the following 5 days, with 2 days (48h) between recordings. The maze was thoroughly cleaned with 70% ethanol between tests.
2.1.16 Morris water maze The Morris water maze is a well-established test for determining spatial learning and memory in rodents. It consists of a large circular bath (PVC plastic, diameter 160cm) filled with water (room temperature) in which a platform of clear Perspex can be placed in one of four positions (NE, SE, SW, NW) just under the water level (2.5cm submerged). For experiments described in chapter 7, normal, non-fluorescent laboratory lighting were used (90 lux). Also visual cues in the form of laminated
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posters of basic black shapes on a white background were mounted at the north, south, east and west ends of the bath. A position for the platform was randomly assigned to each animal and this remained constant for that animal over all days of the trial. The testing protocol consisted of 4 consecutive trials per animal per day over 4 consecutive days. Thus, each rat repeated the same trial 16 times inclusive of the fourth day. For the first trial, rats were placed in the water bath at the north end and were forced to swim for 90s. Rats usually began to swim around the entire bath exploring their aquatic environment. If in this first trial the rat had not found the platform in that time, it was guided there by following the researchers hand above the water. Once the rat had found the platform, it was left there for 30s to observe its surroundings and the position of the platform in relation to the visual cues. It was then removed from the platform and dried in a towel for 90s and the test repeated, however, this time the rat was placed in the water at the east end on the bath. Again rats were forced to swim, however, having been shown the platform they were expected to find it more easily. If, however, the rat had not found the platform by 90s in the bath, they were again shown it and left on it for 30s and again towel dried for 90s prior to repetition. For the third and fourth trials, rats were placed in the south and west ends of the bath respectively. In such a manner, the visual cues were important in locating the platform and not the position which the rat entered the bath. The four trials were repeated for each rat on the same day within 15min. This daily protocol was repeated for each rat for a total of four consecutive days each. Again, Ethovision and an over-head camera were used to track the swim path of the rat in the bath and both the distance and the time taken to find the platform were recorded.
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2.1.17 Light-dark box The light-dark box is used to measure anxiety and fear levels in rats. It consists of two rectangular Perspex chambers, one white (27cm x 27cm x 28cm) and one black (27cm x 9cm x 28cm), joined together on the matching sides. The two chambers are connected with a small open doorway (9cm x 9cm) allowing the rat to freely enter and exit either chamber. For experiments outlined in Chapter 7, the larger white chamber was illuminated to 1000 lux using a desk lamp and all other lighting in the behavioural suite was turned off so that the dark chamber had an illumination of only 10 lux. The rat was placed in the light box facing the doorway to the dark chamber and left in the box for 5min. A video camera mounted on the ceiling recorded the movements of the rat in between the light and the dark chambers. Data was manually analysed by a blinded reviewer for time spent in each half of the box, number of risk assessment (operationally defined as two paws in another chamber; i.e. peering through the doorway) and number of entries into each chamber (all four paws in a chamber). For this series of experiments, Ethovision was incapable of determining the movements of the rat in the white box, as it functions on the rat being lighter in shade than the background. Thus, due to the brightness emitted by the white Perspex box, the rat could not be electronically tracked. Following each trial, the box was cleaned thoroughly with 70% ethanol.
2.1.18 Sucrose consumption test The sucrose consumption test is used to study depression and anhedonia (the lack of deriving pleasure) in rodents, based on the satisfaction the animal derives from drinking sweet liquids. The test covers 4 consecutive days of trials, during which the consumption of a sucrose solution supplied in the absence of fresh water is recorded.
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For experiments outlined in Chapter 7, 4 rats were recorded simultaneously, thus 4 experimental cages similar to their home cages were placed on the floor within the view range of the ceiling-mounted video camera. The sucrose solution (20% w/v sucrose in drinking water) was then placed in drinking bottles and weighed. Prior to placing the rats in the experimental cages, each was first given a taste of sucrose by placing a small drop of the solution on their tongue, and the bottle of sucrose in the normal position of the water bottle in the rat cage. Rats were left in the box and video recorded for 15min. Following the trial, the water bottles were reweighed and the consumed volume calculated. This was repeated at the same approximate time 4 days consecutively. Videos were manually analysed for approaches of each rat to the water bottle (operationally defined as a touch, sniff or taste of the drinking nozzle).
Tissue collection and processing 2.1.19 Transcardial perfusion and rat brain fixation. Once end-point of experimentation was achieved (i.e. fully-kindled or 8 weeks poststatus epilepticus), the process of brain fixation was done using a standard transcardial perfusion protocol. Rats were terminally anaesthetised using Pentobarbitone (Lethabarb, 300mg/kg i.p) then placed in a fume cupboard on a corkboard in a stainless steel tray with its ventral surface facing up. After dissection the descending thoracic aorta was located, clamped using curved haemostats with the heart still beating. A peristaltic pump with silicon tubing (i.d. 1.6mm, o.d. 4.8mm) was used for transcardial perfusion. One end of the tubing was placed into a beaker containing 0.1M phosphate buffered saline (0.1M PBS pH 7.4, 4C) and the other, via an attached blunted 26G needle, carefully inserted through the apex of the heart into the left ventricle and pushed superiorly, through the aortic valve until the tip was seen to
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enter the ascending aorta. The tip proceeded for approximately 1mm into the aorta, not reaching the aortic arch. Extreme care was taken not to pierce the ventricular septum, atrial wall or the aortic wall. The needle was stabilised in the apex of the heart by clamping with haemostats around the cardiac muscle of the apex and the needle. At this point the peristaltic pump was turned on at the lowest speed and the right atrium of the rat heart snipped open using fine dissection scissors and the pump speed was turned up to the level that was previously determined to be closest to normal systolic-diastolic pressures of the rat (approx. 56 ml/min). 100ml of cold (4C) 0.1M PBS was pumped through the rat to evacuate the circulatory system of blood, followed by 400ml of cold 4% paraformaldehyde (PFA, pH=7.4, 4C) at the same pumping rate.
Following PFA perfusion, the pump was switched off and the needle removed from the heart and the aortic clamp removed. The rat, now stiff in the upper body, was turned over, with the dorsal surface up and the brain carefully dissected. If a successful perfusion had been performed, no blood was visible in the cortical vessels and the brain was firm and not mushy. The excised brain was placed in 4% PFA (4C, pH, 7.4) and refrigerated overnight for post-fixation, to improve the preservation of the tissue.
2.1.20 Cryoprotection and tissue storage Following the overnight post-fix step, the brain was transferred to a 20% sucrose/0.1M PBS (4C, pH=7.4) solution and refrigerated for 48h for cryoprotection. The brain was hereafter transferred to a plastic mould filled with freezing compound (OCT, Tissuetek, USA) and left to stand for a few minutes to allow the freezing
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compound to surround the entire brain. The mould was then transferred to a 100ml plastic beaker containing ice-cold isopentane cooled over liquid nitrogen. The isopentane was at the correct temperature for freezing the brain when it had begun to form white (frozen) patches in the bottom of the beaker. The beaker with isopentane remained over liquid nitrogen whilst the mould containing the brain was placed in it and frozen. The level of isopentane in the beaker was such that the mould was not completely submerged, so that its superior surface remained exposed to the air. Freezing was monitored by observing the embedding compound in the mould surrounding the brain to turn white from outside to in. If the mould was allowed to be submerged in the isopentane, monitoring was not possible as the superior surface of the mould was rapidly frozen and the progression of the freezing to the centre of the block could not be observed. This often led to misjudging the freezing progression resulting in severe freezing damage of the cells, particularly in the central structures such as the thalamus. Once the entire surface of the mould appeared frozen (white), usually within 2min, the mould was removed from the ice-cold isopentane and rapidly transferred to the -80C freezer for storage.
2.1.21 Cryostat sectioning Sections of the entire thalamic-hippocampal region (approx., bregma 0.4mm to 6.0mm [348]) of the rat brain were cut on a cryostat (Leica, Germany) at 20m, stuck to 1% gelatinised microscope slides and allowed to air dry for 30min; a time optimised to minimise autoimmunofluorescence. Three sections per slide were collected. Consecutive sections were collected on adjacent slides and used for immunohistochemistry, thionin or Timms staining. Slides for histology (thionin or Timms staining) were not washed in 0.1M PBS immediately, but allowed to air dry
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completely and stored, unstained at -80C for later use. Generally every fifth and sixth slide was used for histological purposes, all others being used for
immunohistochemistry.
Histology and immunohistochemistry 2.1.22 Antibodies and antigens Primary antibodies used for immunohistochemistry experiments were as follows; monoclonal mouse-anti-human PAR2, (SAM-11, Santa-Cruz, CA. USA); polyclonal rabbit-(anti)-human-trypsin, raised against human (pancreatic) cationic trypsin (PRSS1, Biodesign, ME, USA); polyclonal bovine--rabbit glial fibrillary-associated protein (GFAP, Dako, Denmark) and polyclonal goat--human tissue plasminogen activator (tPA/PLAT, Abcam, UK).
Secondary antibodies used were; donkey--Mouse Alexa488 (green fluorescent conjugate) for PAR2: donkey--rabbit Alexa594 (red fluorescent conjugate) for trypsin and GFAP (Molecular Probes, Invitrogen, Australia) and donkey--goat AMCA (Blue fluorescent conjugate) (Millipore, Australia) for tPA (see Table 2.3 for summary). The fluorophores used had very little excitation and emission spectra overlap. Any overlap was able to be reduced using specific filters on the fluorescence microscope and highly spectra-specific confocal microscopy.
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Table 2.3 Primary and secondary antibodies used
Table 2.3 Summary table of primary and secondary antibodies used in immunohistochemistry experiments. Show the host species of both primary (1) and secondary (2) antibodies, their dilutions and the fluorophores and their corresponding colour used for each antigen in the immunohistochemistry study. Ms; mouse. Rb; rabbit. Gt; goat. Hu; human. Bo; bovine. Do; donkey. ; anti. (i.e. Ms--Hu; Mouseanti-human; an antibody raised in a mouse against a human antigen.
Pre-absorption experiments testing the specificity of the polyclonal antibodies (anti( )-trypsin and -tPA) were performed using trypsinogen (Sigma, Australia) as well as purified recombinant motopsin (supplied by Shinichi Mitsui, Kochi Medical School, Nankoku, Jap`an). The tPA antigen was unavailable from the supplier as the antibody was deemed highly specific. The motopsin antigen was used as a nonspecific protease to determine the cross reactivity of both trypsin and tPA antibodies, as all serine proteases share approximately 40% homology (determined from Blast searches) and motopsin has been shown to have no cross reactivity with tPA [111].
2.1.23 Immunohistochemistry protocol Slides were washed 3 times (10min each) in 0.1M PBS to remove excess embedding compound. They were then exposed to a pre-block solution of 10% normal horse serum (NHS) in antibody diluent (0.1M PBS-0.1% tritonX100) for 1h at room temperature, after which, slides were rinsed 0.1M PBS. Primary antibodies were diluted in 4% NHS in diluent and placed on each sample. Dilutions were; -PAR2,
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1:50; -trypsin, 1:200; -GFAP, 1:500; -tPA, 1:50. Slides were placed in a dark humidity chamber and left for 12-16h at room temperature, after which they were washed 3 times (10min each) in 0.1M PBS and exposed to secondary antibody (60 l per Section; Donkey--Mouse Alexa488, 1:500 for PAR2; donkey--rabbit Alexa594, 1:500 for trypsin and GFAP; donkey--goat AMCA, 1:200 for tPA) (See also Table 2.3). Slides were placed in a dark humidity chamber for 1.5h at room temperature. Slides were then washed 3 times (10min each) in 0.1M PBS and coverslipped using phosphate buffered glycerol (pH=8.6) as a mounting medium and sealed with clear-coat nail varnish. Every step following the addition of the secondary antibody for incubation was done in low light conditions to prevent photo-bleaching of the fluorophores. Completed slides were stored in a slide box at 4C.
For double and triple labelling experiments, protocols were as above, except instead of being mounted in phosphate buffered glycerol following the secondary antibody incubation, the slices were exposed to the second primary antibody over a second night, and the remainder of the protocol repeated entailing the specificities for each antibody described above. Slices were incubated in the -trypsin or -GFAP antibodies over the first night, washed in PBS, and then exposed to -PAR2 over a second night and exposed to the corresponding secondary antibody as described above. Since both the GFAP and trypsin antibodies used in the current experiment were exclusively raised in the same host species (rabbit), double labelling experiments of trypsin and GFAP could not be performed.
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For the triple labeling experiments (i.e. trypsin, PAR2 and tPA), -trypsin and tPA (final conc. 1:200 and 1:50 respectively) were incubated together over the first night and -PAR2 was incubated over the second night. All protocols following the overnight primary antibody incubations (i.e. washing and secondary antibody) were identical for such antibody.
2.1.24 Positive and negative controls Pancreatic tissue was harvested from the same perfused rats used to collect the brain and used as a positive control for the antibodies. All immunohistochemical protocols were equivalent for the pancreas as those described for the brain. Negative controls consisted of omitting the primary antibody from the immunohistochemistry protocol, replacing it with the antibody diluent (4% NHS) only.
2.1.25 Pre-absorption experiments The protocol of pre-absorption of the primary antibodies with the antigens trypsinogen and motopsin were as prescribed by Shinichi Mitsui, who kindly supplied the full length recombinant motopsin antigen. Briefly, 10 g of either trypsinogen (Sigma) or motopsin recombinant antigen were preincubated with 1.0 g of either trypsin or -tPA antibody in 4% NHS/diluent at room temperature for 30min prior to placing the solution on the tissue for overnight incubation. The
immunohistochemistry protocol was identical thereafter.
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2.1.26 Thionin staining protocol Thionin staining was performed for histological examination of the electrode placement. Slides were thawed, air dried and cleared twice in xylene for 1min, rehydrated in methanol (2x100%, 90% 50% 1min each) and immersed in a thionin solution (0.1% in acetate buffer) for 30min. Following this, slides were washed in dH2O and run through a dehydration sequence of methanol (50%, 90%, 2x100%, 1min each), cleared in xylene (2 x 1min), mounted in dibutyl phthalate xylene, coverslipped and allowed to dry.
2.1.27 Timms staining protocol Mossy fibre sprouting was examined in electrically kindled brains from i.c.v. injected animals using Timms staining procedures, a technique that labels chelatable zinc ions present in growing synaptic terminals of the mossy fibre pathway. The technique uses silver staining methods to detect the presence of zinc in the synapses after its sulphonation. The Timms protocol was initially adapted from that described in the literature [360-362] by a colleague, Dr Bianca Jupp. The modified protocol consists of a non-perfusion sulphide treatment step using hydrogen sulphide gas followed by autometallographic development using silver nitrate.
Glass and plastic ware was rinsed in hydrogen peroxide to remove oxidants prior to staining procedures. Slides were loaded in plastic staining racks and transferred to a 6l glass-desiccator with a beaker containing a 400 ml, 0.1% (w/v) sodium sulphide solution adjusted to pH 7.4 with hydrochloric acid inside the desiccator causing the formation of hydrogen sulphide gas. The desiccator was evacuated of air using an electric vacuum pump (Millipore) thereby allowing pure hydrogen sulphide to
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smoke the slides. Sections were left in the desiccator at 4C for 12h overnight. The reaction was stopped by release of the vacuum seal in the fume cupboard.
Following the overnight sulphide treatment, sections were run through a rehydration sequence consisting of 15min in 100% ethanol, followed by 70% and 50% ethanol (2min each) and finally dH2O (also 2min). The auto-metallographic developer consisted of 120ml Arabic gum solution, 20ml sodium citrate buffer and 60ml hydroquinine solution. Silver nitrate (0.5ml) was added to the developer solution in low light conditions and stirred using a plastic spatula just prior to submersion of the slide rack in the developer. Slides were submerged in the developer solution and left in the dark for 90min at 37C, after which they were then rinsed under running tap water for 5min and dipped in dH2O for 10s before fixation in 5% (w/v) sodium thiosulphate solution for 10min. Sections were then dehydrated through a sequence of dH2O, 50%, 70% and 100% ethanol for 2min each, cleared in xylene and coverslipped with dibutyl phthalate xylene mounting-medium.
Microscopy and image acquisition 2.1.28 Bright field and fluorescence microscopy All light and fluorescence microscopy was performed on the same upright microscope (Axioskop 2, mot plus, with ebq100 isolated fluorescence burner, supporting Cy5.5, FITC, GFP and Rhodamine filter sets, Zeiss, Germany). Bright field images were only used for thionin and Timms stained sections. Fluorescently stained sections were visualised and imaged on either the upright fluorescence microscope or a confocal laser scanning microscope. For fluorescence microscopy, images were taken in grey scale at 100x, 200x or 400x using a digital camera (Axiocam Mrm, Zeiss,
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Germany) and captured using imaging software (Axiovision 4.4 Zeiss, Germany). Images were automatically coloured by the imaging software, with specific colours appropriately corresponding to each fluorophore. Illuminance and photographic constraints were kept uniform during each microscopy procedure, in particular for those images taken for quantitative analysis.
2.1.29 Confocal microscopy A confocal laser scanning microscope (fully automated Olympus IX81 inverted microscope, with VF1000 confocal equipment) was used for triple labelled section image acquisition as far superior specificity of fluorescence signals could be obtained, thereby allowing the isolation of specific small ranges of excitation and emission wavelengths and as such prevent any potential overlap between emission spectra.
Quantitation of data 2.1.30 Quantitation of kindling EEG data All EEG records were blinded by giving a false name to each file. ADT stimulus strengths were compared in non-injected and peptide-injected situations within and between treatment groups. All EEG records were visually analysed by counting seconds of post-stimulus AD/seizure. The stimulus artifact, as recorded on the EEG, acted as marker for the stimulus. The duration of each stimulus induced AD/seizure, the total number, total duration (including possible secondary seizure plus the latency between first and second seizure) and class of each seizure were recorded. The total number of stimulations required to induce 5 class V seizures, or a fully-kindled state (referred to as kindling rate) was recorded and compared between groups.
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2.1.31 Quantitation of 24h video-EEG data from kainic acid rats The amplifier and software package used in this experiment only uses the bipolar electrode for EEG recordings, therefore only one (intra-amygdala) EEG trace per rat was returned. Thus, cortical synchronisation of seizure activity was not able to be determined, which made EEG analysis quite difficult. Many apparent seizures on EEG were no more than a grooming artifact as confirmed by replaying the video, however, the resolution of the infrared camera, especially during the night-cycle, was not clear enough to observe seizure classes of low order (i.e. class I and II). If a suspected seizure on EEG was located that did not result in clear seizure behaviours, the EEG was more tightly scrutinised, often with the aid of independent researchers. EEGs that were not clear as to whether seizures or artifacts occurred from both video and EEG were discarded from analysis. Occasionally, the video and chart (EEG) recordings failed at some point during the 24h recordings, especially at night when the rats were not checked regularly. In cases where the video recording crashed, seizure analysis relied solely on EEG and, as such, a class of seizure could not be determined. Nevertheless, a true seizure on EEG was often text book and distinguishable from baseline and artifactual EEGs in its wave-form and frequency evolution, usually following a distinct and predictable progression (see Figure 2.7).
2.1.32 Quantitation of immunofluorescence and mossy fibre sprouting. Fluorescence intensity (FI) was measured in single stained sections only, thereby avoiding any epifluorescent bleed-through from another fluorophore. Axiovision 4.4 software (Zeiss, Germany) was used to measure optical densities of the regions of interest (ROI). ROIs were CA1, CA2, CA3, CA3c and dentate gyrus of the hippocampus and in the M1/M2 region of the motor cortex (see Figure 2.8). The
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stratum moleculare-lacunosum (SML) of the hippocampus proper devoid of staining was used as a reference or background fluorescence value. Methods used were similar to those previously described [32-36]. An ROI was imaged and isolated by drawing a box around the region and the optical FIs measured using the software (average greyscale pixel intensity per unit area). The final FI of each ROI was determined by division of the background value (Equation 2.2). FIs were measured in the 6 ROIs described in 3-5 slices of each brain, and final densities were expressed as an average per region.
FI final =
F ROI I FI SML
Equation 2.2; Final fluorescence intensity (FI final) ratio is a product of the fluorescence
intensity (FI) of the region of interest (ROI) and the background (SML)
Optical density (OD) of Timms stained Sections for mossy fibre sprouting was measured in a similar manner. Sections were visualised in bright field using the same microscope described above. A box was drawn around the outer molecular layer of the dentate gyrus (stratum moleculare, SM), and the stratum oriens (SO) and stratum radiatum (SR) of the CA3 (Figure 2.9). OD were measured using the same software as described above. The OD of the SM and SO were expressed as a ratio to the background (control) SR, a region that is insensitive to mossy fibre changes [363] (Equation 2.3).
O final = D
O SM or O SO D D O SR D
Equation 2.3; Final optical density (OD final) ratio is a product of the fluorescence intensity
(FI) of the regions of interest (SM or SO) and the background (SR)
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2.1.33 Statistical analysis. Data were analysed using statistical software packages (Microsoft Excel for Mac X, and Statistica 6.0, StatSoft Inc., OK, USA). Significance tests utilised were Students t-tests and ANOVA for analysis of data from two or three groups respectively. Where data was collected from the same animal longitudinally (e.g. behavioural class and EEG seizure afterdischarge length during kindling) a repeated measures ANOVA was performed. Planned comparison post-tests were performed between all groups. Data was expressed as mean s.e.m. The level of significance was set at p0.05 (two tailed) for all tests.
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Figure 2.8 Regions of interest (ROI) used in the immunofluorescence Cortex
CA1 DG
Background CA3c CA3
CA2
Figure 2.8 Regions of interest (ROI) used in the immunofluorescence quantitation experiments. Boxes highlight the ROI from which fluorescence intensity measurements were made
SM SR
SO
Mossy fibre pathway
Figure 2.9 Timms-stained hippocampus and region of interest schema. shows a grey scale image of a Timms stained left hippocampus, illustrating the mossy fibre pathways from the dentate gyrus to the CA3. An ROI was placed in the dentate stratum moleculare (SM) as well as CA3 stratum oriens (SO) and radiatum (SR). Mossy fibre sprouting into the SO and dentate SM was assessed, and normalised to the SR to account for variations in staining. Image was taken at 4X magnification. Scale bar represents 250 m. Image courtesy of David Liu.
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quantitation experiments Figure 2.9 Timms-stained hippocampus and region of interest schema.
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Chapter 3 Localisation of PAR and trypsin in the brain
Chapter 3 Immunohistochemical localisation of PAR2 and trypsin in the rat brain
Introduction In order to allow for plastic changes in the hippocampus, cell adhesion molecules constituting the matrix must be partially digested to allow for formation of novel cellto-cell synaptic connections, and this is achieved by the action of proteases [84, 364]. The subcellular localisation of various proteases has of late often been shown to be in granular-like structures in many cell types. For instance, tPA is stored in vesicles other than Weibel-Palade bodies in vascular endothelium cells [323], trypsin in zymogen granules of pancreatic acinar cells [144] and as trypsin IV (mesotrypsin) in small vesicles of epithelial cell lines from the prostrate, colon and airways [365]. As already discussed in Chapter 1, hippocampal neurons store tPA in granular structures known as large dense core vesicles (LDCVs) in the soma and axons [132, 133, 327], and is even considered to be a marker for LDCVs [132].
Evidence suggests that serine proteases and PARs may have both neurodegenerative and neuroprotective properties in pathological states [40, 44, 63, 67, 87, 109, 110, 154, 197, 204, 245, 285, 286, 309, 320-322, 333, 366, 367]. All four PARs have been located in the rat brain [285, 309, 310, 319, 320]. Of the thrombin receptors, PAR1, PAR3 and PAR4 have all been shown to be expressed in neurons, microglia and astrocytes [137, 319, 321, 325, 326, 328, 330, 368]. Thrombin, the major agonist of these three receptors, and the prerequisites for its activation (i.e. prothrombin and Factor Xa) as well as inhibition (i.e. serpins, see Chapter 1), have been located in the rat brain [115, 117-119, 135, 285, 295, 309, 319, 320, 369-371]. The trypsin receptor,
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PAR2, is expressed on neurons of the hippocampus, cortex, amygdala, thalamus, the hypothalamus and the striatum. PAR2 is also expressed on cultured astrocytes [153, 155, 285, 328, 330] and microglia [135]. Very little work has, however, been published on the regional and cellular expression of trypsin in the brain, even though it has been shown to be present in pyramidal cells in human hippocampi [121] and as the zymogen trypsinogen (I or IV/mesotrypsin) at the mRNA level [122, 309, 372]. The subcellular expression of trypsin and how it relates to that of PAR2 in the brain is not well characterised, yet this knowledge is vital to fully understand the function of this protease, which is no doubt important in the brain. Furthermore, since PAR2 is highly expressed in all regions of the brain, it seems only logical to predict a similar regional distribution pattern for it and trypsin.
The aim of these studies was to use immunohistochemistry to investigate the regional and cellular distribution of trypsin and PAR2 the in the rat brain, with special reference to the hippocampus and the highly expressed central protease, tPA.
Methods All methods for immunohistochemistry are described in detail in Chapter 2. Brief outlines follow here.
3.1.1
Tissue preparation
Female rats (NECs, n=20) were terminally anaesthetised with xylazine (20mg/kg) and ketamine (100mg/kg, i.p.) and transcardially perfused with 100ml phosphate buffered saline (PBS, pH 7.4) followed by 400ml 4% paraformaldehyde (PFA), both at 4C. The brains were removed, post-fixed overnight in 4%PFA at 4C then cryoprotected
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in 20% sucrose-PBS at 4C for 48h before being embedded in freezing compound (OCT, Tissuetek) and snap frozen over liquid nitrogen.
3.1.2
Immunohistochemistry
Coronal, cryostat cut sections (20m) of hippocampal/thalamic regions were exposed to pre-block solution for 1h, incubated for 16h in primary antibodies in 4%NHS/diluent [Ms--Hu PAR2 (SAM-11) 1:50; Rb--Hu (pancreatic) trypsin (PRSS1), 1:200; Bo--Rb GFAP, 1:500; Gt- Hu tissue plasminogen activator
(PLAT; tPA) 1:50], then exposed to secondary antibody for 1.5h [PAR2: Do- -Ms IgG Alexa-488 (1:500); trypsin/GFAP: Do- -Rb IgG Alexa-594 (1:500), tPA: Do -Gt IgG AMCA]. Sections were visualised and imaged on either a fluorescence microscope or a confocal laser scanning microscope.
Results 3.1.3 PAR2 in the rat hippocampus
PAR2 was found to be highly expressed in the hippocampus. The size, morphology and anatomical distribution of PAR2-like immunoreactive (IR) cells in all regions examined suggest that the majority, if not all PAR2-like IR cells were neurons and not glial cells. Contrary to other studies utilising either brain slices or cultured conditions [204, 285, 330, 332], PAR2-like IR was not observed in astrocytes, with no overlap occurring between PAR2-positive cells and the astrocytic marker, GFAP, suggesting that astrocytes did not express PAR2 in the brain of rats used in this particular study (Figure 3.2).
In excitatory pyramidal cells throughout the CA1, CA2 and CA3, and CA3c (CA4)
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regions and in granular cells of the dentate gyrus of the hippocampus, PAR2 had a strong expression in the membrane, proximal dendrites and axon hillock, and diffusely throughout the cytoplasm, with no PAR2-like IR detected in the nucleus (Figure 3.1). Pyramidal cell staining was remarkably similar to that previously described [285], however, contrary to an earlier report [319] there were no obvious differences in fluorescence intensity within pyramidal cells between the CA regions of the hippocampus. PAR2-positive interneurons were also scattered throughout the stratum moleculare and alveus, displaying similar membranous and somato-dendriticlike staining as observed in the pyramidal cells (Figure 3.1). Punctate and striated PAR2-like IR staining was also observed in the fimbria hippocampi, most probably representing an axonal/dendritic localisation of PAR2 in the Schaffer collateral and associational-commissural paths (Figure 3.2). Weak staining was detected in the infrapyramidal mossy fibre pathway of the alveus of CA3, but not the supra-pyramidal mossy fibre pathway, with ramified staining in the stratum radiatum of the CA3 at the proximity of the fimbria hippocampi (Figure 3.1).
Granule cells of the dentate gyrus were of round appearance with strong membranous PAR2-like IR, with less apparent (diffuse) cytoplasmic staining as that observed in the pyramidal cell layers. Granule cells displayed little proximal process staining as observed in the pyramidal cells (Figure 3.1). There was no PAR2-like IR observed in the dentate hilus other than that in pyramidal and inter-neurons. The stratum lacunosum-moleculare of the hippocampal sulcus was completely devoid of PAR2like IR.
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Figure 3.1 PAR2 is highly expressed in all regions of the rat brain
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Figure 3.2 PAR2 expression in white matter tracts of the rat brain
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3.1.4
PAR2 expression in the cortex, thalamus, striatum, amygdala and white matter tracts
PAR2-like IR was observed in all layers of the cortex, with similar cellular-like staining as observed in the hippocampal pyramidal cells. Cortical neurons generally expressed PAR2 in the membrane and diffusely throughout the soma and proximal dendrites. No cortical layer or region was generally more distinctly labelled than another, all layers showing similar degrees of neuronal staining. In some instances, long apical dendrites were distinctly labelled in the internal pyramidal layer (layer 5) which extended towards layer 1 (Figure 3.1).
In the thalamus, PAR2-like IR was common in neurons, with a somato-dendritic cellular distribution much like observed in the hippocampus and cortex. However, this thalamic PAR2-like staining was generally of lesser intensity than that observed in these other structures. Also, much like observed in the cortex, no particular nucleus of the thalamus was more distinctly labelled than another. Neurons of the magnocellular region of the paraventricular nuclei also displayed distinct PAR2-like IR.
The nuclei reticularis thalami (NRT) had an obvious reticular PAR2-like IR that distinctly lacked cell somata, only showing diffuse process-like staining forming a net-like structure (Figure 3.2). The external medullary lamina was generally void of specific PAR2 staining.
In the amygdala, neurons showed intense somatic PAR2-like IR, with less distinct membrane-like staining as observed in the cortex and hippocampus and for some larger ovoid-shaped cells, the PAR2-like IR had a granular appearance. The intensity
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of PAR2-like IR in this region was consistantly higher than that of the thalamus, suggesting a higher requirement for PAR2 in this limbic region (Figure 3.1).
The striatum/caudate putamen showed similar PAR2 staining to the NRT, with a reticular-like staining pattern being more prominent than any distinct somatic PAR2like IR. Some cell bodies scattered throughout the reticular network, however, weakly expressed PAR2 (data not shown).
Punctate PAR2-like IR was detected in white matter tracts, particularly in the corpus callosum, external and internal capsule, the cingulum, the stratum radiatum and fimbri hippocampi, and stria medularis and the stria terminalis, which represents an axonaldendritic PAR2 expression. The cingulum in particular showed distinct process-like PAR2 staining radiating into the primary and secondary motor cortices from the external capsule, merging into the distinct neuronal PAR2-like IR of the cortex described above (Figure 3.2).
3.1.5
Trypsin expression in the rat hippocampus
Much like that described for PAR2, the hippocampus had an abundant trypsin expression. Unlike PAR2, however, trypsin expression was found in cells with morphologies resembling both neurons and in smaller stellate cells likely to be astrocytes, particularly in the cortex and hippocampus. High levels of trypsin-like IR were detected in the CA1, CA2, CA3 and CA3c pyramidal cell layers, usually appearing as diffuse staining throughout the cytoplasm, but often with a granular-like appearance (Figure 3.3). Little trypsin-like IR was observed in processes of the infraor supra-pyramidal layer of the mossy fibres unlike that shown for PAR2, however,
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the mossy fibres in the stratum radiatum of the CA3 showed punctate trypsin-like IR. Furthermore, stellate cells resembling astrocytes were distinctly labelled in the stratum oriens, stratum moleculare and dentate hilus (Figure 3.3). Trypsin-like IR positive interneurons were scattered throughout the stratum moleculare together with trypsin-positive glial-like cells, which were also observed in the stratum oriens and alveus. Much like PAR2, punctate and striated trypsin-like IR was observed in the fimbria hippocampi, which again probably represents trypsin expression in neuronal process of the Schaffer collateral path and associational-commissural pathway (Figure 3.4). The stratum lacunosum around the hippocampal sulcus was devoid of staining.
Granule cells of the dentate gyrus were strongly trypsin-IR positive, showing a distinctly round morphology with a particularly dense somatic trypsin staining (Figure 3.3). Generally, these cells appeared more intensely labelled than the pyramidal cells, interneurons and glial-like cells also trypsin-positive. Little process-like trypsin-IR was observed to arise from the granular layer to the mossy fibre pathway.
Confocal microscopy revealed trypsin-like IR was confined to large granular-like structures throughout the cytoplasm of pyramidal neurons (estimated diameter 50200nm), suggesting trypsin, like other proteases previously described, may be stored within vesicles of these pyramidal cells (Figure 3.4).
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3.1.6
Trypsin expression in the cortex, thalamus, striatum, amygdala and white matter tracts
All regions of the cortex appeared trypsin-positive. Similar to that observed in pyramidal and granule cells of the hippocampus, trypsin-like IR generally appeared somatic. Again, staining was intense within cellular structures resembling both pyramidal neurons and glia. In neurons, trypsin staining was diffuse throughout the soma, even appearing in granular-like structures, which again was most evident using confocal microscopy (Figure 3.4). There were no distinct differences observed in trypsin-like staining between layers or regions of the cortex, however, cells around the longitudinal fissure were often more strongly labelled than neighbouring cells (Figure 3.3).
In the thalamus, trypsin-like IR was observed in cells with both neuronal and glial morphologies, generally displaying a somatic-like staining pattern (Figure 3.3). Much like that observed in the PAR2 localisation study, trypsin staining here was less intense than the hippocampus and cortex. No specific nuclei of the thalamus displayed more intense staining than another. The NRT was weakly trypsin-IR positive, displaying a reticulated punctate staining pattern.
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Figure 3.3 Trypsin is highly expresses in all regions of the rat brain
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Figure 3.4 Trypsin expression in white matter tracts and neuronal vesicles
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Trypsin-like IR in the amygdala was intense in scattered neurons with both pyramidal-like and interneuron-like morphologies (not shown). Cells in this region had a somato-dendritic trypsin-like IR, usually with a granular-like staining pattern. Little trypsin staining was observed in the striatum/cordate putamen, however, like in the NRT, it appeared punctate and reticulated.
Trypsin-like IR was also detected at low levels (both intensity and density) in the white matter tracts, in particular the corpus callosum, cingulum, external capsule, fornix and fimbria hippocampi. Staining in these regions was generally punctate and striated (Figure 3.4).
3.1.7
Trypsin and tPA colocalise in PAR2-positive neurons
All neurons that were positive for PAR2-like IR were also positive for trypsin-like IR in all regions of the brain examined, although at varying levels of intensity between cells within the same regions. In pyramidal cells of the hippocampal CA regions, granule cells of the dentate gyrus and neurons of the cortex (Figure 3.5), thalamus and amygdala (not shown), PAR2-like IR was localised to the plasma membrane and diffusely throughout the cytoplasm. In contrast, trypsin-like IR was generally localised to the soma of these same cells often displaying a granular-like appearance with varying intensities (Figure 3.5). This different subcellular staining pattern of PAR2 and trypsin was best demonstrated with confocal microscopy, which revealed trypsin-IR occurred in distinct large granular-like structures throughout the soma of neurons (CA3 pyramidal cell shown, Figure 3.6) whereas PAR2-IR staining was localised in the plasma membrane and diffusely throughout the cell soma. Importantly, the granular-like pattern of trypsin-like IR was replicated with tPA-like
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IR (Figure 3.6), strongly suggesting that trypsin is stored together with tPA, most likely in LDCVs [132, 133] of PAR2-positive neurons.
3.1.8
PAR2 and trypsin in the pancreas
Pancreatic tissue used as a positive control revealed intense PAR2-like IR in the membrane of acinar cells with trypsin-like IR throughout the pancreas in both acinar cells and ducts (Figure 3.7). PAR2-like IR also lined the ducts positive for trypsin. These results suggests both the trypsin and PAR2 antibodies are most likely specific to the antigens to which they were raised, as staining was similar to that previously described [121, 304, 318, 373].
3.1.9
Primary antibody pre-absorption
Pre-absorption of the trypsin antibody with purified trypsinogen appropriately blocked all trypsin-like IR, indicative of a specific binding of the antibody to the trypsinogen antigen. Preblocking tPA antibody with trypsinogen had no effect on staining, suggesting there was no cross-reactivity between the tPA antibody and trypsinogen antigen. Motopsin pre-absorption also had no effect on either trypsin-IR or tPA-IR. Therefore, the staining observed was most likely specific to each antibody, and not the result of a non-specific binding of either antibody to proteins sharing only approximately 40% homology, such as trypsin, tPA and motopsin.
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Figure 3.5 PAR2 and trypsin colocalise in all regions of the rat brain
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Figure 3.6 Trypsin and tPA colocalise to LDCVs in PAR2 positive neurons
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Figure 3.7 PAR2 and trypsin expression in the pancreas
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Discussion The data presented in this Chapter demonstrate that PAR2 and trypsin are highly coexpressed within neurons of the rat brain, with the highest levels being detected in plastic regions of the limbic system such as the hippocampus. All neurons positive for PAR2 were positive for trypsin, which appeared within granular-like structures together with tPA. Trypsin, but not PAR2, was also present in cells with a distinct stellate glial-like morphology. Trypsin and tPA were also co-stored in LDCVs within PAR2-positive neurons of rat hippocampus. Based on previous literature describing roles for proteases in the brain, these findings indicate that both trypsin and PAR2 may have roles in facilitating and regulating neuronal plasticity and synaptic reorganisation related to memory and learning. However, given the nature of proteases in the brain, trypsin and PAR2 may also be involved in pathogenesis related to neuroinflammatory conditions such as epilepsy [40, 43, 45, 63, 64, 73, 86, 154, 374] multiple sclerosis [122, 126, 204, 375] and Alzheimers disease [90, 115, 205, 246].
This study is the first to characterise trypsin expression in the brain specifically in relation to PAR2. The staining of PAR2 in neurons showed a distinct somatodendritic and plasmalemmal pattern. The relatively high level of PAR2-like IR in the limbic structures, in particular in the hippocampus and amygdala, suggests a high requirement for this receptor in highly plastic regions, much more so than structures with lesser plasticity, such as the thalamus. The presence of trypsin within PAR2positive neurons in the brain in these same limbic structures suggests that like other proteases such as tPA, motopsin and thrombin, trypsin may have important roles in hippocampal functioning. Furthermore, that trypsin is co-stored with tPA [133], the
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protease inhibitor neuroserpin [152] and possibly other proteases, in vesicles likely to be LDCVs, suggests these proteases and inhibitors may be released as a package and may work together to facilitate local neuronal plasticity and potentially coordinate cellular mechanisms related to memory, such as matrix remodelling and LTP. Activation of trypsin may occur as in the gut, since enterokinase has been detected in the brain at the mRNA level [143]. Perhaps this is stored in separate vesicles to the zymogen, much as done in the pancreas.
The high levels of trypsin-like IR noted in the granule cells of the dentate gyrus may reflect the high level of plasticity encountered in this region. The mossy fibre pathway is a highly plastic region connecting the dentate gyrus to the Schaffer collaterals of the CA3 region which, during epileptogenesis, becomes aberrantly plastic in a process known as mossy fibre spouting that is most likely to be dependent on the function of proteases like tPA [67, 376]. The high levels of trypsin-IR encountered here may reflect a similar involvement of trypsin in plastic processes. Perhaps like tPA [40, 53, 64, 72, 88], trypsin is also up-regulated during brain insults and may thus be involved in epileptogenic phenomena such as aberrant LTP, mossy-fibre sprouting and cell losses. This question will be further investigated throughout the course of this thesis (see Chapter 6).
The predominant plasmalemmal expression of PAR2 in neurons of the hippocampus and the co-expression of trypsin with tPA in LDCVs within these cells raises the possibility of an autocrine feedback type mechanism to regulate the extracellularlyactivated enzymes involved in plasticity changes, as previously suggested [259]. The apparent granular-like PAR2-like staining pattern in the somata of these hippocampal
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neurons possibly indicates a high receptor turnover [285, 377-380], and/or intracellular storage of newly synthesised receptors ready for shipment to the membrane when required. Such mechanisms could be taken as further evidence that these protease-expressing neurons require full-time monitoring by PAR2. That is, such cells maintain high levels of plasmalemma PAR2 expression, and in this sense, may provide neuroprotection against proteolysis. The possibility that PAR2 acts as the detector for multiple, co-released proteases will be addressed further in the General Discussion (Chapter 8).
If trypsin is involved in facilitating synaptic plasticity related to memory and learning (like tPA), then it would be expected to be located around synaptic boutons. Similarly, if PAR2 functions as an autocrine regulator of trypsin (and possibly tPA and other proteases), then it too would be expected to be found in the same region. The punctate staining of both trypsin and PAR2 on neuronal processes supports this proposal and suggests that either there are clusters of PAR2 on dendritic spines (or boutons), or an anterograde shipment of packages of PAR2 and trypsin through the axon/dendrite to more distal cell structures at the pre- and/or post-synapse is occurring. How proteases stored in LDCVs, which are apparently too large to fit through the lumen of an axon/dendrite, are shipped to these distal structures is unknown. It may, however, involve secondary vesicular transport mechanisms such as piece-meal, where smaller protease-containing shuttle vesicles are cleaved from LDCVs and shipped as a more transportable package [381]. Questions such as these require further investigation using more robust microscopic techniques, such as scanning electron microscopy and potentially electrophysiology that together may isolate a function for trypsin and PAR2 at the synapse.
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The lack of PAR2 staining in astrocytes in this study is in conflict with other reports that have demonstrated PAR2 in glial cells under culture conditions [204, 285, 330] and in isolated rat brain slices [285]. Perhaps this reflects differences in strain of animal or experimental conditions used between studies. The one study which did describe an astrocytic localisation for PAR2 in brain slices did not, however, demonstrate this with a photomicrograph [285], making it difficult to compare its results with those of the present study. Although the earlier study did provide photomicrographs sharing strikingly similar PAR2-like IR of pyramidal cells in the hippocampus [285] as that presented here, they did not reveal cells with a distinct stellate, astrocytic morphology. Perhaps astrocytes express such low levels of PAR2 in vivo under normal circumstances that they are undetectable using
immunohistochemistry. Furthermore, in culture, astrocytes may bolster their intrinsic PAR2 expression, perhaps as a response to the stress of cell extraction and the culture process. Perhaps cultured cells are in an inflammatory state, evidenced by the upregulation of PAR2 and possibly other molecules involved in this stress-related situation. Finally, another possible reason for the lack of PAR2 on these astrocytes is that in vivo they function as scavengers, or janitors, of molecules such as glutamate, cytokines and perhaps proteases. Thus, the trypsin detected here in astrocytes may have been in the process of degradation after being scavenged by astrocytes following its release from neurons. This may suggest that cells which functionally require proteases, such as neurons, also express PAR2, but those which do not (i.e. astrocytes) may have no requirement for PAR2, which may explain the lack of PAR2 in astrocytes in vivo.
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The pre-absorption experiments described here indicate that both the tPA and trypsin antibodies used were most likely specific to their antigen. Since tPA, trypsin and motopsin share approximately 40% homology (Blast search
(http://www.ncbi.nlm.nih.gov/blast/ Blast.cgi)) [382] making up the catalytic triad of the active site [382], some cross-reactivity of antibody binding between trypsin and tPA may be expected. However, it is generally accepted that that greater than 85% homology is necessary before cross-reactivity using polyclonal antibodies can become problematic (Abcam technical staff, written correspondence). Thus, pre-blocking with motopsin that shares 40% homology with both tPA and trypsin, was considered a useful means of determining any non-specific binding of either trypsin or tPA antibodies to such a similar protease. Unfortunately, we could not get access to the specific tPA antigen to which the antibody was raised in order to determine whether the trypsin antibody binds to the tPA antigen. By default, however, since both tPA and motopsin share the same homologous regions to trypsin [382] and preblock with trypsin antibody and motopsin antigen revealed no cross reactivity of any antibody. This suggests that the trypsin staining was specific to trypsin(ogen) and not to any like proteases such as tPA and motopsin. Likewise, tPA binding was unaffected by preabsorption with either trypsin(ogen) or motopsin antigen, indicating that the staining observed in the current study was most likely specific for both trypsin and tPA respectively.
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One potential problem with the interpretation of the trypsin localisation data is that the trypsin(ogen) detected in the brain cannot be unequivocally identified as cationic (pancreatic) trypsin (PRSS1, trypsin-I) to which the antibody is raised and not the previously described (at the mRNA level) trypsin(ogen)-IV (mesotrypsin)[12, 20]. As trypsin-I and trypsin-IV share 90 to 97% homology (Blast search) it seems highly likely that the trypsin antibody used here labels both isoforms. This, however, may not be of concern, as even though still controversial [383], trypsin-IV most likely activates PAR2 [365]. Given the high homology and the cellular localisation of trypsin-like IR in regard to PAR2, it seems logical to predict that whatever trypsin isoform was detected here, it would be able to activate PAR2. Obviously due to the high degree of homology between the trypsin (I and IV) molecules it will be difficult to isolate each isoform using polyclonal antibodies. Thus, techniques other than immunohistochemistry will be required to accurately scrutinise which isoform was most likely detected in the current study. In summary, two novel findings arose from this study. First, trypsin and its receptor, PAR2, were found to be highly expressed and colocalised to neurons of many regions of the rat brain, with particularly high expression of both in plastic regions of the limbic system such as the hippocampus. The second novel finding was that trypsin is stored in LDCVs together with tPA within PAR2-positive neurons, suggesting that both enzymes (and possibly other proteases and inhibitors like neuroserpin) are released together as a neuromodulatory package in response to common stimuli. In addition to these two important findings, the study also found that trypsin, but not PAR2, was expressed in astrocytes. This finding demonstrates that not all cells that contain trypsin necessarily co-express PAR2. Perhaps normally in vivo astrocytes provide another level of control for extracellular trypsin by scavenging it and other
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enzymes from the matrix following release from neurons. Therefore, in conclusion, the co-expression of trypsin and PAR2 in neurons of plastic regions of the brain suggests proteases like trypsin (and tPA) have not only have important functional roles in memory and inflammatory diseases such as epilepsy, but the activity of these potentially harmful proteases may be tightly regulated by the neurons themselves via a PAR2-dependent feedback mechanism and phagocytotic astrocytes.
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Chapter 4 Pharmacokinmetics of SLIGRL
Chapter 4 The pharmacokinetics of SLIGRL in cerebrospinal fluid
Introduction Many studies have used SLIGRL or other PAR peptides in vivo, most recently concentrating on peripheral neurogenic responses to inflammation, nociception and hyperalgesia in response to localised injection (such as intraplantar or intradermal) of the peptide at various concentrations [278, 281, 283, 284, 290, 294, 297, 384]. These studies all focused on roles for PAR2 in peripheral nerves and most assumed no direct influence of the PAR2 agonists on the CNS. One study used intrathecal (directly in to the spinal CSF) administration of the PAR1 and PAR2 peptides showing a spinal nerve role in thermal hyperalgesia [385]. The authors of that study, however, did not comment on the potential for these peptides to influence any higher brain structures. Presumably, when injected intrathecally, a peptide such as SLIGRL that is highly soluble in aqueous solutions, has the potential to affect the entire brain and spinal chord due to rostral and caudal CSF flow. In the study mentioned above [385], no pharmacokinetic analysis of SLIGRL in CSF was reported, even though CSF microdialysis was performed for purposes of PGE2 analysis. One key study that did address how SLIGRL may affect the CNS, utilised an intraparenchymal delivery of the PAR2 peptide into the striatum of rats [309], and as such the observed effects were most likely localised to the region of injection [386]. That study [309], however, also did not describe any pharmacokinetics of peptide. Furthermore, the small volumes administered would be expected to diffuse only small distances from the site of injection and not enter CSF to influence whole brain function [386].
It has not been previously reported whether PAR peptides such as SLIGRL
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redistribute between body compartments after administration via any route, and whether they remain active after redistribution. It is also not clear if SLIGRL enters plasma following non-systemic injection (i.e. i.p. or s.c.), and whether it enters the CSF from plasma per se. Even though at present it may be speculative, such systemic administration of a PAR peptide at the very least may result in a CNS-mediated response, as small peptides are known to cross freely from plasma to CSF [387].
For pharmacokinetic analysis of any compound in a whole animal it is necessary to collect samples of biological fluids such as plasma and CSF, both prior to and serially after administration of the compound. This allows the determination of the dynamic changes in the concentration of the compound over time and its movement throughout body compartments. Plasma collection techniques are relatively simple, such as collecting venous or arterial blood. However, CSF collection is less trivial, particularly from rodents. In the human, a lumbar puncture, or spinal tap is a commonly used technique for CSF collection and even though somewhat painful and uncomfortable for the patient, large volumes are relatively easy to obtain by a physician due to the considerable volume of CSF in the human and the large intravertebral space from which samples are obtained. In rodents, however, samples are difficult to obtain due to the much smaller volumes and spaces from which the samples are obtained. An adult rat (i.e. 300g), for instance, has a volume of CSF of approximately 540l [388]. This not only encompasses about 0.3% of total body water, but it also resides in a very small compartment of the ventricular system of the brain, the cisterna magna and the intravertebral space. Most studies describing drug concentrations in CSF of the rat utilise the cisterna magna for CSF microdialysis, a procedure known as cisternal puncture [389-393] due to a relatively large volume of
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CSF of up to 150 l that is able to be collected [391]. In an anaesthetised or awake rat, samples are able to be withdrawn by introducing a small gauge needle into the cisterna magna via the posterior atlanto-occiptial membrane and through the foramen magna [389]. This technique, however, can lead to damage to the brain stem or cerebellum, making serial collection of CSF for longitudinal studies rather difficult. Furthermore, such samples are often contaminated with blood, particularly during serial sampling, where the same injection site is used repeatedly. Thus, many researchers surgically introduce a cannula into the cisterna magna, making it possible to collect volumes from a freely moving rat [392]. However, due to the position of the cannula in close proximity to the brain stem, the movements of the rats head can cause damage to this particularly delicate region. Other routes of CSF collection are possible in rats, such as a lumbar puncture [385, 394], and from the fourth [394] or lateral [395, 396] ventricles of the brain. These latter bilateral lakes of CSF in the rat are of small volume (approx. 10 l) but are much less precarious for the rat during CSF collection than the cisterna magna, as the cannula is firmly in place and well away from moving structures such as the atlanto-occipital joint near the brain stem. Furthermore, as the rat has a CSF production/turnover rate of approximately 2.2 l/min [397], it is highly likely that small volumes collected from the lateral ventricles are replaced within minutes, making serial sampling possible [395, 396]. The surgical techniques of lateral ventricular cannulation have been well established by our laboratory and are therefore potentially a viable source of CSF for pharmacokinetic analysis.
Analytical techniques of the collected biological fluids for pharmacokinetic studies generally utilise highly specialised and accurate spectroscopic or chromatographic
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technologies. Liquid chromatography coupled mass spectrometry (LCMS) is an analytical technique that couples the separation capabilities of liquid chromatography with mass spectrometry analysis techniques for detection of small molecule drugs, metabolites and peptides in nonvolatile liquids such as biological fluids including plasma and CSF. The mass spectrometry (MS) component is used due to high sensitivity and specificity for molecular weights, particularly in tandem MS-MS situations, where the mass spectrometry step is repeated multiple times. Thus, relative amounts and potentially the structure of specific molecular fragments can be determined for compounds with a small molecular weight. For this reason, LCMS is routinely used for pharmacokinetic studies of drugs and peptides, giving an indication of relative concentration and temporal changes in concentration (half-life) of the compound/drug in question in a biological fluid. The PAR2 agonist peptide SLIGRL is a six amino acid (carboxyl-terminally amidated) peptide with a molecular weight of 657.8, and as such is a good candidate for LCMS-MS analysis and detection of levels in CSF.
Therefore the purpose of this study was to determine the pharmacokinetics of SLIGRL in CSF, specifically whether the peptide is detectable in such biological fluids using LCMS analysis, and whether SLIGRL can enter the CSF following a peripheral administration. Further, we wished to confirm whether the lateral ventricles are a viable source for serial CSF sampling. Ultimately, we wished to determine a half-life for SLIGRL.
Methods All methods have been described in detail in Chapter 2
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4.1.1
Animals, surgery and confirmation of cannula placement
Surgical implantations in female NEC rats (n=14, 12-14 weeks) were as previously described (Chapter 2.4), with some minor alterations. Briefly, rats were implanted with an i.c.v. cannula in the lateral ventricle, at the same coordinates described previously, via which SLIGRL was injected and CSF extracted. Bipolar electrodes were omitted in these experiments. All rats with an i.c.v. cannula were subjected to the angiotensin II test to confirm correct placement, as previously described in Chapter 2.5.
4.1.2
i.c.v. peptide injection
The injection protocol for i.c.v. SLIGRL administration was as previously described in Chapter 2.3.
4.1.3
CSF extraction
A total of seven serial samples of CSF were extracted from the embedded i.c.v. cannula. Time points of extraction were:- 5min prior to peptide injection, and 5, 10, 20, 45, 60 and 90min post-injection following i.c.v. injection (n=1), and 5min prior to peptide injection and 10, 20, 45, 60 and 90min following i.p. peptide injection experiments (n=3). The first post-injection sample (t=+5min) was not collected following i.p. administration to allow for SLIGRL absorption and redistribution in the circulation and CSF. All serial samples were collected prior to preparation of the samples for LCMS analysis.
4.1.4
Preparation of CSF samples
A total of 77 CSF samples were collected from 14 animals. Of these, 4 animals were
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injected with SLIGRL (i.c.v., n=1; i.p., n=3). Samples from one additional animal were pooled for LCMS confirmation by spiking the sample with SLIGRL (see below, section 4.2.7). All other CSF samples were used for purposes not related to this thesis, but did validate the use of the lateral ventricle as a CSF source.
4.1.5
LCMS-MS
LCMS analyses were performed by a contract service laboratory at the Bio21 Institute (Melbourne, Victoria, Australia). The LCMS machine (Agilent 1100 LCMS ion-trap SL) eluted 8 l of each of the diluted CSF samples. The first sample collected (t=5min) was the first to be analysed, followed by the final sample (i.e. t=+90min) and each previously collected sample thereafter in reverse order. Thus, the highest (expected) concentration was run through the column last in order to avoid contamination of the column with high concentrations of SLIGRL. The column was flushed with acetonitrile following each experiment to remove any residual SLIGRL.
4.1.6
Confirmation of SLIGRL detection using LCMS
In order for the LCMS to specifically detect SLIGRL, it needed to first be confirmed that the peptide could be detected at its molecular weight (657.8MW). This was done by spiking 20 l samples of micro-filtered water, saline and clean (neat) CSF (pooled from multiple extractions, n=1) with known concentrations (0.0, 4.2, 8.3 and 12.5 M) of SLIGRL and running these through the LCMS to determine the finger print (peak intensity and elution time relative to concentration) for each spiked sample.
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4.1.7
Calculation of half-life (t1/2) of SLIGRL
Relative concentrations of SLIGRL were represented by the area under the curve (AUC) generated by the LCMS machine. Thus, the apparent concentration of the drug was a function of peak intensity and time-of-flight, or elution duration. All postinjection sample area values (relative concentrations) were normalised to baseline (pre-injection) values by division.
For t1/2 calculation, the normalised AUC values were expressed as the natural log (ln) and plotted against time. t1/2 of SLIGRL was calculated by linear regression using the following formulae;
ln[C m ax] ln[2] t1 / 2 = w herek = C m in k

Equation 4.1; Half-life (t1/2) calculation. C max = Maximum concentration (AUC) collected, C min = Minimum concentration (AUC) collected, = time interval between max and min AUC., k = rate constant.
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Results 4.1.8 Reliability of CSF extraction from lateral ventricles
On average, the volume of CSF collected from the lateral ventricle over all time points and animals was 4.1 0.2 l (n=14 animals, total 77 samples), which did not vary significantly over the serially-collected samples throughout the experiment (Figure 4.1). Furthermore, the samples collected were clean and blood free, being transparent and straw-yellow in colour, as well as not showing any precipitates in the microfuge tubes following the precipitation step. The volumes of CSF collected, even though small, were sufficiently large enough following dilution for LCMS analysis, generally being of a volume around 21 l after dilution. Thus, serial sampling from the lateral ventricle of a freely-moving rat proved both an accurate and a reliable technique to obtain sufficient volumes of CSF for LCMS analysis at all time points studied.
4.1.9
Confirmation of SLIGRL detection using LCMS
The solutions (micro-filtered water, saline and pooled CSF) spiked with known SLIGRL concentrations (0.0, 4.2, 8.3 and 12.0 M) rendered extremely high signals on the LCMS at the 657.5MW range (results not shown). There were no distinct differences in elution duration (consistently recorded at approximately 21min) or peak intensity of SLIGRL measured between corresponding concentrations of the different spiked solutions, suggesting that all three solutions (micro-filtered water, saline and spiked CSF) shared a similar SLIGRL solubility and that the LCMS gave consistent results. Also, the size of the peak intensity of the signal generated by the LCMS corresponded well to the relative concentration of the spiked sample. These consistent results and the detection of a compound at the 657.5MW band thereby confirmed that
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Figure 4.1 CSF can be extracted from the lateral ventricles
(min)
Figure 4.1 CSF is reliably extracted for the lateral ventricle of conscious rats. Average CSF volumes collected from the lateral ventricles of the rat brain both prior to and following peptide administration (both i.c.v. and i.p.). Dotted line represents the average volume collected (i.e. 4.1 0.2 L, n=14 animals, total of 77 samples).
Figure 4.2 Example of an original LCMS curves of SLIGRL in CSF following i.c.v. administration. upper traces show the results of the MS component of each run. Concentration of SLIGRL is relative to the area under the curve. Dark blue; t=+5min, Black; t=+10min, Red; t=+20min, Green; t=+45min, Brown; t=+60min, Light blue; t=+90min. Pre-injected sample (t=-5min) is not visible on the graph as the peak intensity is too low. Lower trace shows the average peptide spike at the 657.5MW range, suggesting the compound detected is most likely to be SLIGRL
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Figure 4.2 Original LCMS curve of SLIGRL in CSF
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SLIGRL could be readily detected in CSF with a finger-print 657.5MW and elution duration of 21min. The high signals generated in these confirmatory experiments were the initial reason behind diluting the CSF sample further with micro-filtered water after the acetonitrile dilution step.
4.1.10 SLIGRL in CSF following i.c.v. administration A proof-of-principle experiment was conducted in an unanaesthetised rat for CSF collection before and after direct SLIGRL injection into the ventricle (0.15mg/kg i.c.v.) at the time points described. The LCMS record for this single experiment is shown in Figure 4.2 and Figure 4.3 depicting the AUC plotted against time of sample collection. No peak was detected at the SLIGRL finger-print in CSF prior to SLIGRL injection (i.e. t=-5min). Five minutes following SLIGRL injection (t=+5min), a large peak was detected at the SLIGRL finger-print found previously and was subsequently detected in all further serial samples collected. The AUC progressively decreased in a biphasic manner from t=+5 to t=+90min), returning approximately to baseline levels by this final time point (Figure 4.3). The ln(AUC) was plotted against time of sample collection which revealed an approximately linear relationship, suggesting first-order kinetics of SLIGRL decay in CSF with a calculated half-life (t1/2) of 27.2min (t=5min and t=90min (n=1)) (Figure 4.4).
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Injec Figure 4.3 SLIGRL can be detected in CSF t Figure 4.4 SLIGRL decays with first-order kinetics Redistribution phase
Clearance phase
(min)
Figure 4.3 SLIGRL can be detected in CSF following central administration. Appearance of SLIGRL in CSF following i.c.v. administration (0.15mg/kg). The graph represents the area under the curve (AUC) of LCMS peak intensity returned for each sample versus time of CSF sample taken. Clearly shows that SLIGRL rapidly increases in CSF following administration, then rapidly decays biphasically a redistribution phase tapering into a clearance phase (n=1).
(min)
Figure 4.4 SLIGRL decays from CSF with first order kinetics. Natural log (ln) of the area under the curve (AUC, normalised to baseline samples) of SLIGRL in CSF, after i.c.v. administration, reaching peak intensity at 5min post-injection. The clearance phase shows a distinct logarithmic decay, as designated by the linear relationship and the line of best fit. The t1/2 calculated from this single experiment was 27.2min (n=1).
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This preliminary data from one rat suggests that SLIGRL can be reliably detected in CSF collected from the lateral ventricle of a freely-moving rat following i.c.v. injection using LCMS, and that SLIGRL decays in CSF to approximate baseline levels within 90min after injection with a t1/2 of 20-30min.
4.1.11 Detection of SLIGRL in CSF following peripheral (i.p.) injection Unlike in the i.c.v. SLIGRL injection result above, a small but modest peak was detected at the SLIGRL finger-print in the pre-injected (t=-5min) CSF samples following intraperitoneal (i.p.) SLIGRL administration (1.5mg/kg). In the first postinjection sample (t=+10min) a large SLIGRL spike was detected at 657.5MW, which gave an average AUC of 14.9 2.9 fold greater than the baseline sample (n=3). The SLIGRL peak intensity and AUC rapidly declined over the following 90min, returning to approximate baseline levels by this final time point (Figure 4.5). Like for the i.c.v. experiments, SLIGRL was found to rapidly decay in CSF following i.p. administration as clearly demonstrated by both the average AUC data (Figure 4.5) and the plot of ln(AUC) against sample collection time (Figure 4.6). Unlike the i.c.v. experiment, however, this decay curve for SLIGRL in CSF after i.p. injection was monophasic (Figure 4.5). SLIGRL again cleared the CSF with linear first-order kinetics and a t1/2 of 25.2 3.1min (n=3).
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Injec t
Figure 4.5 SLIGRL crosses from blood to CSF
clearance phase only
(min)
Figure 4.5 SLIGRL crosses the blood CSF barrier following peripheral administration. SLIGRL appears in CSF within 10min after i.p. administration and decays logarithmically over the following 90 minutes. Graph shows the area under the curve (AUC, normalised to baseline levels) of the peak samples returned by the LCMS as a function of time of CSF sample collected (n=3).
(min)
Figure 4.6 SLIGRL decays from CSF with first order kinetics Natural log (ln) of the area under the curve (AUC) of SLIGRL in CSF after i.p administration, from 10min post-injection. Linear regression analysis of this clearance phase shows a first order pharmacokinetics with t1/2 25.16 3.1min (n=3).
Figure 4.6 SLGRIL decays from CSF with first order kinetics
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Discussion There are three major findings from this study. The first was the confirmation that CSF can be reliably obtained from the lateral ventricle of a freely-moving rat in sufficient quantities for LCMS analysis. As such, the method used here is suitable for serial collection of CSF for pharmacokinetic analysis of compounds in CSF. The second finding from this present study was that SLIGRL appears to be able to cross the blood-CSF barrier within 10min following a peripheral (i.p.) injection. Finally, the study found that SLIGRL rapidly clears from CSF with first-order pharmacokinetics after i.p. administration with a t1/2 of 25.2 3.1min.
There are only a few previous studies in which the lateral ventricle was used as a source of CSF for pharmacokinetic analysis [395, 396]. As discussed earlier, the majority of studies use the cisterna magna for CSF analysis via microdialysis, using both puncture and cannulation techniques [389-393]. Whilst these methods may allow greater volumes of CSF to be collected than the current study [16, 19], they present other experimental problems for serial collection, such as post-extraction hypovolaemia in the cisterna magna cannulation model [398] and risk of blood contamination in the puncture methods. In the present study, clean (blood-free) serial CSF samples of approximately 4l were reliably and efficiently extracted from the lateral ventricle of an unanaesthetised, freely moving rat with little stress or discomfort to the rat or researcher. Although small, the volumes were sufficiently large enough for LCMS analysis. These findings confirm the use of the lateral ventricles as a viable source for serial CSF collection [395, 396]. The decline in levels of SLIGRL in CSF after direct i.c.v. injection was biphasic. The initial phase of SLIGRL decay in CSF following the i.c.v. injection (i.e. between
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samples t=+5 and t=+10min) is indicative of a rapid redistribution phase, as shown by the rapid decay in SLIGRL concentration after the peak. This rapid decay is most likely due to spatial diffusion and caudal spread of the peptide throughout the entire ventricular system and spinal column, as well as the penetration of the peptide into the brain parenchyma [387, 399]. The secondary phase of the decay in SLIGRL (i.e. between samples t=+10 and t=+90min) represents the clearance phase, more accurately representing the true pharmacokinetics of SLIGRL in CSF than the redistribution phase and is therefore a more accurate measure of half-life (t1/2). Even so, the estimated half-life (t1/2= 27.2min) was similar to that obtained from the later i.p. experiments.
From the i.p. experiments, it is clear that SLIGRL crosses the blood-CSF barrier following peripheral administration, although unlike the single i.c.v. injected experiment, no redistribution phase was observed. Presumably the redistribution occurred at the site of injection (the peritoneum) prior to being absorbed into circulation. Once in the circulation, SLIGRL appears to have rapidly entered the CSF by mechanisms, although speculative, most likely primarily involving simple diffusion, since transport of peptides to the brain parenchyma from the CSF is mainly passive [387]. As there is a free communication between interstitial fluid of the brain and CSF, and the blood-CSF and blood-brain barriers have similar peptide transport mechanisms [387], it can be presumed that the SLIGRL concentration in the interstitial fluid of the brain is similar to that in CSF. Thus, it can be expected that SLIGRL in the CSF also enters the brain where it may activate centrally expressed PAR2 (Chapter 3). Although we did not account for damage done to the blood-brain barrier during cannulation, the collection of blood free CSF samples suggests little or
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no blood entered the CSF, therefore vascular damage and a leaking blood-brain barrier seems unlikely to explain the presence of SLIGRL in CSF following peripheral administration.
Clearance of small molecules and peptides like SLIGRL from CSF most likely depends on active metabolism and transportation to other compartments, such as back into plasma [387] (Figure 4.6). This is because the concentrations of brain-accessible compounds are generally lower in CSF than in plasma, due to what is known as the CSF-sink [387], where the system must work against a concentration gradient using active transport mechanisms to remove compounds from CSF. Specific active peptide transporters such as PEPT2/3 and PHT1/2 [400] specific to hexamer peptides like SLIGRL may be involved in these removal processes.
As there is little soluble protein in CSF (0.3%), protein binding of SLIGRL most likely does not explain the rapid decay in SLIGRL in the CSF following administration. Presumably, the peptide returns to the plasma or continuously diffuses in to the brain, where the concentration is expected to be similar to that in CSF (Figure 4.7). Alternatively, many proteases such as cathepsins, carboxypeptidases and aminopeptidases exist in apical brush border, vascular and epithelial membranes of the choroid plexus which produces CSF [387], so these may metabolise SLIGRL in CSF, and also contribute to its clearance (Figure 4.7). Perhaps it is worth reiterating here that the SLIGRL used in all experiments reported in this thesis was carboxylterminally amidated (i.e. SLIGRL-NH2) [260]. Given the presence of
carboxypeptidases in the brush border of the choroid plexus, it is possible that a smaller t1/2 value would have been obtained for non-carboxyl amidated SLIGRL.
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Likewise, perhaps if more stable PAR2 agonists such as 2-furoyl-LIGRL [266] or SLIGRLI [270] were used, a longer t1/2 may have been obtained, which may also account for the greater efficacy of these compounds. These experiments can be performed in future research.
The detection of a SLIGRL-like compound in the pre-injected samples tested in the i.p. injection route (n=3) is intriguing. Perhaps an endogenous SLIGRL-like compound was present in CSF, or that some SLIGRL had remained in the LCMS column from previous experimental runs. Alternatively, the CSF extraction line used to collect the sample from the rat (see section 2.3.1 and 2.6) may have been contaminated. Since the line was meticulously flushed prior to and between all sample collections, this possibility seems unlikely, as SLIGRL is highly soluble in water. Furthermore, since both the neat, un-spiked CSF samples in the confirmation runs and the pre-injected sample (t=-5min) taken in the i.c.v.-treated animal were clear of SLIGRL finger-print, the presence of a SLIGRL-like compound (with a molecular weight of 657.5) in the CSF also seems unlikely. This leaves column contamination as the most likely explanation for the appearance of SLIGRL in this CSF sample.
In summary, the novel findings of this study suggest that the PAR2 peptide agonist SLIGRL enters the CSF of a rat within 10min following i.p. injection and decays with first-order pharmacokinetics with a t1/2 of approximately 25min. This study also clearly confirmed that the lateral ventricle is a reliable source of CSF for serial CSF sampling from freely-moving rats. The prediction arising from these experiments is that peripheral administration of SLIGRL is able to activate PAR2 expressed on central neurons to cause centrally-mediated effects. Some of these effects will be
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described in the following chapters of this thesis, in particular in relation to hippocampal plasticity, such as learning memory and epilepsy.
Figure 4.7 Passage of peptides across the blood-brain barrier CSF CSFBB Brain
Choroid plexus with apical brush border, containing proteases
BCSFB
BBB
Plasma
Figure 4.7 Passage of peptides across the blood-brain barrier (BBB), blood-CSF barrier (BCSFB), and CSF-brain barrier (CSFBB). Presumably, SLIGRL is freely diffusible into both the CSF and brain from plasma, and also freely diffuses between the CSF and the brain. Removal of peptides from both the CSF and brain back into plasma is, however, rate-limiting, requiring active transport. The BCSFB of the choroid plexus contains an apical brush border with proteases which may cleave SLIGRL, thus potentially reducing its CSF concentration. Thick arrows denote passive (free) diffusion, thin arrows denote active transport. Derived from Smith et al. 2004 [384].
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Chapter 5 Neurotoxicity of SLIGRL
Chapter 5 The behavioural neurotoxicity of centrally administered SLIGRL
Introduction Even though both PAR2 and its physiological agonist trypsin are highly expressed in neurons of the rat brain (see Chapter 3), very little is understood about the effects of central administration of PAR2 peptides, like SLIGRL, in an in vivo system and whether such peptides have any effects on behaviour.
It has been reported that trypsin applied directly to isolated brain slices induced epileptiform hyperexcitability in hippocampal CA1 pyramidal neurons [145], and activation of PAR2 using SLIGRL has been shown to be toxic to hippocampal pyramidal neurons in vitro in a concentration-dependent manner [320], suggesting SLIGRL may have neurotoxic effects in vitro. One key in vivo study utilised intraparenchymal (striatal) micro-injections of SLIGRL in rats to investigate neuronal survival in a HIV-associated dementia [309] where it was found that PAR2 appeared to have a similar level of neuroprotection as that reported in rat models of stroke (transient middle cerebral artery occlusion) [333]. Whilst both studies suggested that PAR2 influences experimentally-induced pathological neurotoxicity, neither described any PAR2 and/or SLIGRL-induced behavioural neurotoxicity per se. Furthermore, intraparenchymal injections of small volumes (i.e. 0.2 l) of a compound like SLIGRL would not be expected to diffuse more than 0.5mm from the injection epicentre [386]. Thus, only localised effects of such a compound delivered via this technique would be expected. Administration of the same compound directly into the cerebrospinal fluid (CSF), however, should result in a more global CNS response [386], affecting other structures, such as the hippocampus, amygdala, limbic cortex
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and hypothalamus involved in complex emotional behaviour.
Since PAR2 and trypsin are highly expressed in the brain (Chapter 3) and trypsin [145] and SLIGRL [320] have dose-dependent effects on the excitability and survival of pyramidal neurons, the aim of this study was to investigate whether centrallyapplied SLIGRL, at various doses, induces any behavioural changes (neurotoxic or other) in conscious rats. This was done not only to characterise any dose-dependent neurotoxic effects SLIGRL has in vivo, but also to determine a safe SLIGRL dose to apply centrally for use in later experiments investigating the function of PAR2 in the brain during epileptogenesis.
Methods All methods have been described in detail in Chapter 2 5.1.1 Animals, surgery and cannula position confirmation.
GAERS and NEC rats (13 weeks, 180-250g) of both sexes were used for these experiments. Surgeries in NECs were as previously described (Chapter 2). GAERS surgical protocols were similar, except the intra-amygdala bipolar electrode was omitted. Confirmation of the correct placement of the i.c.v. cannula using AngII was also performed on all animals, as previously described (Chapter 2). GAERS and NECs were used in this study to determine whether there was any behavioural differences in response to PAR2 between strains.
5.1.2
Drugs, peptides and i.c.v. injection
The PAR2 peptides (SLIGRL and LRGILS (control)) were injected at five different concentrations (0.0, 0.015, 0.15, 1.5 and 3mg/kg, i.c.v.). Ketamine (0.75, 7.5, 75 and
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150 mg/kg in saline, i.p.) was used for confirmation of the neurotoxicity coefficient scale (described below and Chapter 2). i.c.v. injections were also as previously described (Chapter 2). For the peptide injections, each rat received all four doses of the assigned peptide in a randomised fashion with 48h between trials. Rats in the groups that were administered ketamine received one single dose only.
5.1.3
Experimental design and EEG monitoring
All rats were EEG monitored during trials as previously described (see Chapter 2.7). After a 15min habituation period, baseline EEG recordings and visual behavioural monitoring commenced. Following 30min of baseline recording, rats were injected with peptide (SLIGRL, LRGILS i.c.v.), saline ((vehicle) i.c.v.) or ketamine (i.p.) as described previously. A 90min post-treatment recording period followed, during which visual behavioural monitoring continued (Figure 5.1).
Figure 5.1 Schematic of neurotoxicity experiment
Figure 5.1 Schematic of neurotoxicity experimental protocol.
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5.1.4
Behavioural monitoring, assessment and scoring
During the entire duration of the experiment, animals were visually monitored for changes in behaviours and scored according to the method described in Chapter 2 (see Table 2.1). The highest neurotoxic behaviour displayed by a particular rat in response to any given dose of peptide or ketamine was used in the analysis. The time spent in the observed behavioural state was also scored accordingly (see Table 2.2), and Equation 2.1 (see Chapter 2) was used to calculate the neurotoxicity coefficient score. No statistical analyses were performed in the current study.
Results 5.1.5 Confirmation of the scoring system in NEC rats; ketamine causes neurotoxicity At the lowest dose (0.75mg/kg, n=3), ketamine induced minimal neurotoxic behaviours, however, normal exploratory behaviours were often substituted with excessive locomotor activity and hyperactivity associated with bouts of behavioural arrest, shivering and the occasional loss of balance. Such behaviours lasted up to 30min.
At the 7.5mg/kg dose, rats (n=3) experienced frequent loss of balance, occasional writhing behaviours, atonia and mild sedation. Such behaviours were observed to last as long as 60min following administration.
At the 75mg/kg dose of ketamine (n=3), more pronounced neurological deficits were observed including severe sedation, lapses in consciousness, generalised loss of locomotor activity, atonia and catatonia. These behaviours lasted approximately
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60min and were often associated with rapid and forced breathing and reduced algesia. At this dose, however, ketamine alone failed to induce complete anaesthesia, as it does in combination with Xylasil for surgical anaesthesia (see Chapter 2).
At the highest dose (150mg/kg, n=3), ketamine induced loss of consciousness, partial or complete anaesthesia, loss of nociception and complete behavioural arrest, generally within 15min of administration. No seizure-like activity or other neurotoxic behaviours such as writhing and barrel rolling were observed. These ketamineinduced effects lasted as long as 90min. Since complete anaesthesia had been achieved at the highest dose, further doses were not tested.
The relationship between ketamine dose and neurological toxicity shown in Figure 5.2 confirmed the validity of the neurological scoring scale developed.
5.1.6
SLIGRL (i.c.v.) induces dose-dependant neurotoxic behaviours
5.1.6.1 NEC rats Saline alone (2-4 l, i.c.v., n=5) caused no changes in behaviour or EEG from baseline over the 90min experimental period.
At the lowest dose used (0.015mg.kg, n=5), SLIGRL induced changes in behaviour such as mild hyperactivity and apparently enhanced exploratory behaviours, with rats frequently attempting to escape the holding cage more so than prior to injection. No changes were observed in normal baseline EEG (Figure 5.4). Observed behaviours were generally short lived (less than 30min).
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A 10-fold higher dose of SLIGRL (0.15mg/kg, i.c.v., n=5) induced similar levels of hyperactivity and occasional wild running behaviours as the first dose, but these were occasionally associated with bouts of behavioural arrest and an inclination to pause and stare. Again no distinct EEG changes were observed. The duration of the observed behaviours were generally longer lived than for the lowest dose, lasting around 60min, appearing within 5min of administration.
A further 10-fold increase in dose of SLIGRL (1.5mg/kg, i.c.v. n=5) induced obvious neurotoxic behaviours, such loss of coordination, ataxia, myoclonic jerks, spasticity, writhing and swimming-like behaviours. On one occasion, barrel-rolling behaviours were observed. All rats developed catatonic-like states, associated with sedation and nociceptive hypersensitivity/hyperalgesia, and even losses of consciousness. Rats usually displayed rapid and forced breathing. Neurotoxic behaviours appeared within 5min following injection, reached a maximum after 10min and then lasted without further change for at least another 40min. Normal behaviour had generally returned by 90min following injections at this dose. There were also obvious changes on the EEG patterns, with intermittent, synchronised oscillatory slow-wave forms of 1-2Hz (delta-like rhythms, Figure 5.4) which coincided with the catatonic-like states observed in the rats. Such EEG waveforms are commonly associated with neurotoxic states such as coma [401]. Once normal behaviours had returned (approx. 90min postinjection), normal baseline EEG also returned.
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The highest dose of SLIGRL (3mg/kg i.c.v., n=5) caused severe neurotoxic behaviours such as complete loss of consciousness, severe ataxia, atonia and catatonia, writhing, barrel-rolling and often generalised seizures (comparable to behavioural seizure Class II [339], Chapter 2), the latter invariably associated with epileptiform spiking on EEG (2-5Hz, Figure 5.4). Such EEG epileptiform activities usually appeared within 10min after SLIGRL administration, however, they did not always translate to seizure-like manifestations, which may have reflected the atonic/catatonic states of the rats. Such epileptiform discharges were often very frequent (up to 6/min) in the first 30min following administration. Interictally, the delta-like EEG rhythms described above were also observed (Figure 5.4), and continued intermittently during the entire post-injection recording period, only phasing out after 90min. The behaviours lasted up to 130min1, however, the most severe of the behaviours (seizure-like EEG, barrel-rolling) had all but disappeared by 40min, resulting in a catatonic state only, associated with the described delta-like EEG. The relationship between SLIGRL dose and toxicity observed is shown on Figure 5.3.
From the dose-response curve (Figure 5.3) we suggest that the maximum tolerated dose of SLIGRL, given i.c.v. prior to the onset of significant neurotoxic-like behaviours is 0.15mg/kg.
At this dose, monitoring continued well past 90min as behavioural deficits were still observed. Rats were thus allowed to recover completely prior to discontinuation of monitoring.
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5.1.6.2 GAERS In the GEARS, the lower doses of SLIGRL (0.015 and 0.15mg/kg) did not effect the expression of normal spike-wave discharges (SWDs) associated with their genetic condition (not shown). However, during the convulsive and catatonic-like states induced by the higher doses of SLIGRL (1.5mg/kg; in particular 3.0mg/kg), SWDs all but vanished, being replaced with the generalised seizure-spiking and the delta-like EEG waveform described above. These apparent SLIGRL-induced, seizure-like discharges however were of much lower frequency and generally lacked the wave element of normal SWDs with only spike-discharges apparent (Figure 5.4). No other obvious differences in response to SLIGRL were observed between the GAERS and NEC cohorts.
In both cohorts (NEC and GAERS), central administration of the control peptide LRGILS induced no observable changes in normal rodent behaviours, EEG-wave forms or expression of SWDs (in GAERS) at any of the doses tested over the 90min post-injection period compared to baseline (Figure 5.3). Thus, like saline, LRGILS induced no aberrant behavioural effects as observed in the SLIGRL groups, suggesting that the SLIGRL responses were specific and most probably PAR2 mediated. These data also confirm the use of LRGILS as an appropriate control for the later in vivo experiments on epilepsy and learning [244, 291, 375, 402, 403].
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Figure 5.2 Dose-response curve to ketamine
Figure 5.2 Dose-response curve to ketamine (i.p.) in the conscious rat. The graph shows a distinct dose-dependent increase in neurotoxicity after ketamine injection (n=8). These data validate the use of the neurological coefficient scoring system used in this project (n=3 at each time point).
Figure 5.3 Dose-response curve to SLIGRL (i.c.v.) in the conscious rats (GAERS and NEC combined). The graph distinctly shows a dose-dependent neurotoxic effect specific to SLIGRL (n=5) when injected centrally, comparable to that of ketamine (Figure 5.2). LRGILS (n=5) has no effect at the same doses. The maximal tolerated dose was taken to be 0.15mg/kg (arrow. LRGILS n=5, SLIGRL n=5).
Figure 5.3 Dose-response curve to SLIGRL

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B Figure 5.4 Examples of EEG patterns caused by SLIGRL

1 sec
1 sec
C
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D
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E
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F
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Figure 5.4 Examples of EEG changes associated with i.c.v. SLIGRL administration in conscious NECs. A; normal baseline (beta-wave) EEG recorded prior to SLIGRL administration. B; normal spike-wave discharge (SWD) recorded from a GAERS during baseline recording. C and D; delta-wave-like EEG activity recorded from SLIGRL-treated animals (1.5 and 3.0 mg/kg i.c.v., respectively). E and F; SLIGRL (3.0mg/kg i.c.v.) induced seizure activity recorded on EEG, recorded approx. 6 and 15min post-injection, respectively. X-axis shows time base (hr:min:sec); all Y-axes (arbitrary amplitude) are of equivalent scale.
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All animals completely recovered from these toxicity experiments, and no rat displayed any behavioural or locomotor deficits 24h following any of the SLIGRL doses. Thus, SLIGRL appeared not to have induced any lasting neurotoxic deficits. By contrast, ketamine-treated rats, particularly those given 15mg/kg, were still rather sedate and petulant the day following experimentation, but these animals all completely recovered after this time.
Discussion The results of this study clearly indicate that SLIGRL causes dose-dependent neurotoxic dyskinesias when given i.c.v. to freely-moving rats. The maximum tolerated dose of SLIGRL prior to onset of such neurotoxic behaviours was found to be 0.15mg/kg (i.c.v.), higher doses inducing marked neurotoxic-like behaviours such as catatonia, loss of consciousness and generalised seizure. Neither saline nor the control peptide LRGILS induced any changes in behaviours at any doses tested. Therefore, this appears to be the first report of any centrally-mediated dyskinesias induced by PAR2 activation.
As previously described [404, 405], the dose-dependant changes in ketamine-induced neurotoxic-like behaviours observed here using the adapted neurotoxicity coefficient scoring system not only confirmed its accuracy but also validated its use in further research investigating neurotoxic behaviours induced by drug administration.
A simple arithmetic coefficient that encompasses both the neurotoxic behaviour and its duration has distinct advantages over other scoring systems that only use a behavioural score. For example, in the present study, 0.015mg/kg and 0.15mg/kg
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doses of SLIGRL induced similar behavioural deficits in the rat, but the duration of the behaviour was longer at the higher dose. If these behaviours had been given neurological scores only, no difference in toxicity would have been observed. The neurological coefficient, however, incorporates the duration of the behaviour and thus, the higher SLIGRL dose resulted in a higher over-all neurotoxicity coefficient, establishing it to be a more toxic dose. On the other hand, the current doseneurotoxicity coefficient curve alone cannot be used to determine actual neurological scores. For example, a neurotoxicity coefficient of approximately 4 at the lowest dose for SLIGRL (Figure 5.4) does not discriminate between an animal with a neurological score of 4 (thus high toxicity) with a short duration (i.e. duration score of 1; 0-20min (Table 2.2)) and one with a neurological score of 1 (the lowest score) with a long duration (i.e. duration score of 4; >60min (Table 2.2)). Regardless of this apparent limitation, the use of coefficients of neurotoxicity was deemed to provide a more robust method of assessing safe doses of SLIGRL (i.e. the maximal tolerated dose) for use in experiments described in the following Chapter of this thesis.
Neurotoxic-like behaviours are induced by many compounds [356-358, 405-408], and seizures per se can be elicited by many drugs [33, 35, 196, 409-411]. Both responses are usually dose-dependent [412] and often rely on intraneuronal calcium transients [413]. Sustained high concentrations of intracellular calcium are signals for apoptosis [414], especially in relation to glutamate neurotoxicity [415] and since glutamate is known to mediate seizures [191, 195] it seems likely that such seizures are calciumdependent. The major response following activation of neuronal PAR2 is a release in intracellular calcium [277, 285, 286, 309, 320]. Therefore the observed neurotoxiclike behaviours and seizure induced by SLIGRL at the higher doses in this study may
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be directly proportional to intracellular calcium responses in neurons [285, 320].
From a behavioural neurotoxic perspective, the toxicity findings reported for cultured hippocampal neurons [320] appear to correlate well with the current in vivo toxicity findings on behaviour. For example, a 200g rat with an approximate CSF volume of 390 l [388], injected with SLIGRL (2l) directly into the CSF at a dose of 0.15mg/kg, will have a theoretical immediate CSF concentration of 88 M after diffusion throughout the CSF. This concentration is just less than the minimum ( 100 M) inducing neurotoxicity in vitro [320]. Any concentration we administered thereafter in the current study induced neurotoxic behaviours (i.e. 1.5 and 3.0mg/kg), thus, similar to that observed at high doses in vitro, SLIGRL does in fact induce neurotoxic behavioural effects in vivo. Further investigations are required to determine if higher doses of SLIGRL induce neuronal death in vivo, as shown in vitro.
The t1/2 of SLIGRL in CSF (following s.c. administration) is approximately 25min (see Chapter 4). Thus, any neurotoxic behaviours induced by SLIGRL, at any dose, would be expected to rapidly decline, as the concentration of SLIGRL in CSF would be halved approximately every 30min. Comparatively, this is what was encountered behaviourally. For example, the mild behavioural changes observed with 0.15mg.kg SLIGRL generally lasted no longer than 30min. Presumably, 30min following injection of SLIGRL administration into the CSF, the available concentration is halved (i.e. from 88 M to 44 M) and thus too low to induce any behavioural neurotoxicity (in vitro). At a dose of 1.5mg/kg, a theoretical CSF-SLIGRL concentration of approximately 880 M would be achieved, and 440 M after
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30min; 220 M, after 60min; 110 M after 90min; and 55 M after 120min. Again, these concentrations of SLIGRL in CSF correlate well with the behavioural observations in this study, as rats dosed with 1.5mg/kg SLIGRL (i.c.v.) completely recovered by 120min when the theoretical SLIGRL CSF concentration would be approximately 55 M, correlating well with previous in vitro studies described [320]. These data suggest that, like in vitro, activation of PAR2 appears to induce neuronal hyperexcitability which manifests as neurotoxic behaviours such as catatonia and seizure in vivo.
In conclusion, from both observational and theoretical perspectives, the results from the current study, and the previous pharmacokinetic study described in Chapter 4, indicate that SLIGRL induces dose-dependent neurotoxic effects at concentrations above the maximal-tolerated dose of 0.15mg/kg (i.c.v.). This dose was therefore chosen to be used in the subsequent in-vivo experiments investigating the potential anti-epileptogenic, behavioural and neurocognitive effects of SLIGRIL.
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Chapter 6 PAR2 in Epileptogenesis
Chapter 6 Anticonvulsant and anti-epileptogenic effects of PAR2 and
SLIGRL in the electrical kindling and kainic acid models of TLE
Introduction Recently, inflammatory insults to the brain involving both proteases and cytokines have been associated with seizure and epilepsy, both in human patients and animal models [41, 43, 51, 53, 55, 56, 58, 61, 63, 68, 71, 88, 154, 155, 202, 238, 239, 243, 297, 374]. Low levels of proteases including tPA [40, 42, 73], neuropsin [87], motopsin [111], thrombin [245] and trypsin [121] (see Chapter 3) are normally present in limbic regions of the brain like the hippocampus and are associated with neuronal plasticity and matrix remodeling related to synaptogenesis, LTP, LTD and memory formation [84, 85, 87, 99, 109]. Increased levels of tPA in hippocampal pyramidal neurons, however, correlate with pro-epileptogenic changes such as neuronal loss [42], LTP [64], mossy-fibre sprouting [67] and generation of seizure [40, 43, 64, 67, 72, 154]. Furthermore, inhibition or loss of tPA (i.e. tPA gene knockout) significantly attenuates experimental seizure induction and epileptogenesis [42, 43, 129, 154, 155, 258]. Therefore, proteases not only have important roles in neuronal plasticity relating to memory formation, but they also appear to be involved in the generation of seizure and the development of acquired TLE.
PAR2 and trypsin are highly colocalised in neurons of the brain, particularly in limbic regions associated with epilepsy (cortex, hippocampus and amygdala) (Chapter 3). PAR2 also is up-regulated in neurons of the hippocampus after severe cerebral ischaemic injury [319], radiation exposure [416], in HIV-associated dementia [309]
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and multiple sclerosis [204], and in response to pro-inflammatory cytokines IL-1 and TNF [309, 377, 417]. Therefore, PAR2 may be involved in inflammatory events in the brain.
Very little is understood about trypsin and PAR2 in the brain, most studies have concentrated on the thrombin receptors PAR1, PAR3 and PAR4. Furthermore, most of this research has focused on Parkinsons disease, Alzheimers disease, stroke and vascular trauma. Recent research has shown, however, that PAR2 may be neuroprotective during inflammatory insults to the brain [246, 266, 309, 333]. Perhaps, like acinar cells of the pancreas [304, 313, 314, 318], PAR2 may act to regulate trypsin expression in neurons of the brain, particularly in highly plastic limbic structures. Given the expression of both PAR2 and trypsin in these regions (Chapter 3), PAR2 may have a neuroprotective function against protease-driven inflammation, seizure and epilepsy.
Armed with the knowledge that (1) PAR2 and trypsin are highly expressed in the brain (Chapter 3), (2) proteases have strong influences in epileptogenesis, (3) SLIGRL crosses the blood-CSF barrier following peripheral administration (Chapter 4) and that (4) SLIGRL has centrally-mediated neurotoxic effects (Chapter 5), the aims of the studies described in this chapter were to test the effects of both central (i.v.c.) and peripheral (s.c.) administration of SLIGRL on epileptogenesis in the electrical amygdala kindling and kainic acid models of seizure and epilepsy in the rat.
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Materials and Methods All methods are described in detail in Chapter 2.
6.1.1
Animals and post-operative care.
Briefly, female Non-Epileptic Controls (NEC) Wistar rats (n=84) were used for these studies. Post-surgery, animals were individually housed as previously described and were allowed to recover for seven days prior to any experimentation.
6.1.2
Drugs and peptides.
In the electrical kindling and kainic acid studies, SLIGRL and LRGILS were administered at the maximal tolerated dose of SLIGRL (0.15mg/kg, i.c.v.) as determined in Chapter 5. For peripheral administration of SLIGRL in the kindling model, a dose of 1.5mg/kg (s.c.) was used based on the dose that inhibited ulcerative colitis and stomach ulcer in rats and mice when given the same route (Devlin and Cocks, unpublished data; [302]).
6.1.3
Surgeries
Briefly, two custom-built EEG electrodes, two nickel-alloy jewelers (anchor) screws, a guide cannula and, a twisted steel bipolar electrode were inserted through designated burr holes in the skull. All co-ordinates were obtained from the Rat Brain Atlas of Paxinos and Watson (2005) (see Chapter 2) [348]. All animals were subject to confirmation experiments for the patency and cannula location as well as bipolar electrode location, as described in Chapter 2.
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6.1.4
Afterdischarge threshold (ADT) determination in the electrical amygdala kindling model
Seven days post-surgery, the ADT of each rat (n=49) was determined as described in Chapter 2. Twenty-four hours later, rats were divided into treatment groups for i.c.v. injection of either SLIGRL (n=18) or LRGILS (n=14, 2-4 l (adjusted to weight), 0.15mg/kg) allowing 10min for uptake. The other cohort of rats received subcutaneous (s.c.) peptide (SLIGRL n=7, 1.5mg/kg. LRGILS n=5 1.5mg/kg. or saline n=5), allowing 20min for uptake. The ADT protocol was then repeated in order to determine whether the PAR2 peptide treatment had any effect on the ADT of individual rats.
6.1.5
Electrical amygdala kindling
Twenty-four hours after the second (post-peptide treated) ADT protocol, rats from both the i.c.v. and the s.c. cohorts were again injected with the same dose of the corresponding PAR2 peptides followed by the uptake period described above for each cohort. Rats were then connected to the kindling apparatus and a 30-60s baseline EEG was recorded before a kindling stimulus (1s train of 1ms biphasic square wave pulses, 60Hz) was applied at the original (pre-peptide injected) ADT recorded. This entire protocol was repeated twice daily, 4h apart, 5 days per week until a fully-kindled state was achieved, or 80 stimulations, depending on which came first.
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6.1.6
Kainic acid-induced status epilepticus
After recovery from surgery, rats in the kainic acid cohort (n=31) were divided into three groups for treatment with either SLIGRL (0.15mg/kg i.c.v., n=13), LRGILS (0.15mg/kg i.c.v., n=10) or equi-volume vehicle (sterile saline, n=8). Rats were allowed to move freely in their cage for 10min before being injected with kainic acid (15mg/kg in saline, i.p.) initially and half-doses (i.e. 7.5mg/kg) every 60min thereafter until status epilepticus was achieved (see Chapter 2.6). No more than 4 doses were administered (i.e. 60mg/kg kainic acid total). Rats remained in status epilepticus for 4h from onset. Seizures were stopped with diazepam (4mg/kg in saline, i.p.).
6.1.7
Daily peptide administrations
The day following status epilepticus, rats in the kainic acid cohort were injected with the corresponding peptide (SLIGRL or LRGILS, 0.15mg/kg, i.c.v.) or vehicle (Chapter 2). This was repeated daily at the same approximate time (i.e. 10-11:00a.m.) for 8 weeks. When being recorded (24h protocol, see below), the EEG record was stopped and rats unhooked from the EEG leads prior to injection. Following the injection, rats were reconnected to the EEG leads and the EEG restarted.
6.1.8
Twenty-four hour recording protocol
Seven days post-status epilepticus, rats began the once-weekly 24h video-EEG recording protocol to assess the frequency and nature of spontaneous seizures as described in Chapter 2. Unfortunately, not all rats reached the eight weeks of 24h recording initially hoped for due to losses of caps, resulting in early termination. 6.1.9 Immunohistochemistry and histology
Immunohistochemistry and Timms staining were performed on the brains of the
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i.c.v.-treated kindled animals as previously described (Chapter 2). Cell counts were not performed because of the known lack of association between induced seizures, cell loss and hippocampal sclerosis in the amygdala kindling model [185, 221]. Also, as there was no ultimate difference in the endpoint of the other cohorts (i.e. s.c. kindled, and i.v.c. kainic acid animals) regardless of treatment, all being considered epileptic, no histology was performed on these tissues as no gross histopathological differences were expected.
Results 6.1.10 i.c.v. SLIGRL elevates ADT during kindling For the two combined groups of rats in this cohort, the average (baseline) pre-peptide treated stimulus strength inducing ADT was 154.812.2 A (n=32). Once divided into the respective peptide treatment groups (SLIGRL; n=18, LRGILS; n=14), there were no significant differences observed between the pre-injected ADTs (p=0.41, Figure 6.1).
In the SLIGRL-treated cohort, 66.7% of animals showed an increased ADT after peptide treatment compared to baseline, 11.1% showed a decrease and 22.2% showed no change in ADT. The average ADT after SLIGRL treatment was 222.2 20.0A, significantly raising the ADT by 54.9 24.1% (p<0.05 compared to same animals in uninjected situations). After LRGILS treatment, only 7.1% of rats showed an increased ADT, 71.4% showed a decreased ADT and 21.5% showed no change in ADT. The post-treatment average ADT in the control group was 105.79.7A, significantly reducing it by 23.5 6.6% after injection (p<0.05 compared to the same animals in the uninjected situation). Thus, SLIGRL significantly increased the ADT
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compared to both baseline and LRGILS-treated situations (p<0.05, Figure 6.1).
6.1.11 i.c.v. SLIGRL administration prevents kindling-induced seizure As kindling stimuli were repeated, there was a progressively increasing AD duration (ADD) after each consecutive stimulus in control (LRGILS-treated) animals, until a characteristic seizure-like discharge on EEG and behavioural seizure was elicited, much as described in other electrical amygdala kindling experiments [353, 418-420]. When treated with LRGILS, rats responded to repeated kindling stimulations with a progressively increasing ADD following stimulation until reaching a plateau after 25 stimulations (average) and a fully-kindled state of five class V seizures was achieved much as frequently described in similar experiments [11, 15, 16, 337, 339, 353, 419]. These animals showed an average ADD of 91.2 19.7s over the entire kindling protocol (n=8) (Figure 6.1).
By contrast, the SLIGRL-treated animals (n=12) failed to show a significant evolution in ADD upon repeated kindling stimulations, the average ADD remaining constant at an average of 25.2 10.1s in up to 80 stimulations (p<0.01 repeated measures ANOVA). These data, depicted in Figure 6.1 clearly shows the lack of ADD evolution in SLIGRL-treated animals compared to LRGILS animals (Figure 6.1).
The latency to the first seizure was also significantly increased in the SLIGRL group compared to LRGILS, expressing their first seizure, usually of class I, within an average of 25.2 12.5 stimulations (Figure 6.1). In contrast, the LRGILS rats reached a class I seizure within an average of 5.5 1.8 stimulations (p<0.05). As with the ADD data, the seizure severity remained relatively uniform over the kindling period,
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remaining (on average) a non-convulsive class I-II in the SLIGRL treatment group, most animals treated with SLIGRL failed to express clinical seizures of higher-order (class III-V). The LRGILS-treated animals on the other hand showed a characteristic steady and progressive rise in seizure class until five class V seizures were expressed, characteristic of kindling experiments (Figure 6.1). SLIGRL also significantly reduced the total seizure expression, with control animals showing 65.1 10.7% of all stimulations resulting in seizure (of any class), whereas only 15.4 6.8% of all stimulations resulted in seizure in the SLIGRL-treated group (n=12, p<0.001). The SLIGRL-treated animals thus have a significantly reduced seizure risk, with an average of only 5.1 2.2 seizures compared to control with an average of 18.3 3.2 seizures expressed throughout the duration of the kindling experiment (p<0.005, Figure 6.1). Consequently, 87.5% of the i.c.v. LRGILS-treated animals reached a fully-kindled state with a kindling rate 29.3 3.7 stimulations (n=7). Of the SLIGRLtreated animals only 33.3% reached a fully-kindled state, all others (n=8 of 12) failed to reach a fully-kindled state within 80 stimulations, rarely showing seizures above class II (Figure 6.1). Because the majority of SLIGRL-treated animals did not reach a fully-kindled state, an accurate kindling rate could not be calculated in this cohort.
Ten rats used in the ADT studies had lost their caps prior to the end point of the experiment and thus these were omitted from further analyses, other than changes in ADT. During kindling, there were no differences between the treatment groups in the proportion of stimulations that resulted in an AD on the EEG (data not shown) indicating that the slower kindling rate in the SLIGRL-treated rats was not merely a consequence of an afterdischarge-inhibiting effect.
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6.1.12 s.c. SLIGRL delivery elevates afterdischarge threshold The combined average ADT prior to SLIGRL, LRGILS and saline administration was 195.3 16.3 A, and again as for the i.c.v. cohorts, there was no difference in ADT observed between the three treatment groups. Also, as observed following the i.c.v. administration described above, animals treated s.c. with SLIGRL (n=7) showed a significant 38.8 13.1% increase in ADT (from the baseline pre-treatment level) compared to both LRGILS-(1.5mg/kg s.c., n=5) and vehicle-(saline, n=5, p<0.01 ANOVA) (Figure 6.2). Again, LRGILS and saline treatments showed a small (although not significant), yet expected reduction in ADT. Thus, much like when administered centrally (i.c.v.), peripheral administration of SLIGRL (s.c.) appeared to induce centrally-mediated increases in ADT that correlated well with the results discussed earlier in Chapter 4 that SLIGRL can enter the CSF and brain following peripheral administration, except these data suggest an active molecule does so.
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Figure 6.1 Effect of SLIGRL (i.c.v.) on electrical kindling
C *
Figure 6.1 SLIGRL (i.c.v.) has anti-seizure and anti-epileptogenic properties in the electrical amygdala kindling model in the rat. A; SLIGRL (0.15mg/kg i.c.v., n=12)-treated rats had significantly higher afterdischarge thresholds (ADT) than those treated with control-peptide LRGILS (0.15mg/kg i.c.v, n=8) and the pre-injection ADT (*p<0.01 from LRGILS, #p<0.05 from the pre-injected situation). B; SLIGRL significantly inhibited the evolution of afterdischarge (AD) duration during kindling (p<0.005 repeated measures ANOVA). C; SLIGRL significantly increased the latency to first seizure (p<0.05). D; SLIGRL significantly reduced the progression to convulsive class IV and V seizures throughout kindling (p<0.01, repeated measures ANOVA). E; SLIGRL significantly reduced the percentage of stimulations that result in seizure (p<0.05) F; SLIGRL treatment markedly reduced the number of animals reaching a fully kindled, or epileptic state. Data are expressed as means s.e.m. (LRGILS n=8, SLIGRL n=12).
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6.1.13 s.c. SLIGRL attenuates kindling-induced seizure SLIGRL-treated animals (n=7) displayed a significantly slower progression in ADD and seizure severity prior to reaching a fully-kindled state compared to controls (LRGILS, n=5; saline; n=5, p<0.05 repeated measures ANOVA, Figure 6.2). Even though not as striking as that observed following central administration (i.c.v.), animals treated with SLIGRL had an attenuated evolution in ADD, with a slower progression to increased duration than both control groups (LRGILS and saline) (p<0.05 repeated measures ANOVA). The average ADDs during the entire kindling experiment was 61.3 4.6s, 88.5 7.3s and 94.1 7.4s for SLIGRL-, LRGILS- and saline-treated animals respectively (p<0.05).
Much like that described above for i.c.v. SLIGRL, s.c. administration of SLIGRL caused a significant increase in latency to reach the first seizure during electrical amygdala kindling. Thus, SLIGRL-treated animals displayed their first seizure, usually of class I, in an average of 18.0 5.4 stimulations, whereas LRGILS- and saline-treated animals displayed their first seizure with 5.4 2.0 and 3.0 0.7 stimulations respectively (p<0.05, Figure 6.2). Following induction of the first seizure, the progression to higher order seizures was also significantly attenuated in the SLIGRL group (p<0.05, Figure 6.2).
Seizure frequency was also reduced in the SLIGRL animals, with 50.9 8.1% of stimulations resulting in seizure, compared to 75.7 6.9% and 82.0 6.8% in the LRGILS and saline treated animals respectively (p<0.05, Figure 6.3), however, no differences in the total number of seizures experienced throughout the kindling
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experiment were observed in this cohort. Thus, animals treated with LRGILS and saline had mean kindling rates of 22.0 1.0 stimulations and 16.8 1.5 stimulations respectively (difference not significant) to reach a fully-kindled state. In contrast, s.c. SLIGRL treatment again was associated with a significantly greater kindling rate requiring 35.2 4.8 stimulations (p<0.05, Figure 6.2). Unlike the central (i.c.v.) peptide administration experiments, all animals (100%) from all groups reached a fully-kindled state within 60 stimulations.
There were no differences between the treatment groups in the proportion of stimulations that resulted in an AD on the EEG thereby indicating that the slower kindling rate in the s.c. SLIGRL-treated rats was not a consequence of an afterdischarge-inhibiting effect.
Overall, in both i.c.v. and s.c. cohorts, there was a strong correlation between seizure duration and severity (R2=0.72, not shown), seizures of higher order generally having a longer duration.
6.1.14 i.c.v. SLIGRL administration epilepticus
attenuates kainic acid induced status
In the kainic acid-induced status epilepticus study, rats pre-treated with LRGILS (0.15mg/ml i.c.v., n=10) or saline (equi-volume i.c.v., n=8) required 4.5 0.5mg/kg and 5.4 0.6mg/kg kainic acid respectively to induce status epilepticus (difference not significant). Strikingly, those pre-treated with SLIGRL (0.15mg/ml i.c.v., n=13) required 7.3 0.5mg/kg kainic acid, which was significantly more than both controls (p<0.05, Figure 6.3). Once rats were in status epilepticus, however, there were no
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differences in the seizure progression and behavioural manifestations observed, with all rats showing a characteristic progression of status epilepticus from class I to class V-like manifestations, often experiencing pop-corn and Hokey-pokey seizures (see Chapter 2). Usually severe behavioural manifestations lasted no longer than 2h in all groups, which may have reflected the exhausted physiological state of the rat following severe seizures experienced during status epilepticus. Electrographic seizures were, however, still present.
6.1.15 i.c.v. SLIGRL increases latency of spontaneous seizure and reduces seizure duration following status epilepticus The weekly 24h video-EEG recording experiments for the status epilepticus study showed there was a significantly increased latency to the first recorded spontaneous seizure after status epilepticus with kainic acid in the SLIGRL-treated rats compared to controls (SLIGRL, 4.3 0.7 weeks, n=5; LRGILS, 2.0 0.0 weeks; n=3. Saline, 1.8 0.5 weeks; n=4; doses as shown above; p<0.05, Figure 6.3), much as was observed in the kindling studies. SLIGRL-treated rats also displayed a significantly shorter average seizure duration throughout the entire 8 week protocol (SLIGRL, 42.8 2.6s, n=5; LRGILS, 74.9 19.1s, n=3; Saline, 68.5 8.6s, n=4; p<0.05, Figure 6.3). There were, however, no significant differences in the frequency of recorded spontaneous seizures in the 8 week recording period (SLIGRL, 3.1 1.1 seizures/week, n=5; LRGILS, 1.7 0.3 seizures/week n=3; saline, 2.5 0.7 seizures/week, n=4). An example of a typical spontaneous seizure is shown in Figure 2.7.
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Figure 6.2 SLIGRL (s.c.) has anti-seizure and anti-epileptogenic properties in the electrical amygdala kindling model in the rat. A; SLIGRL (1.5mg/kg, n=7) treatment significantly raised ADT compared to LRGILS (1.5mg/kg s.c., n=5, *p<0.05 from LRGILS, #p<0.05 from the preinjected situation). B; SLIGRL significantly attenuated evolution in AD duration during kindling (p<0.05, repeated measures ANOVA). C; SLIGRL significantly increased latency to first seizure (*p<0.05). D; SLIGRL treatment significantly slowed seizure spreading to convulsive class IV and V seizures throughout kindling (p<0.05 repeated measures ANOVA). E; SLIGRL significantly reduced the number of stimulations that result in seizure (*p<0.05). F; SLIGRL significantly attenuates the progression to a fullykindled state (*p<0.05). G; Rats treated with SLIGRL had significantly slower kindling rates than those treated with LRGILS (*p<0.05). Data are expressed as means s.e.m.(saline n=5, LRGILS n=5, SLIGRL n=7).
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Figure 6.2 Effect of SLIGL (s.c.) on electrical kindling
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Due to some animals dying following kainic acid-induced status epilepticus or as a result of during spontaneous seizures, unrelated infections (see also General Discussion, Chapter 8) or the loss of their headpieces prior to the 8 week experimental end point, only a small animal numbers in each group remained at the completion of the experiment (SLIGRL n=5, LRGILS n=3, saline n=4). Thus, not only is the physiological data limited, there were also too few brains left for accurate histological analysis. Furthermore, given the end points of the 3 treatment groups were ultimately equivalent with respect to spontaneous seizure frequency and an epileptic state per se, the laborious task of immunohistochemistry and histology seemed futile as major differences in brain pathologies were not expected. Repetition of the experiment was unfortunately not possible due to time constraints, however, this is essential to fully understand the consequences of SLIGRL treatment on kainic acid-induced epileptogenesis.
6.1.16 PAR2 and trypsin colocalise in neurons in the brain As extensively described in the immunohistochemistry experiments of this thesis (Chapter 3), high levels of both PAR2 and trypsin were co-expressed in neurons of the hippocampus, cortex, thalamus and amygdala (see Chapter 3 for images). Briefly, in the hippocampus, PAR2-like IR was highly expressed in pyramidal cells of the CA1, CA2, CA3 and CA3c regions and throughout the granular cell layer of the dentate gyrus (Figure 3.1). Again no PAR2-like IR was detected in astrocytes.
Intense trypsin-like IR occurred within pyramidal cells of the hippocampal CA1, CA2, CA3 and CA3c regions, in particular, in the granular cell layer of the dentate gyrus as well as in the cortex, thalamus and amygdala. Trypsin-like IR was generally
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granular-like in the soma and, like PAR2, often appeared in the axon hillock and basal processes of neurons. Trypsin-like IR was detected in cells with glial morphology.
6.1.17 PAR2 and trypsin increase expression during kindling. Fluorescence intensity (FI) analysis of the PAR2-like IR in sham animals revealed no differences in PAR2 expression between the cortex and the CA1, CA2, CA3, CA3c and dentate gyrus regions of the hippocampus. No other brain regions were analysed. None of the hippocampal and cortical regions from the brains of kindled, i.c.v. peptide-treated rats displayed any significant changes in PAR2 expression compared to sham suggesting that neither kindling nor peptide administration had any effect of the basal PAR2 expression in the brain (Figure 6.4).
In the brains taken from the fully-kindled animals treated with LRGILS, there was an average near 50% increase of trypsin expression from sham control levels in all regions analysed, with the greatest change occurring in the granular cell layer of the dentate gyrus (Figure 6.4). Thus, like described for other proteases such as tPA [40, 42, 64, 72, 73, 86, 127], trypsin expression increased in the hippocampus following pro-epileptogenic stimuli. In contrast, expression of trypsin in SLIGRL-treated kindled animals was no different from that of sham-kindled rats in all regions analysed except for the dentate gyrus, which showed a modest but significant 27.3% increase in trypsin-FI from sham in the granule cell layer (p<0.05).
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6.1.18 SLIGRL treatment does not affect mossy fibre sprouting Both left and right hemispheres of the hippocampus showed significant mossy fibre sprouting in both peptide treatment cohorts in the i.c.v. kindling groups, with significantly greater Timms stained optical density (OD) in the outer molecular layer of the dentate gyrus post-kindling compared to sham (Figure 6.4 and 6.6). There were no differences in mossy fibre sprouting between ipsilateral (right) and contralateral (left) kindled hemispheres, suggesting that electrical amygdala kindling affected both hippocampi equally with respect to mossy fibre sprouting. Also, there were no significant differences in mossy fibre sprouting OD between SLIGRL- and LRGILStreated animals, with both groups showing equivalent amounts of Timms staining in the mossy fibre pathway (Figure 6.4).
There was no correlation between trypsin-FI in the dentate gyrus and mossy fibre sprouting-OD in the same region (R2=0.046, data not shown), suggesting that trypsin may not be involved in the process of mossy fibre sprouting. Discussion Two main findings emerged from this study of TLE in the amygdala electrical kindling and kainic acid models of seizure and epilepsy in the rat. First, SLIGRL administered either centrally or peripherally, attenuated both seizure development and progression to an epileptic state, particularly in the kindling model (Chapter 5). The second, and possibly mechanistically related, finding was that centrally-applied SLIGRL virtually abolished the approximate doubling in hippocampal trypsin immunofluorescence that occurred in response to kindling. The elevation in ADT following SLIGRL treatment is consistent with that seen in kindling animal models
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treated with anti-convulsant medications currently used in clinical practice, such as carbamazepine, valproate and phenytoin [15, 16, 353], indicating potential antiseizure properties of SLIGRL. By contrast, the increased latency to first seizure and the reduced progression of both the behavioural and the electrographic seizure severity and duration in all models, and the reduced number reaching a fully-kindled epileptic state in the i.c.v. kindling model is more indicative of anti-epileptogenic properties of SLIGRL [26, 339]. These data suggest that rats treated with SLIGRL require a greater insult to induce epilepsy.
As discussed in Chaper 5 of this thesis, where it was found that high doses of SLIGRL (i.c.v.) induced neurotoxic behaviours, it was expected in the present study that SLIGRL at the dose used may induce neuronal hyperexcitability [285, 320], behavioural hyperactivity (see Chapter 5) and, as such, accelerate seizure and epilepsy. The exact opposite, however, occurred with SLIGRL reducing seizure risk and attenuating epileptogenesis. The mechanism for the anti-epileptogenic effects of PAR2 activation could not be definitively established by the work in this thesis, but may involve down-stream effects other than calcium liberation, such as an inhibition of enzyme and protein synthesis via ERK/MAPK pathways [304, 314, 318], thus restricting trypsin production and expression as demonstrated in the FI data (Figure 6.5).
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Figure 6.3 SLIGRL (i.c.v.) has anti-epileptogenic properties in the kainate model in the rat. A; SLIGRL (0.15mg/kg i.c.v., n=13)-treated rats required significantly more kainic acid to induce status epilepticus (SE) than controls (LRGILS 0.15mg/kg n=10 i.c.v., saline n=8; *p<0.05). B; SLIGRL significantly increased latency to first recorded spontaneous seizure (weeks) following SE (*p<0.05). C; SLIGRL significantly reduced average seizure duration over the 8 week recording period (*p<0.05 ANOVA ). Data are expressed as means s.e.m. (saline n=4, LRGILS n=3, SLIGRL n=5).
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Figure 6.4 PAR2 Figure 6.3 Effect of SLIGRL on status epilepticusRL (i.c.v.) has anti-epileptogenic and trypsin immunofluorescence in the hippocampus
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Figure 6.5 Kindling induced trypsin IR
Figure 6.4 The expression of trypsin, but not PAR2, is up-regulated in the hippocampus during electrical amygdala kindling in the rat. Quantitative immunohistochemistry of relative fluorescence intensities (FI) of hippocampal pyramidal cells in electrical amygdala kindled animals. A; SLIGRL or LRGILS had no effect on PAR2 FI increases in CA1, CA2, CA3, CA3c and dentate gyrus (DG) region of the hippocampus at the end point of kindling. B; trypsin FI increased significantly in all regions of the hippocampus only in LRGILStreated animals in response to kindling compared to both sham and SLIGRL treated animals. Trypsin only significantly increased in DG in SLIGRL treated animals. C; kindling increases mossy fibre sprouting (optical density, OD) in the stratum moleculare of the dentate gyrus equally in both hemispheres of the brain. This was unaffected by SLIGRL (* p<0.05 from sham (SLIGRL n=5, LRGILS, n=6, Sham=4)). Data are expressed as means s.e.m. (sham n=4, LRGILS n=6, SLIGRL n=5).
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B * * * * * *
C * * * *
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Figure 6.6 Timms staining in the hippocampus g-induced trypsin expression
Figure 6.5 Examples of kindling-induced trypsin expression in the rat hippocampus. Fluorescence micrographs of hippocampal sections showing trypsin-like epi-fluorescence in the CA1 region of the hippocampus from animals in the different treatment groups described in Figure 6.1. A; Sham kindled, B; fully-kindled and LRGILS-treated, C; kindled and SLIGRL-treated. Varying levels of trypsin-like epi-fluorescence can be seen, with most intense staining observed in the LRGILS-treated animals (i.e. B). SLIGRL-treated animals had levels comparable to sham. Fluorescence intensity levels of both PAR2 and trypsin in the hippocampus are shown in Figure 6.4.
ML
GCL
GCL
Figure 6.6 Examples of Timms-stained hippocampus in control and epileptic (fully-kindled) rats. A; normal dentate gyrus (DG) and mossy fibre pathway staining of a non-kindled (sham) animal. B; kindled (LRGILS) animals show dense Timms-stained band (i.e. OD) in the outer stratum moleculare (ML) of the DG adjacent to the granular cell layer (GCL) of the hippocampus (white arrow heads). Note the small finite fibres stretching between through the GCL (arrows, see insert) forming a thick band in the outer ML. No differences in OD of the mossy fibre pathway were observed between SLIGRL- and LRGILS-treated rats (see also Figure 6.4).
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Alternatively it may also involve as yet unrecognised PAR2 effects on neuronal hyperpolarisation such as activation of calcium-activated potassium channels or nonspecific effects of SLIGRL on NK1-like receptor mediated chloride ion transport, such as proposed in the mouse airway epithelium [266]. These data, however, do clearly suggest that at the maximal-tolerated dose (0.15mg/kg i.c.v. Chapter 5) used in the present study, SLIGRL is cytoprotective against a potentially epileptogenic brain insult, paralleling the findings in other disease models [244, 309, 333].
From the immunohistochemistry experiments performed here and in Chapter 3, it can be proposed that enzymes like trypsin and tPA, and possibly others like neuropsin [87], motopsin [111] myelencephalon-specific protease [127] and certain serpins [152], are stored together and are likely to be released to locally lyse the extracellular matrix for normal synapotgenesis. During inflammatatory events, such as kindling, however, elevated levels of these proteases like tPA, and now trypsin, may induce matrix and neuronal damage that can contribute to epileptogenesis [40, 43, 64, 67, 72, 73]. These increases in protease expression in the hippocampus thus coincide with epileptogenic damage to the brain, such as cell loss [257, 421], most of which is known to occur in the latent period following status epilepticus [45]. Thus, various proteases, now including trypsin, appear to have central roles in seizure generation and epileptogenesis and may as such be good targets for potential anti-epileptogenic therapeutics. Furthermore, the finding that inhibition of the kindling-induced trypsin expression with SLIGRL correlated with the inhibition of the progression to an epileptic-like state is in good agreement with previous studies that show reduced tPA expression or function correlates with inhibition of epileptogenesis [42, 72, 129, 154]. Taking into consideration this effect of PAR2 activation on trypsin expression, it can
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now be suggested that PAR2 expressed by protease-secreting neurons may provide important feedback mechanisms to regulate the extracellular activities of digestive enzymes, one of which is trypsin, but probably tPA as well, considering its coexpression with trypsin in LDCVs (Chapter 3). Thus much like in the pancreas [304, 314, 318], perhaps similar mechanisms exist for neurons in the brain that store and release protease packages, where PAR2 activation may induce both feedback and feed-forward mechanisms to effectively rid pyramidal neurons of excessive trypsin [304, 314] (see also Chapter 1). The most likely mechanism behind this neuroprotection is therefore the demonstrated reduced trypsin expression, and presumably release, from hippocampal pyramidal neurons following SLIGRL treatment. Thus, SLIGRL appears to be anti-epileptogenic via this mechanism.
The relationship between ADT and trypsin expression is intriguing. Perhaps the increasing trypsin levels during kindling, as discussed earlier, are able to potentiate NMDA receptor signaling in such a way that cells become depolarised and hyperexcitable, leading to a reduced seizure threshold and a lower ADT. Why the ADD is lower however following SLGRIL treatement, is not understood, but may be related to lower trypsin expression and a reduced propensity for NMDA receptor driven hyperexcitability, thus even though they do occur, the seizure are of shorter duration.
Unlike in other neurological trauma models such as ischaemia, experimental autoimmune encephalitis, HIV infection and radiation exposure [204, 285, 309, 319, 326, 333, 416], PAR2 expression did not increase following electrical amygdala kindling as shown in the current study. The reasons for these different outcomes are
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unknown. It is possible, however, that PAR2 up-regulation occurs only in response to high levels of inflammatory mediators of specific types and patterns that do not occur during electrical kindling-induced epileptogenesis. Since we found up-regulation of trypsin expression but not PAR2, proteolysis may overcome the PAR2 regulatory function. If so, then this PAR2-dependent control mechanism may be rendered futile as excessive and unmonitored proteolytic changes occur that are associated with epileptogenesis. Using SLIGRL to mimic the action of trypsin without the proteolytic effects may therefore prove to be a useful means of activating such feedback mechanisms in inflammatory conditions without damaging the matrix or neurons of the brain, thereby activating the demonstrated neuroprotective effects.
As previously discussed, a dose of SLIGRL of greater than 100M can induce widespread neuronal death in culture [320] and high doses of SLIGRL injected peripherally depolarise sensory nerves [290, 296, 297]. The data presented here thus appears to contradict these studies but coincide with the results presented in Chapter 5, that at this maximal tolerated dose, SLIGRL did not cause neurotoxicity. We now extend this to suggest that this dose of SLIGRL caused neuroprotection. Perhaps in future studies using neurons in culture, lower concentrations of SLIGRL may prove to be neuroprotective, and not neurotoxic. These differences are most likely related to dose-dependent PAR2-induced intracellular calcium overloads at higher doses of SLIGRL causing neurotoxicity [320].
The lesser degree of inhibition of epileptogenesis following s.c. SLIGRL compared to that after i.c.v. treatment most likely indicates a lower brain-accessible concentration of the peptide when administered via this route. We chose the dose of 1.5mg/kg for
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the systemic delivery (s.c.) from our and previous studies that have shown this dose effectively inhibited ulcerative colitis and stomach ulcer in rats and mice (Devlin and Cocks, unpublished data;[302]). As outlined in Chapter 4 of this thesis, SLIGRL appears to cross the blood brain barrier following peripheral injection at this dose. The present study now shows that it appears to do so as an active molecule. As such, systemically-administered PAR2 peptides may be valuable new therapeutics not only for epilepsy, but possibly also for other CNS inflammatory disorders that involve excessive release of neuromodulatory enzymes. We did not investigate the expression of tPA following kindling and PAR2 peptide administration in the current experiments as it was not the purpose of the study. It would, however, be important to do so in future research, as tPA is clearly involved in memory, seizure and epileptic pathogenesis.
The lack of any significant differences in the degree of mossy fibre sprouting between the kindled peptide-treated groups, may indicate that mossy fibre sprouting is an epiphenomenon to epileptogenesis and not a causative factor of seizure per se [219, 220, 222, 232]. Whilst the increase in trypsin in the dentate gyrus where mossy fibre sprouting occurs in both peptide-treated groups (i.c.v.) suggests trypsin may be involved in mossy fibre sprouting, this seems unlikely as there was no correlation between trypsin fluorescence intensity and mossy fibre sprouting optical density in this region. Therefore mossy fibre sprouting may rely on proteases other than trypsin, such as tPA [67].
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As shown in Chapter 5, LRIGLS had no recordable central effects in any of the experimental cohorts, similar to saline. Therefore, the inclusion of a saline control in the initial i.c.v. kindling experiment described in the present chapter was deemed unnecessary. Due to some speculation from peers, however, a saline control group was included in the follow-up s.c. kindling and the i.c.v. kainic acid cohorts. The results here are therefore in consensus with Chapter 5, that LRGILS has no specific effects at the dose used.
Apart from the implications for possible new treatments for epilepsy, the present study perhaps highlights a more general physiological role for PAR2. Previously our group offered an alternative to the widely held view that PAR2 is pro-inflammatory [259, 275]; that is that PAR2 act as sentries for extracellular trypsin and trypsin-like enzymes and as such constitute the afferent input for servo-defence mechanisms to regulate paracellular responses to potentially destructive digestive enzymes present in their immediate extracellular environment. The current study now allows this paradigm to be modified; cells that synthesise, store and release proteolytic enzymes, one of which is trypsin or a trypsin-like enzyme will also express PAR2.
In summary, the results of this study indicate that activation of PAR2 inhibits seizures and attenuates epileptogenesis in both the kindling and the kainic acid models of epilepsy. Furthermore, the study has shown that the presence of SLIGRL in CSF both before and during kindling appears to prevent the up-regulation of neuronal trypsin expression. We propose that PAR2 acts as a regulator of proteolytic-dependent processes in plastic regions of the brain like the hippocampus and as such contributes to the synaptic integrity in these important limbic regions. From these data, it is
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proposed that in certain pathological conditions, such as epileptogenic brain insults, excessive synthesis and release of trypsin and other proteolytic enzymes overwhelms this normal control system. Therefore, administration of non-proteolytic PAR2 agonists, like SLIGRL, to prevent this stimulus-induced increase in trypsin expression (and potentially other proteases) may have important implications for the treatment of other neurodegenerative CNS disorders like epilepsy, but may also include stroke [333], Alzheimers disease [246] [205] and multiple sclerosis [103, 139, 375], all of which exhibit protease-driven pathological neurodegradation. In this sense, SLIGRL may be a useful anti-epileptogenic agent.
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Chapter 7 PAR2 in learning and memory
7
Introduction
Chapter 7 The involvement of PAR2 in fear-aversive learning
The concept of protease-mediated memory formation is a well supported neurobiological theory. As described in detail in the General Introduction of this thesis (Chapter 1), recent evidence suggests that the serine proteases tPA [158-160], neuropsin [109] and thrombin [161, 162] are essential in NMDA-driven LTP and LTD [64, 67, 84, 87, 99, 108, 128, 163, 167-170] and are therefore important in learning, memory and emotional reward behaviour, particularly in the development of stress-induced anxiety-like behaviours [102, 131, 159]. These behaviours are thought to be related to protease-dependent synaptic remodelling and structural changes, such as dendritic spine-loss [164-166] and enhancement of NMDA receptor signalling (i.e. LTP). Thus, serine proteases may facilitate cellular learning and may be critically involved in the development of, in particular, fear-motivated learning and memory following stressful situations [159].
Given the involvement of serine proteases in learning and memory, the question arises as to whether PARs are also involved. Thrombin, for instance, has recently been found to enhance LTP [65] but also the PAR1 peptide affects NMDA receptor signalling in hippocampal neurons [66]. This suggests a specific PAR1-mediated response and not merely a thrombin interaction with NMDA receptor. Additionally, in vivo studies in PAR1 gene k/o mice found deficits in fear-mediated emotional learning in response to painful stimuli [161] and that PAR1 may also be important in the regulation of nicotine and morphine reward emotions by facilitating the release of dopamine in the nucleus accumbens [160, 162]. These studies suggest that PARs may
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have important roles in protease-mediated acquisition, consolidation and/or retrieval of fear-related associative memories.
As shown in Chapter 3 of this thesis, trypsin colocalises with tPA in LDCVs of PAR2-positive pyramidal neurons of the hippocampus [422] and activation of PAR2 with SLIGRL results in a reduction of trypsin immunoreactivity in these pyramidal cells that correlates with an attenuated kindling- and kainate-induced epileptogenesis both following central (i.c.v.) and peripheral (s.c.) administration of the peptide (Chapter 6). As proposed in Chapter 3, SLIGRL-mediated PAR2 activation may induce feedback mechanisms acting to reduce neuronal trypsin over-expression, as described in other tissue types [304, 314, 317, 318]. Therefore, if proteases are related to memory formation and trypsin is expressed in the hippocampus together with PAR2, then both trypsin and PAR2 may have roles in learning and memory. This premise is supported by the results shown in Chapter 5 of this thesis where centrallyadministered SLIGRL at the maximum tolerated dose appeared to induce increased exploratory behaviours. In addition, when injected peripherally, SLIGRL was found to cross the blood-CSF barrier (see Chapter 4) and appeared to activate centrallyexpressed PAR2 linked to this feedback mechanism to regulate the expression of trypsin, and potentially other co-stored proteases such as tPA (see Chapters 3 and 6). If this is the case, then changes in normal protease-dependent plastic processes, such as learning and memory in a whole animal following a peripheral injection of SLIGRL may be expected
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Therefore, the purpose of the current study was to investigate the effects of SLIGRL on locomotion, anxiety-like behaviours, learning and memory in genetic absence epileptic rats of Strasbourg (GAERS) using ethological assays such as the elevated plus maze, the open field test, sucrose-consumption test, the light dark box and the Morris water maze. Materials and methods All methods are previously described in greater detail in Chapter 2 of this thesis.
7.1.1
Animals
Genetic absence epileptic rats of Strasbourg (GAERS, 12-14 weeks of age (180200g)) of both sexes were housed as previously described (19-24C, 12 hour light/dark cycle, food and water supplied ad libitum) in boxes of two and three animals. GAERS were used on account of their higher levels of anxiety compared to their non-epileptic controls [347]. 7.1.2 Peptides
SLIGRL and LRGILS diluted in 0.9% sterile saline were injected subcutaneously (inter-scapular, 1.5 mg/kg s.c.) 20min prior to exposure to ethological tests. Vehicletreated (control) animals received an equivalent volume bolus of sterile saline.
7.1.3
Behavioural testing protocols
Three cohorts of rats were used, the first (n=28) for the elevated plus maze and the
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open field test, the second for the sucrose consumption test and the light-dark-box (n=11) and the third for the Morris water maze (n=15). The first and second cohorts were exposed to each behavioural test three times on alternating days (i.e. with 48h between repetitions), 20min following administration of SLIGRL, LRGILS or saline. The sucrose consumption and Morris water maze tests were performed on consecutive days. Animals were randomly allocated treatments and all were age matched to their controls. For all tests, rats were brought into the behavioural testing facility 30min prior to experimentation to allow for acclimatisation to the specific conditions in the room. Following this acclimatisation period, rats were injected with either SLIGRL (1.5mg/kg s.c.), LRGILS (1.5mg/kg s.c.), or saline. Each rat received the same treatment 20min prior to each repeated test. During testing, the rats movements were tracked and recorded using a video camera mounted to the ceiling connected to a PC running EthoVision software (Noldus, The Netherlands). All experiments were performed in the same behavioural testing facility at standardised lighting conditions at the same time of day (a.m.) in order to minimise diurnal variations. Specific protocols of each ethological test are given in Chapter 2.7.
Results 7.1.4 SLIGRL does not affect rat behaviour on the open field test
SLIGRL (n=8) treatment had no effect on any parameters measured on the open field test compared to controls (LRGILS 1.5mg/kg s.c., n=8, or saline n=8). On the first trial, all rats, regardless of treatment, spent similar total times in the centre field, travelled similar distances in the centre field and the entire arena and entered the centre field a similar number of times. These behaviours were also examined on a minute-by-minute basis, and again there were no differences between the treatment
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and control groups.
There were no learning behaviours associated with open field test-retest situations, as has been described previously [423], with all rats showing similar locomotor activity and spending similar times in the centre field as the previous trial, again with no significant difference between treatment groups (Figure 7.1). When the data was analysed as either total time (over the entire 10min trial) or minute-by-minute, trends of all parameters were not statistically different to the previous trials. The trends for SLIGRL-treated animals to enter the centre field more often and to cover larger distances in the centre than controls were not significant. These results clearly suggest that SLIGRL is not anxiolytic.
7.1.5
SLIGRL has no effect on light-dark box behaviours
SLIGRL treatment (n=5) had no significant effects on time spent, entries or risk assessments into the light chamber of the dark box compared to controls (LRGILS n=3, saline n=3) in all trials (Figure 7.2). No significant passive avoidance learning behaviours to the light chamber were observed across the three trials. Therefore, SLIGRL had no specific effects on the behaviour of a rat in the light dark box (Figure 7.2). These results are in support of the open field test data, that SLIGRL does not possess any anxiolytic properties.
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Figure 7.1 SLIGRL has no effect on the behaviours of rats on the B A open field test.
Figure 7.1 SLIGRL has no effect on the behaviours of rats on the open field test. A; total time in spent centre arena. Shows there are no significant differences in the centre field time in the first trial nor was there any centre arena avoidance upon retesting in any of the treatment groups. B; there were also no differences in the entries into the centre field across all trials regardless of treatment. C; there were also no significant differences in the total distance travelled across all trials regardless of treatment. These data suggest SLIGRL has no effect on anxiety or locomotion in rats. There were also no learning behaviours upon retesting on the open field test (saline n=8, LRGILS n=8, SLIGRL n=8).
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7.1.6
SLIGRL has no effect on sucrose consumption
Over the 4 days of testing, there was a trend to increase sucrose consumption in the control rats over the consecutive trials (SLIGRL n=7, LRGILS n=6, saline n=5, repeated measures ANOVA), which is in agreement with previous studies [347]. Volumes of sucrose consumed within days, however, were not significantly different between treatment groups (Figure 7.3). There were also no significant differences in the approaches the rat made to the drinking nozzle (data not shown). Therefore, SLIGRL treatment had no effect on the pleasure derived from drinking sucrose and thus, apparently, had no effect on anhedonia and was therefore not considered antidepressive.
7.1.7
SLIGRL prevents fear avoidance learning on the elevated plus maze
Administration of SLIGRL (n=10) had no significant effect on any of the parameters measured on the elevated plus maze in the first trial compared to controls (LRGILS (n=9) and saline (n=9)). Thus, total open-arm durations, distances travelled, open and closed arm entries and risk assessments were similar regardless of treatment. On a minute-by-minute analysis, all animals tended to initially explore the open arms, then after the first 5min, spent very little time on the open arms, the majority being spent in the closed arms (Figure 7.4). This was also reflected in the distance travelled on the open arms (Figure 7.4). The final minutes of the first trial were usually spent resting/grooming in the closed arms, neither entering nor moving any appreciable distances.
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igure 7.2 SLIGRL has no effect on light-dark box behaviours
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Figure 7.3 SLIGRL has no effect on sucrose consumption in rats
Figure 7.2 SLIGRL has no effect on rat behaviours in the light-dark box. No significant variation was observed within trial or between trials in any of the treatment groups in A; time spent in light arena, B; Total entries into the arena and C; risk assessments into the light arena. This suggests SLIGRL has no effect on anxiety behaviours (saline n=3, LRGILS n=3, SLIGRL n=5).
Figure 7.3 SLIGRL has no effect on sucrose consumption in rats. All treatment groups showed a pattern of progressively consuming more sucrose over the four days of trials. No significant differences were observed between treatment groups (saline n=5, LRGILS n=6, SLIGRL n=7).
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In a retest situation in the elevated plus maze (trial 2, Figure 7.5), much like what has been extensively described in the literature [359, 424-428], control rats (LRGILS or saline) spent only the first minute exploring the open arms, before retreating to the closed arms for the remainder of the trial. Thus, in the retest situation (trial 2), control rats showed a reduction in total open arm time over the 10min trial (p<0.05) (Figure 7.5), a significantly increased closed arm time (p<0.05) and reduced general locomotive behaviours (i.e. open arm and total distance travelled, total open arm and closed arm entries) compared to the first trial (p<0.05) (Figure 7.5).
During the third retest trial, control animals spent even less time in the open arms in the first minutes of the trial compared to trial 2, being in the open arms for only a very brief period of time in the first minute then retreating to the closed arms for the remainder of the trial, however this was not statistically different from trial 2. This same trend was also observed in general locomotor activities (entries and distance travelled), with rats invariably avoiding the open arms of the elevated plus maze throughout the entire third trial (Figure 7.5). None of these changes, however, were observed in risk assessments, which remained statistically similar over all three trials.
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.4 Minute-by-minute analysis on the elevated plus maze
Figure 7.4 Minute-by-minute analysis of the rats behaviour in the first trial of the elevated plus maze. Shows similar progressive open arm avoidance over the 10 minute trial between all treatment groups in both time spent (A) and distance travelled (B) on the open arms. These data suggests SLIGRL is not anxiolytic or sedative (saline n=9, LRGILS n=9, SLIGRL n=10).
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Interestingly, none of these retest behavioural alterations were observed in the SLIGRL-treated animals on trial 2 and trial 3, which showed equivalent maze behaviours in each retest situation compared to trial 1 of the same treatment group (Figure 7.5). On a minute-by-minute basis, there were no significant differences in open arm or closed arm durations, or general locomotor activities between all three trials. Therefore, rats treated with SLIGRL did not retreat to the dark after the first minutes of the trial, as seen in controls, but persisted to explore the open arms as if it was the first trial experienced (Figure 7.5). This phenomenon was observed in both the second and third trials. In both retest situations, all parameters tested (time, entries, distance) were significantly different between SLIGRL and control groups (p<0.05), except for risk assessments (Figure 7.5), which remained similar throughout all trials between all treatment groups. Therefore, SLIGRL-treatment appeared to affect memory acquisition, retention and/or retrieval. In consensus with the data obtained from the open field test and the light-dark box, SLIGRL was not anxiolytic.
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Figure 7.5 SLIGRL impairs fear-avoidance on the elevated plus maze
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Figure 7.5 SLIGRL impairs fear-avoidance learning on the elevated plus maze. A; total time spent on the open arm across the three trial. No significant differences were observed between groups in the first trial. In the second and third trials both control groups clearly avoided the open arms, whereas SLIGRL treated animals spent equivalent time on the open arms as the first trial. This was reflected in B; where the closed arm time increased in both control groups (LRGILS and saline) but remained equivalent in SLIGRL treated groups. C; control rats clearly avoided entering the open arms on retest situations but SLIGRL had equivalent behaviours to the first trial. This was also reflected in D; the total distance travelled (both closed and open arms, where rats in the SLIGRL group covered more distance in the open arms. E; There were no differences in the risk assessments between groups within trials, however there was a significant reduction in all groups on retesting compared to the first trial. (ANOVA planned comparison between groups *p<0.05, between trials #p<0.05) (saline n=9, LRGILS n=9, SLIGRL n=10).
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On the second day, control rats continued this trend in all trials (trials 5-8), being particularly evident in the first trial (trial 5), where a similar time and distance covered to find the platform as the final trial the previous day (trial 4) was observed. As trials continued over all 4 days (16 trials), control rats became very familiar with the location of the platform and thus progressively took the quickest route to the platform (Figure 7.6). This data shows quite clearly that the rats learned the location of the platform in relation to the visual cues and remembered these both within days and over the four day duration of the experiment.
7.1.8
Effect of SLIGRL administration on Morris water maze behaviours
Given there were no differences between LRGILS and saline in all the previous trials, a saline group was excluded from these experiments (see also Chapters 5 and 6 of this thesis).
All animals from both treatment groups (SLIGRL n=8, LRGILS n=7) behaved similarly in the Morris water maze during the first day of the trials. In the first trial of the experiment (trial 1), no rat successfully found the platform, as was expected, and so all animals were guided there. In the subsequent trials (trials 2-4), both the time taken and distance covered to find the platform progressively decreased on average in both groups (Figure 7.6).
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Day 1 Day 2 PAR2 and trypsin in the Day 3 brain Day 4
Figure 7.6 SLIGRL impairs behaviours of the Morris water maze
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Figure 7.6 SLIGRL impairs learning behaviours on the Morris water maze. A; time taken to find platform in consecutive trials. Rats in day 1, regardless of treatment, rapidly learned the location of the platform. In trial 5 (day 2), control rats found the platform in the same time as the last trial of the previous day (trial 4), whereas SLIGRL-treated rats behaved equivalent to the first trial of the first day (trial 1). SLIGRL rats did however quickly find the platform on retesting within each day. This trend was also seen in the third day, where control rats progressively learned the location of the platform, whereas those treated with SLIGRL again appeared to have a deficit in a memory of the platform location in the first trial of day 3 (trial 9). B; these findings were reflected in the distance travelled. (*p<0.05) (LRGILS n=7, SLIGRL n=8). .
In stark contrast to the control rats, on the first trial of the second day (Trial 5), rats treated with SLIGRL had no recollection of the location of the platform, spending the majority of the 90s swimming around the bath looking for the platform, much as they
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did in the first trial of the first day (trial 1) (Figure 7.6). Once the platform was shown to the SLIGRL-treated rats, however, they responded in an equivalent manner to the controls in the remaining trials of the same day (trial 6-8) (Figure 7.6), suggesting that their acute working memory was intact, but the longer term memory established the previous day was markedly impaired. This same trend was observed in the first trial of the third day (trial 9), and although the difference was not as dramatic as that observed on the second day, it was still significantly different from controls in both time and distance parameters (Figure 7.6). By the forth day, SLIGRL and LRGILS animals had equivalent behaviours on the water maze (Figure 7.6).
Discussion The results of the current study clearly demonstrate for the first time that peripheral administration of SLIGRL, and therefore PAR2 activation, has profound effects on motivational, or fear-aversive, learning in rats. In both models of motivational learning (i.e. the elevated plus maze and Morris water maze), rats treated with SLIGRL appeared to have a deficit in the formation and/or recollection of a memory formed during a previous exposure to the same unpleasant, or fear-inducing stimuli (i.e. open spaces and water). SLIGRL did not induce any hyperactivity or sedation, nor did it induce any anxiolytic effects in either the open field test, light-dark box or elevated plus maze. Therefore, it appears that SLIGRL, most likely via PAR2 activation, may have very specific roles in either the formation or recollection of motivational memories.
Even though largely unrecognised, the test-retest paradigm of the elevated plus maze is a good model for conditioned learning in the rat [359, 425-429]. The behaviour of a
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rodent on the elevated plus maze is the result of conflict between its intrinsic tendency to avoid open spaces (fear-avoidance) and a natural curiosity to explore novel environments. Therefore, the first test is a novel experience for the rat and as such, it will tend to spend some time exploring the entire maze, particularly in the first five minutes of the trial [359], as observed here. This first test, in its own right, is thus a very good measure of anxiety in rats; the longer spent exploring the unfamiliar and potentially dangerous and fear-inducing open arms, the less anxious the animal [359]. Repetition of the test (i.e. removal of the novelty) either on a daily or even weekly basis, however, results in a progressive reduction in open arm exploration [427], indicating an open-arm avoidance behaviour learned through experience [359, 427]. Even in the presence of clinical anxiolytics, such as midazolam, where rats spend more time in the open in the first trial, they show no differences to controls in the second trial [429]. This phenomenon is known as one trial tolerance. If memory acquisition is, however, disturbed, such as with saporin injection into the medial septum, such fear-aversive memories are prevented and a rat will behave in the second trial much like it did in the first [428] .
In accordance with such earlier studies [359, 425-429] the results of trial 1 of the elevated plus maze in the current study show that placing a rat in a novel environment, results in an initial tendency to explore the entire apparatus, gradually reverting to the closed arms and avoiding the potentially dangerous open arms. During trial 2 on the elevated plus maze, control rats clearly avoided the open arms, a phenomenon that was also observed to the same extent in the third trial. This is evidence that the control rats had learned from experience to avoid the open arms of the elevated plus maze. Whether such learning is a fear-avoidance represented by an
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aversion to the potentially dangerous open arms as previously suggested [359], or simply a non-conflict behaviour resulting from removal of the novel element of the maze per se is, at this stage uncertain. Risk assessments, however, is a good measure of anxiety levels in the rat [359]. Therefore as there were no significant differences in risk assessment behaviours on the elevated plus maze between treatment groups over the three trials, it seems reasonable to assume that there are no differences in the basal anxiety state of the rats in each treatment group. This is in agreement with the results of the open field test and light dark box. Only a reduced locomotor activity was observed in open arm of the elevated plus maze in control animals on retesting, which may reflect a memory of a previous fearful experience on the open arms and a subsequent avoidance thereafter. Rats may intuitively peer out into the open arms of the elevated plus maze to survey their surroundings (i.e. risk assessment), but they know what is there and therefore are naturally programmed to avoid entry into the area, which is what SLIGRL animals failed to do. This behaviour therefore appears to relate to a motivational, or fear-avoidance, memory [359] and not simply a removal of the novelty of each maze.
The fact that the first trial of the elevated plus maze, open field test and light-dark box showed no differences in any of the measurement parameters between treatment groups indicates that SLIGRL did not induce any anxiolytic effects in rats on these assays [359, 428, 429]. Sedation or hyperactivity would be expected to be observed in the first trial alone [359], so since there were no differences in any first trial behaviours measured, particularly locomotion and risk assessment, such potential sedative or hyperactive effects of SLIGRL can be ruled-out. This is interesting to note, as in previous studies outlined in this thesis (Chapter 5), it was observed that
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i.c.v. SLIGRL at doses equal to, or lower than, 0.15mg/kg induced mild hyperactivity. This may be related to the route of administration and the brain-accessible concentration of the peptide following i.p. administration, much as also described in Chapter 6.
The sucrose consumption test data showed that SLIGRL does not possess any distinct effects on the pleasure derived from drinking sucrose and therefore it is unlikely that SLIGRL has any effect on anhedonia, a symptom of depression.
Much like previous studies [423], we did not observe any fear-aversive-like learning behaviours of rats on re-testing in the open field, as described by the entries into the centre field. The lack of difference in the centre entries in the first trial alone thus corroborates with the findings of the first test of the elevated plus maze, that SLIGRL does not possess any anxiolytic properties. We did not, however, observe habituation in any of the experimental groups between the first and second tests as demonstrated by the lack of reduction in locomotor activity, which is in conflict with previous findings [423, 430, 431]. Perhaps the reason for this apparent discrepancy between the test and re-test habituation observed in these previous studies and those from our study is related to the different strains of rat used in each study. We used GAERS because of their high basal levels of anxiety [347], which we considered may have been useful to detect any anxiolytic effects of SLIGRL. That we observed no such effects in the first tests of either the open field or elevated plus maze argues strongly that SLIGRL is not anxiolytic in rats, at least in GAERS, and that the SLIGRLdependent changes in hippocampal plasticity in both epileptogenesis [422] and motivational learning (this study) are unlikely to affect anxiety. Perhaps such failure
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to develop neophobia during habituation indicates the open field does not provide enough stressful input to affect the higher levels of anxiety already present in GAERS.
The results of the Morris water maze clearly show learning behaviour of control rats in relation to the location of the platform. SLIGRL treatment induced a deficit in learning or memory recall, where much like that observed in the elevated plus maze, rats behaved in ways comparable to the first trial on both the second and third day of trial as the first trial on the first day. These results clearly indicate that SLIGRL treatment inhibited either the formation of a memory of the platform location from the previous day, or prevented recall of the memory of it on the second day. SLIGRL rats still learned the platform location within a day, as observed within all four days of trials, but had a between-day deficit. Thus the working (within day) memory may be intact, but the longer term (between day) memory may be impaired.
Multiple exposures to SLIGRL and the Morris water maze, but not the elevated plus maze, ultimately did result in accurate learning in the long-term (over the 4 days of trials). Why such long-term learning behaviour was not observed in the SLIGRLtreated rats on the elevated plus maze is unknown. It may, however, relate to the degree of fearful/motivational stimuli of each assay. On the elevated plus maze, a rat passively avoided the open arms as it has the option to retreat to the closed arms, whereas the Morris water maze being a forced unpleasant experience provides a much stronger motivation to learn, to, in essence, get out of the water as quickly as possible. Thus, the ability to learn in the long term in the Morris water maze but not the elevated plus maze may be related to the degree of motivation determined by the
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degree of stress induced in the different models. Nevertheless, these data of this chapter strongly suggest that PAR2 may have important roles in longer-term memory formation, consolidation and/or retrieval.
The involvement of PARs in learning and memory is generally unknown, however, few recent studies have suggested that the thrombin receptor PAR1 has roles in emotionally-motivated learning, with PAR1 gene k/o mice showing deficits in behavioural learning and passive and fear avoidance [161]. Furthermore, PAR1 has been shown to be involved in the regulation of nicotine reward [160] The results of the current study are somewhat in consensus with these previous PAR1 studies [160, 161], all of which suggest that like proteases, PARs have important roles in emotional learning and memory acquisition. However, PAR1 gene k/o mice were found to have similar cognitive fear-avoidance learning deficits against painful stimuli [161] as demonstrated here with PAR2 activation. These conflicting results may be explained by the differences in ethological assays, differences in PAR1 and PAR2 functioning, or differences in species used in the studies. Alternatively, PARs are well known to have roles in pain and nociception [278, 284, 290, 297, 331, 384, 432], and, as such, it is possible that the lack of PAR1 in mice results in reduced pain perception, which may thus account for the lack of pain avoidance learning in the PAR1 gene k/o mice. Put simply, mice lacking PAR1 may have a much higher tolerance to painful stimuli than their wild-type controls and therefore may not learn to avoid the painful stimuli on retesting.
If the proposed PAR2-mediated feedback mechanism exists (see Chapters 1 and 6), then perhaps the normal trypsin levels in the brain are artificially reduced by SLIGRL
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interaction with PAR2, leaving very little for release when required, such that the vital enzyme-driven plastic processes for memory formation (LTP and matrix reorganisation) and retention cannot occur. Even though the mechanisms behind the PAR2-induced learning deficits remain speculative, the findings of this chapter support those with that discussed in Chapter 6 of this thesis, that describe roles for PAR2 in neuronal plasticity and neuronal excitability,
Anecdotal observations suggest that PAR2 gene k/o mice are more tumultuous and aggressive than their wild-type counterparts (Van der Buuse, Devlin, Balzary and Cocks, personal communication). If true, then these traits appear to coincide with the data of this chapter that PAR2 is involved in fearful and emotional behaviours. Perhaps animals lacking the PAR2 gene have these behavioural characteristics as a consequence of a disrupted protease-driven motivational learning, which would otherwise be monitored and regulated by PAR2. If so, then such mice may not be well capable of learning fearful situations and, as a consequence, may be more aggressive and domineering.
Other mechanisms may also account for this observed inhibition in fear-motivated learning by SLIGRL. For example, PAR1 and PAR2 have both been suggested to alter NMDA receptor signalling [66, 145, 384]. Therefore, since NMDA receptors are crucial for learning and memory, PAR2 activation with SLIGRL could induce a heightened degree of NMDA receptor signalling and as such potentially improve learning capability, as described previously in tPA over-expressing mice [163]. The exact opposite, however, occurred with SLIGRL treatment in the current study. Thus PAR2-mediated NMDA receptor potentiation seems an unlikely explanation for the
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observed learning deficits following SLIGRL treatment. Another possible explanation for the ability of SLIGRL to inhibit fear-aversion learning is that the peptides ability to interact with non-PAR receptors, such as NK1-like receptor that have been shown to induce changes in chloride ion secretions as previously described in other tissue types [266].
In conclusion, trypsin and PAR2 appear to have distinct roles in the acquisition of fear-motivated learning in the rat but not in anxiety-like or depressive behaviours. The results may not only have implications in the understanding of fear-aversion learning, but also help further unravel the precise roles for PAR2 and trypsin in the limbic structures of the brain.
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Chapter 8 Final Discussion
Chapter 8 General Discussion
The high levels of expression of both trypsin and PAR2 described here in plastic, limbic structures of the brain, such as the hippocampus, suggests that trypsin like other proteases (e.g. tPA), is involved in neuronal plasticity. The in-vivo results suggest that trypsin may not only be necessary for normal functions like memory formation, but also be associated with diseases of the hippocampus such as epilepsy which could be regarded as a condition resulting from pathological neuroplasticity. Furthermore, the colocalisation of trypsin with tPA in LDCVs of PAR2-positive pyramidal neurons in the hippocampus not only suggests that both enzymes, and probably others, are released together as a neuromodulatory package, but also that their extracellular activities are monitored and regulated by PAR2. A long-held hypothesis of those in the laboratory where these experiments were carried out, is that PAR2 acts as a sentry for trypsin expression [259]. The results of the current experiments now extend this theory whereby cells that produce and secrete trypsin, and most likely other proteases (e.g. tPA), for extracellular digestive purposes, also express PAR2. Cell types that can be included in this category are pancreatic acinar cells [304, 313, 314, 318] (see also Chapter 3), mast cells [260], and cells forming barrier epithelium such as keratinocytes [277], airway [273, 275], urinogenital epithelium [288], vascular endothelium [433, 434] and now as we have shown, neurons of the brain (Chapter 3 and 6). However, cells that do not functionally require and/or release proteases (i.e. perhaps astrocytes) may not need PAR2 and under normal in vivo conditions (see Chapter 3).
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The two major findings from this thesis were that peripheral administration of SLIGRL prevented epileptogenesis and short-term fear-aversive memory in conscious rats, both of which are functions considered to be dependent on plasticity in the hippocampus. From these two findings, it is hypothesised that PAR2 has vital roles in such hippocampal functioning
Whilst arguments exist regarding the selectivity of SLIGRL [266, 270], the premise of a gate-keeper receptor for powerful digestive enzymes like trypsin and tPA makes biological sense. Furthermore, only one receptor may to be required to regulate these multiple enzymes, as proposed here. Precedence to this can be found in the pancreas, where levels of multiple digestive enzymes such as trypsin, chymotrypsin and certain carboxypeptidases, are all potentially controlled by PAR2 [121, 144, 304, 313, 314, 318].
Acinar cells of the pancreas and neurons in the hippocampus secrete powerful proteases for very different purposes, the former for digestion of ingested proteins in the intestine, and the latter for plasticity-dependent learning and memory formation. The pancreas copes with the problem of local active enzymes and thus cellular damage (e.g. pancreatitis), by secreting its enzymes as inactive zymogens via the pancreatic duct to the duodenum where they are activated by enterokinase. The brain, however, has no convenient duct system. Thus, whether such proteases are released in the brain as zymogen precursors is unknown, although tPA is produced and secreted as an active protease as there is no known inactive tPA precursor [435]. Given the greater fidelity required in the brain for neuronal enzymes in functions like learning and memory compared to the remote protein digestion in the gut for pancreatic
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enzymes, it seems logical to assume that such proteases are stored within LDCVs as active, ready-to-use forms, even though enterokinase has been demonstrated to be present and active in the brain [142, 143]. That these enzymes dont degrade the cells they are stored in most likely indicates zymogen and lysozyme activators remain localised separate vesicles that render them inactive until release, much like in the acinar cells of the panceras, presumably also being regulated by co-stored inhibitors like neuroserpin.
The comparison between the pancreas and the brain is further identifiable with respect to how each organ copes with the presence of excessive local active enzymes. The pancreas appears to utilise two mechanisms to achieve this protection, both of which are dependent on PAR2. The first involves concomitant PAR2-mediated activation of acinar cell degranulation and epithelial cell fluid secretion to effectively wash out the offending enzymes [304]. The second mechanism used by acinar cells to evade inappropriately activated enzymes involves negative feedback via PAR2 to shut down ERK1/2 nuclear trafficking to effectively prevent further synthesis of trypsin [314] and most likely other enzymes. In addition, activation of PAR2 on sensory nerves innervating the pancreas sends pain signals to the brain [436] that may help to mitigate any behaviours that could aggravate or prolong this early inflammation. Hence, in the pancreas, PAR2 may act as a global monitor for localised inflammation.
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As alluded to above, there is no portal system in the brain like the pancreatic duct to wash out over-produced active enzymes, nor are there any sensory nerves. Therefore, the most likely means available to regulate the activities of released enzymes would be via autocrine and paracrine PAR2-dependent feedback mechanisms, perhaps similar to the mechanisms described above for the pancreas. The finding in this thesis that SLIGRL prevented the over-expression of trypsin in hippocampal neurons in response to electrical amygdala kindling supports the proposition that PAR2 is acting as a control mechanism, and may point to a new therapeutic target for the prevention of epilepsy. PAR2 may therefore also have roles in other neurological diseases potentially involving protease activity, such as Alzheimers disease [91, 103, 139], multiple sclerosis [103, 116, 126, 139, 204], amyotrophic lateral sclerosis [103, 139], dementia [309] and stroke [139, 149, 437].
An additional mechanism for controlling neurally-secreted active enzymes in the brain may involve scavenging by astrocytes like that for neurotransmitters and oxygen radicals and as such may explain the trypsin detected in these cells in this study. Thus, astrocytes may not produce this protease per se but, but rather gather trypsin from the matrix after release by neurons, thus contributing to the cleaning up of excessive proteases. Before this is discussed, it is perhaps pertinent to consider the lack of PAR2 in these cells as found in the current study in the context of previous that claims that PAR2 are pro-inflammatory. Contrary to reports showing PAR2 expression in isolated astrocytes under cell culture conditions [285, 326, 330, 332], the present study failed to find any PAR2 staining in astrocytes in situ, although these cells showed avid trypsin expression. The reason why no PAR2-like immunoreactivity was detected is unknown. Perhaps these cells naturally express such low levels of this
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receptor in situ that they are undetectable using the immunohistochemistry technique utilised here. If astrocytes do have a low level of PAR2 expression, it was not affected by electrical amygdala kindling. This, together with the parallel and similar observation that the same kindling process also did not affect neuronal PAR2 expression, may be taken as evidence that PAR2 on neurons, astrocytes and perhaps other cell types as well, are only up-regulated by high, pathological or artificial (e.g. cell culture) levels of inflammation. Conversely if PAR2 are up-regulated during inflammation, then the electrical amygdala kindling model experiments that caused epileptic-like seizures described in this thesis may have only been associated with low levels of inflammation, insufficient to stimulate PAR2 up-regulation in both neurons and astrocytes. Perhaps these findings even bring into question the validity of experiments using either in vitro or in vivo assays showing PAR2 up-regulation under inflammatory conditions. These systems may stimulate artificial and/or pathological states and not pre-disease physiological or even pathophysiological states that can normally be resolved in vivo.
As discussed in the General Introduction (Chapter 1) of this thesis, epilepsy models, including the electrical kindling model, have been considered by some as inflammatory diseases in the brain due to the up-regulation of cytokines and proteases, both from internal (e.g. neurons and glia) and external (e.g. vascular) origins. Perhaps such an inflammation-driven up-regulation of PAR2 may be due to factors not induced by electrical kindling, but which are induced in other disease models, such as HIV-associated dementia [309]. Other markers of inflammation (apart from up-regulation of PARs) such as cytokines (i.e. IL1 and TNF) were not measured in the experiments described in this thesis, so it is difficult to characterise
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the inflammatory profile of electrical kindling. In addition, specifically what constitutes inflammation, particularly in the brain is not clear: - the scarring, neuronal dispersion and/or loss, the stellation of astrocytes, mossy fibre sprouting, excessive synaptogenesis and LTP may all be consequences of excessive, uncontrollable proteolysis caused by hyper-secretion of neurally derived proteases during epileptogenic stimuli such as electrical kindling, with or without the involvement of pro-inflammatory cytokines. In this context, it is worth reiterating that SLIGRL- and PAR2-mediated abolition of this excessive enzyme expression, and presumably release (with trypsin as a possible marker for other co-released enzymes like tPA), corresponded to a near-complete lack of progression of electrical kindling. That is, prevention of excessive release of neuromodulatory proteases prevents epilepsy in response to such epileptogenic stimuli. A corollary of this paradigm is that inhibition of normal neuromodulatory protease release prevents learning and memory in the short-term, perhaps also involving a lack of NMDA receptor potentiation, as well as synaptic remodelling.
It is possible that the novel effects of SLIGRIL demonstrated in this thesis could be independent of activation of central PAR2 per se. Whilst this is the assumed outcome, SLIGRL has been shown to activate mechanisms via receptors other than PAR2, such as the neurokinin (NK) receptors that cause airway epithelial cells to secrete chloride ion [266]. Furthermore, the full reverse peptide, LRGILS, used as a control for SLIGRL in this study may not necessarily rule out other receptors, since LRGILS was still active at the NK1-like receptor, although with approximately 3-10-fold less potency than SLIGRL [266]. However, as shown in Chapter 5, 10- and 20-fold greater concentrations of LRGILS (i.e. 1.5 and 3.0mg/kg) than the maximum-tolerated dose
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of SLIGRL (i.e. 0.15mg/kg) administered centrally had no neurotoxic effects unlike SLIGRL at the same doses. Therefore, the effects of SLIGRL observed on both epileptogenesis and learning and memory were unlikely to be mediated by an NK1like receptor [266], rather indicating a specific PAR2 effect.
The finding that peripherally administered SLIGRL was effective against both kindling and kainate-induced epileptogenesis, as well having effects on fear-aversive memory formation, supports the pharmacokinetic data (Chapter 4) that SLIGRL crosses from blood to brain as an intact and active molecule, which remains active in CSF (with a half-life of approximately 25min). In all experiments, both SLIGRL and LGRILS were carboxyl-terminally amidated, which may have made them less susceptible to cleavage by carboxypeptidases expressed by the brush border of the choroid plexus. Future experiments investigating central roles for PAR2 may need to utilise more stable, specific and potent PAR2 agonists other than SLIGRL, such as 2furoyl-LIGRL [266] or SLIGRLI-NH2 [270]. These novel peptides may have greater half-lives than SLIGRL and may also allow for more specific, longer-lasting and more robust PAR2 effects to be recorded, potentially even using lower doses than those used here. Such compounds may also limit any potential
neurotoxic/neurodegenerative and non-PAR2-mediated effects caused by the higher doses of SLIGRL, as discussed previously. The working hypothesis of the functions of PAR2 and trypsin (and most likely other proteases like tPA) in the brain developed from the results of this thesis is schematically shown in Figure 8.1. An explanation is as follows. Under normal circumstances in the hippocampus, when a stimulus reaches a pyramidal neuron, depolarisation induces exocytosis of trypsin, tPA, neuroserpin etc. from LDCVs into
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the extracellular matrix. These act as a multi-faceted neuromodulatory package, working in concert to precisely orchestrate plastic changes in the hippocampus that underlie cellular learning. This occurs first by matrix re-organisation, facilitating synaptogenesis and the formation of new cell-to-cell connections, and second, by facilitation of LTP by potentiated NMDA receptor signalling. Therefore, proteases provide for a plastic cellular environment that allows neurons to learn and effectively remember the stimulus. Hence, the hippocampus, with thousands of cells within their own proteolytically plastic micro-milieu, can store many working memories within the brain. PAR2 may monitor and precisely control their levels in the extracellular environment.
In the case of an inflammatory insult (Figure 8.1), proteases not only up-regulate in neurons and glia, but also normally blood-bound proteases infiltrate the brain matrix. These enzymes cause excessive damage to the cell matrix and neuronal damage or death, much as observed in epileptogenesis. Furthermore, they contribute to NMDAdriven neuronal depolarisations that facilitate paroxysmal depolarisation shifts, hyperexcitability and ultimately seizure. Therefore, proteases can contribute to
epileptogenesis when in excess, as already well documented. Under these conditions, PAR2 is rendered futile by excessive protease (trypsin) activity. Furthermore, PAR2 does not up-regulate and therefore the balance between PAR2 and trypsin (and other enzymes) may be disrupted. Pre-treatment with SLIGRL, however, confuses neurons that trypsin (and other enzyme) levels are high and that an inflammatory event is present in the matrix. This leads to the observed reduction in intracellular trypsin stores. When such an insult stimuli occurs in the brain (i.e. electrical kindling stimuli) the amount of proteases released is restricted and the potential for protease-driven,
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excessive plasticity is reduced. Hence, epileptogenesis is prevented, as observed in the current experiments.
Therefore, this early hypothesis presented here describes a new dogma relating to PAR2 and trypsin in hippocampal plasticity, both in health and disease; PAR2 is the gatekeeper for extracellular protease released from neurons, during both normal and inflammatory events.
As already mentioned, non-proteolytic PAR2 agonists like SLIGRL may be useful anti-epileptogenic, or neuroprotective drugs to trick the gate-keeper (PAR2) such that proteolytically-driven hyperexcitability and damage to the matrix are reduced following inflammatory trauma in the brain. For example, they may in the future be effective first-line neuroprotective agents in cases of head trauma to prevent epileptogenesis and possibly pathogenesis of other neurological conditions that involve inflammation, even though temporary deficits in learning and memory may occur. Presumably, however, when asked whether they would rather live with epilepsy for the rest of their lives, or lose the memories of the particular traumatic event, most patients would no doubt opt for the latter. Such a temporary memory loss, after all, may not be detrimental to the patients quality of life, but rather may even reduce the psychological trauma experienced by the patient. These results also provide a new concept of the involvement of PAR2 and trypsin in normal and pathogenic plasticity in the hippocampus, which may lead to further unravelling the exact mechanisms of hippocampal functioning.
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Figure 8.1 Hypothesised feedback inhibition of trypsin by PAR2
Figure 8.1 Hypothesised gate-keeper role for PAR2 in the brain. A; a normal pyramidal neuron; trypsin activates PAR2 to reduce production of trypsin and other mediators such as tPA, thus regulating the amount of such mediators available for plasticity. B; following an insult stimuli, excessive trypsin and other mediators are produced and released into the matrix leading to excessive synaptic reorganisation, excessive NMDA receptor (NMDAR) signalling leading to aberrant LTP, hyperexcitability, seizure and epilepsy. Under these conditions PAR2 feedback is ineffective due to excessive proteolysis. C; pre-treatment with SLIGRL artificially tricks cells that trypsin levels are high, thus, restoring the PAR2 feedback mechanism to prevent the up-regulation of trypsin during insult. Thus, excessive amounts of trypsin cannot be released following insult stimuli and the potential for protease-induced excessive synaptic-reorganisation, hyperexcitability and the risk of seizure and epilepsy are all reduced. In normal, non-insult situations, when PAR2 in activated with SLIGRL the extracellular trypsin concentration may be rendered too low, therefore normal physiological plasticity required for memory are also reduced, attenuating the ability to learn. Therefore PAR2 acts as a gate-keeper for neuronal protease expression.
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Future perspectives Perhaps the first new investigation to arise from these studies is to examine the intriguing possibility that trypsin and SLIGRL inhibit LTP via the described feedback-induced reduction in available neuronal trypsin. If this is the case, then the results of both the epilepsy and learning/memory studies may be readily explained, as both of these phenomena are protease and NMDA receptor-driven LTP-dependent. However, this appears paradoxical, since it is known that LTP, particularly LTP2, is dependant on IP3dependent increases in calcium [81], a well-documented response to PAR2 activation [285]. Thus, from a theoretical perspective, PAR2 activation would appear to facilitate LTP2. The demonstrated in vivo results of this thesis, however, describe the opposite, whereby an apparent reduction in LTP-like neuronal plasticity occurs in response to PAR2 activation. Thus, potentially by an as yet unrecognised secondary function, PAR2 activation may result in reduced LTP-like plasticity related to learning, memory and epileptogenesis. This may be related to the dose of SLIGRL used, with higher doses contributing to LTP-like depolarisations, which may account also for the neurotoxic behaviours observed in Chapter 5. By contrast, lower doses of SLIGRL may inhibit LTP, via an as yet unrecognised mechanism. These possibilities will be best investigated in electrophysiological LTP experiments, where the effect of agonists and antagonists of PAR2, NMDA and NK1/2 receptors on LTP can be directly tested.
In conjunction with the in vitro experiments on LTP, it will also be important to examine the effect of PAR2 activation on trypsin storage in pyramidal cells under culture conditions; that is, does it in fact reduce intracellular trypsin stores? As mentioned above, a common cellular response to SLIGRL is G-protein-mediated IP3
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calcium release which is a stimulus for vesicular exocytosis [133], so release of such a proposed neuromodulatory package is not out of the question. Furthermore, PAR2 activation of the MAPK pathway may modulate nuclear molecule trafficking, like that described for ERK1/2 [314] and as such, may also lead inhibition of trypsin production. These possibilities will be investigated in experiments utilising live cell, flow-through confocal microscopy to directly measure changes in trypsinimmunoreactivity in living cells in response to PAR2 activation. It will also be interesting to observe the effects of peripherally administered SLIGRL at various doses in models of epilepsy, learning and memory paradigms and other neurological diseases, to test whether doses higher than those given here have greater efficacies as that observed here. Furthermore, if compounds like SLIGRL are going to be of any clinical value it will be necessary to perform experiments where SLIGRL is administered post-insult to observe whether it has similar, if not the same effects as when given prophylactically. Other PAR2 agonists, such as 2-furoyl-LIGRL and SLIGRLI, as well as reliable PAR2 antagonists could also be used to firstly confirm the data presented here and secondly to indentify a more robust PAR2 agonist with a potentially greater clinical value. Non-peptidic PAR2 agonists which conform to Lipinskis rule of 5 of drug absorption [271] are also being developed, which may prove important tools for PAR2 research. The creation of a robust antagonist for PAR2 would also prove invaluable.
There are many models for neurological insult and trauma beyond those used here (electrical kindling and kainate) which are specific to epilepsy. It would be interesting to investigate the effects of PAR2 agonists on other neurological insult models involving inflammation, such as traumatic brain injury model (TBI), Alzheimers
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disease, multiple sclerosis, amyotrophic lateral sclerosis and stroke. Both TBI and stroke will be utilised in future studies to observe whether PAR2 agonists offer similar protection following acute brain injury. Furthermore, PAR2 antagonists may exacerbate such neurological diseases. Finally, PAR2 gene k/o mice may also prove invaluable, as they may be prone to disease states like epilepsy if the current hypothesis is correct, but possible compensatory changes in protease systems should first be investigated.
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Final Conclusion
The results from this thesis indicate that PAR2 is likely to be important in the regulation of plasticity within the hippocampus, apparently acting to modulate the extracellular actions of trypsin and other enzymes in the brain. Trypsin may therefore to be important not only in learning and memory, but also in neurodegenerative events in the brain during which it and similar proteases cause significant and excessive neuronal plasticity-induced neuronal damage that ultimately lead to the development of seizure and epilepsy. The synthetic PAR2 peptide agonist SLIGRL, which activates PAR2 in the absence of proteolytic activity, has significant plasticity-inhibiting effects in the rat, which not only induce temporary deficits in motivational learning, but also significantly attenuates the progression of epileptogenesis following both central and peripheral injection. The most likely explanation for these effects is a PAR2-mediated prevention of trypsin-over expression. Thus, PAR2 appears to act as a gate-keeper for extracellular proteases that includes trypsin to effectively control intraneuronal levels of their expression, particularly when extracellular levels are high. A possible important clinical outcome of this study is that non-proteolytic agonists of PAR2 may become useful anti-epileptogenic or neuroprotective agents to combat inflammatory insults to the brain which otherwise lead to chronic neurological diseases such as epilepsy.
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We are the music makers, and we are the dreamers of dreams
Willy Wonka, (Charlie and the Chocolate Factory, by Roald Dahl)
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PAR2-Gatekeepers For Proteases in The Brain, Thesis, RJ Lohman 2008 Uq2

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Protease-activated receptor 2; a gate-keeper for protease activity in the brain

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From little things, big things grow

PAR2 and trypsin in the brain

PAR2 and trypsin in the brain

PAR2 and trypsin in the brain

PAR2 and trypsin in the brain

PAR2 and trypsin in the brain

PAR2 and trypsin in the brain

PAR2 and trypsin in the brain

Posters and abstracts

PAR2 and trypsin in the brain

PAR2 and trypsin in the brain

PAR2 and trypsin in the brain

2 General Materials and Methods.........................................................49

PAR2 and trypsin in the brain

3 Immunohistochemical localisation of PAR2 and trypsin in the rat brain.........................................................................................................87

PAR2 and trypsin in the brain

4 The pharmacokinetics of SLIGRL in cerebrospinal fluid.............111

5 The behavioural neurotoxicity of centrally administered SLIGRL 130

PAR2 and trypsin in the brain

7 The involvement of PAR2 in fear-aversive learning......................174

PAR2 and trypsin in the brain

PAR2 and trypsin in the brain

Chapter 1 General Introduction

The burden of epilepsy

PAR2 and trypsin in the brain

Chapter 1 General Introduction

stigma of having a mental illness.

PAR2 and trypsin in the brain

Chapter 1 General Introduction

the most common form of acquired epilepsy.

PAR2 and trypsin in the brain

Chapter 1 General Introduction

Treatment of seizures in epileptic patients

PAR2 and trypsin in the brain

Chapter 1 General Introduction

Temporal lobe epileptogenesis

PAR2 and trypsin in the brain

Chapter 1 General Introduction

PAR2 and trypsin in the brain

Figure 1.1Regions of the human brain

Chapter 1 General Introduction

Figure 1.2 The human hippocampus

PAR2 and trypsin in the brain

Chapter 1 General Introduction

A cellular basis for memory; long-term potentiation

PAR2 and trypsin in the brain

Chapter 1 General Introduction

PAR2 and trypsin in the brain

Figure 1.3 Long-term potentiation

Chapter 1 General Introduction

Tissue plasminogen activator (tPA)

PAR2 and trypsin in the brain

Chapter 1 General Introduction

PAR2 and trypsin in the brain

Chapter 1 General Introduction

PAR2 and trypsin in the brain

Chapter 1 General Introduction

PAR2 and trypsin in the brain

Chapter 1 General Introduction

PAR2 and trypsin in the brain