Form B (per rule 8(a))

APPLICATION FOR PERMISSION FOR ANIMAL EXPERIMENTS

Application to be submitted to sent either to the CPCSEA (address in form A above) or Institutional Animal
Ethics Committee (IAEC).

Part A
*1. Name and address of establishment.
Smt. Kishoritai Bhoyar College of Pharmacy, Behind Railway Station,
New Kamptee, Nagpur. 411002.
*2. Registration number and date of registration.
853/AC/04/CPCSEA, Date- 16/12/2004.
3. Name, address and registration number of breeder from which animals acquired (or to be
acquired) for experiments mentioned in parts B & C
National Center for Laboratory Animal Sciences, National Institute of Nutrition,
Jamia, Osmania, P.O.Hyderabad 50007 (Reg. No: 154/99/CPCSEA).
4. Place where the animals are presently kept (or proposed to be kept).
Animal House of the Smt. Kishoritai Bhoyar College of Pharmacy,
Behind Railway Station, New Kamptee, Nagpur. 411002.
5. Place where the experiment is to be performed (Please provide CPCSEA Reg.Number).
Pharmacology Laboratory, Room No. 308 and 309, Smt. Kishoritai Bhoyar College of
Pharmacy, Behind Railway Station, New Kamptee, Nagpur. 411002. (Reg. No:
853/AC/04/CPCSEA)
6. Date on which the experiment is to commence and duration of experiment.
Date: - Sep. 2012 up to Mar. 2013 Duration: - 7 months
7. Type of research involved (Basic Research/Educational/Regulatory).
Educational Research leading to M.Pharmacy.


Signature
Name and designation of Investigator
Mayur B. Kale
B.Pharm
Date:
Place: Kamptee.
*Applicable only for application to be submitted to CPCSEA
PART B
Protocol form for research proposals to be submitted to the committee / Institutional Animal Ethics
Committee, for new experiments or extensions of ongoing experiments using animals other than non-
human primates.

1. Project / Dissertation / Thesis Title:
Assessment of Role of Agmatine in Diabetes Induced Depression in Rats

2. Principal Investigator / Research Scholar / Research Guide / Advisor:
(i) Research Guide
a. Name: Mr.N.R.Kotagale
b. Designation: Professor in Pharmacology
c. Dept / Div/ Lab: Department of Pharmacology, Post Graduation Laboratory,
Smt. Kishoritai Bhoyar College of Pharmacy, Behind Railway
Station, New Kamptee, Nagpur-441002
d. Telephone No. : 07109-288650
e. Experience: 10 years
(ii) Research Scholar
a. Name: Mayur B. Kale
b. Designation: Student in M.Pharm Pharmacology
c. Dept/Div/Lab: Department of Pharmacology, Post Graduation Laboratory,
Smt. Kishoritai Bhoyar College of Pharmacy, Behind Railway
Station, New Kamptee, Nagpur 441002
d. Telephone No.: 07109-288650
e. Experience :

3. Funding source with complete address (Please attach the proof).
Smt. Kishoritai Bhoyar College of Pharmacy, Behind Railway Station, New Kamptee,
Nagpur- 441002.
4. Duration of the project.
a. Number of months: 6 months.
b. Date of initiation: October 2012
c. Date of completion: March 2013

5. Detailed study plan may be given (Not more than one page)

Objective:
To evaluate the effects of agmatine in diabetes induced depression

Plan of Work:
Sprague Dawley rats will be divided in 6 groups each containing 4 animals.
Sucrose preference test will be carried out prior to the administration of high fat diet.
Diabetes will be induced in the animals by high fat diet and co-administration of alloxan
intra peritonially.
Assessment T2DM will be done by estimation of plasma glucose (PGL), triglycerides
(PTG), total cholesterol (PTC) levels and then the animals with glucose level above 150
mg/kg will be further considered for the experiment.
Depression will be evaluated by observing their activity in sucrose preference test before
and after the administration of agmatine.

Following doses will be given to study the antidepressant activity in the above
mentioned model.
Effect of agmatine on the rats.
Combination of agmatine and I
1
agonist.
Combination of agmatine and I
2
agonist.
Combination of agmatine and I
1
antagonist.
Combination of agmatine and I
2
antagonist.

During the protocol, the blood sample will be collected from each group and will be subjected
to the measurement of blood glucose, triglyceride and cholesterol levels by UV technique.

Experimental Design:

Animal handling
Before starting this protocol, the rats will be housed in a temperature-controlled room with
free access to food and water, were maintained four per cage in a temperature (252
0
C) relative humidity
(5070%) and maintained on a 12:12-h light/dark cycle (lights on 07:0019:00 h) and were submitted to
daily handling for at least 3 days. The weight of the animals at the beginning of the experiments will be
recorded. Each rat will be weighed daily during the protocol and the following recovery period to verify
the influence of Type2 diabetes/or drug administration on weight gain/loss.

1. Estimation of blood glucose, choleserol and triglyceride level;
Blood glucose will be measured by using Glucometer (One Touch Hori-zon, Singapore). Serum
samples will be analyzed for estimation of triglyceride and cholersterol using an auto blood analyzer.
Baseline Sucrose Preference test
The sucrose preference test is a two-bottle choice paradigm. This test has been used
extensively in evaluating stress-induced anhedonia. Mice will be habituated to drink sucrose for five days
and then will be exposed to two bottles (water and 1% sucrose solution) for two days. All the animals will
be simultaneously exposed to CUMS. The amount of water and sucrose solution will be measured at 8:00
and 17:00. The preference for sucrose over water will be used as a measure of response to a naturally
rewarding stimulus. The sum of water and sucrose intake will be defined as total intake and the sucrose
preference will be expressed as the percentage of sucrose intake from the total intake by following
formula:
% sucrose preference = sucrose intake 100/ total intake

Behavioural and biochemical measurements
(As per mentioned above.)

Treatment Groups:
Sr.
No.
Group Dose/Treatment Activity to be assessed/
Biochemical
Parameters
No. of animals
1. Group A Saline( only Handaling) Assessment of
blood glucose,
triglyceride and
cholesterol levels.

Sucrose preference
test.(Anhedonia)
6 1 = 6

High Fat Diet and Alloxan
(100mg/kg)i.p.to each animal on 14
th

day of protocol
6 1 = 6

2. Group B Agmatine (10mg/kg) i.p. for 21 days
throughout the protocol.
6 1 = 6

Agmatine (20mg/kg) i.p. for 21 days
throughout the protocol.

6 1 = 6
Agmatine (40mg/kg) i.p. for 21 days
throughout the protocol.

6 1 = 6
3. Group C Agmatine + Moxonidine(I1 Agonist)
i.p. for 21 days throughout the
protocol.
Assessment of
blood glucose,
triglyceride and
cholesterol levels.

Sucrose preference
test.(Anhedonia)
6 1 = 6
Agmatine + 2-BFI(I2 Agonist)
i.p. for 21 days throughout the
protocol.
6 1 = 6
4. Group D Agmatine + Efaroxan(I
1
antagonist)
i.p. for 21 days throughout the protocol.
6 1 = 6
6 1 = 6
Agmatine+BU224(I
2
antagonist)i.p.
for 21days throughout the protocol

Total Animals Required 54

Sucrose preference test
Sucrose preference test will be performed once in a week.. After overnight water deprivation,
rats will be allowed to access water and sucrose 1% solution according to two-bottle paradigm for 1 hour
and sucrose preference will be measured as indication of anhedonia.


6. Animals required:
a. Species/Common name: Sprague Dawley Rats
b. Age / weight / size: Adult Rat (200-222 g)
c. Gender: Either sex
d. Number to be used (Year-wise breakups and total figures needed to be given): 54
e. Number of days each animal will be used: 21
f. Proposed source of animals: National Center for Laboratory Animal Sciences,
National Institute of Nutrition, Jamia, Osmania,
P.O. Hyderabad 50007 (Reg. No: 154/99/CPCSEA).
7. Rationale for animal usage:
a. Why is animal usage necessary for these studies?
These are preclinical studies that can not be undertaken in human beings at this stage. Hence, it is
necessary to undertake this study on animals.

b. Why are the particular species selected required?
1. Rats are found suitable since these are among the standard laboratory animals that offers
following advantages:
a) Ease of handling
b) Low cost
c) Ease of maintenance
2. Global investigators use rats as standard animals for preclinical research.

c. Why is the estimated number of animals essential?
Proposed protocols and groups required demand the given number of animals and
to generate the reproducible data and statistical interpretation.

d. Are similar experiments conducted in the past? If so, the number of animals used and
results obtained in brief No.

e. If yes, why new experiment is required? Not Applicable.

f. Have similar experiments been made by any other organization agency? If so, their results in
your knowledge.
As per the best of my knowledge, no other organization or agency perform such kind of
experiment using these drugs.

Furnish details of injections schedule
Substances : Drugs (Agmatine), Moxonidine, 2-BFI, Ifaraxon, BU224,
Doses : Agmatine Sulphate (10, 20, 40 mg/kg, i.p.), Moxonidine(0.5mg/kg),
2-BFI(10mg/kg), Ifaraxon(1mg/kg), BU224(10mg/kg),
Alloxan(10mg/kg, i.p.)
Sites : Intra-peritoneal
Volumes : 0.3 to 0.5 ml/Rat

Blood Withdrawal
Volume : 2-3 ml/rat
Sites : Tail-vein
Radiation (dosage and schedules): NO

8. Please provide brief descriptions of similar studies from in vitro / in vivo (from other animal
Models) on same/similar test component or line of research. If, enough information is available, justify
the proposed reasons.
Not applicable.

9. Does the protocol prohibit use of anaesthetic or analgesic for the conduct of painful procedures
(any which cause more pain than that associated with routine injection or blood withdrawal)?
No.
If Yes, explanation and justification
Not applicable.
10. Will survival surgery be done?
No
a. List and description of all such surgical procedures (including methods of asepsis)
b. Names, qualifications and experience levels of operators
Name:
Qualification:
Experience:
c. Description of post-operative care
d. Justification if major survival surgery is to be performed more than once on a single individual
animal.
Not applicable.

11. Methods of disposal post-experimentation
(Euthanasia (Specific method)/ Method of carcass disposal/ Rehabilitation)
The animals after experiments will be disposed in such a way that it will not cause any microbial
contamination to the environment. The dead animals will be incinerated in a closed container in
specifically designated area. Carcass will be disposed off as per the biological waste disposal
requirements.

12. Animals transportation methods:
Any extra institutional transport in envisaged the animals will be transported from Hyderabad to the
animal house of Smt. Kishoritai Bhoyar College of Pharmacy, Kamptee by air-conditioned van.

13. Use of hazardous agents (use of recombinant DNA-based agents or potential human pathogens
requires documented approval of the Institutional Bio safety Committee (IBC). For each categor0y, the
agents and the bio safety level required, appropriate therapeutic measures and the mode of disposal of
contaminated food, animal wastes and carcasses must be identified)
a. Radionuclide NA
b Microorganisms / Biological infectious Agents NA
c. Hazardous chemical or drug NA
d. Recombinant DNA NA
e. Any Other (give name) NA
If, your project involved use of any of the above, attach copy of the minutes of IBC granting
approval. NA.




























Investigator's declaration.

1. I certify that I have determined that the research proposal herein is not unnecessarily duplicative
of previously reported research.

2. I certify that, I am qualified and have experience in the experimentation on animals.

3. For procedures listed under item 11, I certify that I have reviewed the pertinent scientific
literature and have found no valid alternative to any procedure described herein which may
cause less pain or distress.

4. I will obtain approval from the IAEC/CPCSEA before initiating any significant changes in this
study.

5. Certified that performance of experiment will be initiated only upon review and approval of
scientific intent by appropriate expert body (Institutional Scientific Advisory Committee/
funding agency/ other body (to be named).

6. Institutional Bio safety Committee's (IBC) certification of review and concurrence will be taken
(Required for studies utilizing DNA agents of human pathogens).

7. I shall maintain all the records as per format (Form D)

8. I certify that, I will not initiate the study unless approval from CPCSEA received in wiring.
Further, I certify that I will follow the recommendations of CPCSEA.

9. I certify that I will ensure the rehabilitation policies are adopted.


Supervisors Investigator

Dr. C. T. Chopde Shweta T.Bhattacharya
M. Pharm., Ph. D., FIC

Date:




CERTIFICATE


This is certify that the project title ASSESSMENT OF ROLE OF AGMATINE IN DIABETES
INDUCED DEPRESSION IN RATS has been approved by the IAEC.





Name of Chairman/Member Secretary IAEC: Name of CPCSEA nominee:


Dr. R. R. Ugale Dr. Kailash Marwah

Signature with date


Chairman/Member Secretary of IAEC: CPCSEA nominee:




No. of Animals to be used: 54
No. of Animals to be rehabilitated: 54