Neuropharmacology 58 (2010) 722e729

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Neuropharmacology
journal homepage: www.elsevier.com/locate/neuropharm

Thyroid hormone prevents cognitive decit in a mouse model of Alzheimer's disease

Ai Ling Fu*, Cheng Yu Zhou, Xiang Chen
School of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China

a r t i c l e i n f o
Article history: Received 31 May 2009 Received in revised form 21 December 2009 Accepted 21 December 2009 Keywords: Thyroxine Alzheimer's disease Cognition Cholinergic function Free radical Neuronal apoptosis

a b s t r a c t
This study aimed to examine the feasibility of using thyroid hormone (TH) as a therapeutic agent for Alzheimer's disease (AD). Mice were injected intra-hippocampally aggregated amyloid b-peptide (Ab) to produce AD animal model. Intraperitoneal administration of L-thyroxine (L-T4) into Ab-induced AD model mice prevented their cognitive impairment and improved their memory function. The mechanisms of L-T4 treating AD might be associated with regulating cholinergic function, protecting the brains of AD model mice against damage from free radicals, and rescuing hippocampal neurons from apoptosis. The results of the present study indicate that the use of TH has some therapeutic potential in AD. 2009 Elsevier Ltd. All rights reserved.

1. Introduction Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive loss of memory and deterioration of cognitive functions. The neuropathological features of AD brains include neurobrillary tangles and senile plaques, the neurotoxicity of which is believed to be responsible for the neuronal loss in AD patients. Amyloid b-peptide (Ab) is the major component of the senile plaques, and the amount of Ab to form the plaques is correlative with the degree of neuronal damage and cognitive decits (Blennow et al., 2006; Mattson, 2004). Although AD has become the most common cause of dementia diagnosed after the age of 60 today, there is still now not fully elucidated and no efcient method for its treatment. Thyroid hormones (TH), including T3 and T4, are essential for development of mammalian brain and maintenance of optimal cognitive ability in different periods (Bgin et al., 2008; Zhang et al., 2009). In the study of TH in the brain, extraction and quantication of TH in selected regions of the rat brain have suggested that L-T4 can be uptaken by the brain (Pinna et al., 1999). Additionally, previous studies demonstrated that mouse organic anion transporting polypeptide 14 (mOatp14) involves in the uptake of T4 across the bloodebrain barrier (Tohyama et al., 2004), and T4 is

* Corresponding author. Tel.: 86 23 6825 0267. E-mail address: Fuailing1008@yahoo.com.cn (A.L. Fu). 0028-3908/$ e see front matter 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2009.12.020

found to be stereospecic, saturable, and energy dependent transport into a mouse neuroblastoma cell line (Lakshmanan et al., 1990). Furthermore, animal behavioral experiments suggested that thyroxine treatment reverses hypo-thyroidism-induced impairment of hippocampus-dependent cognition in thyroidectomized adult rats, (Alzoubi et al., 2009). In clinical practices, TH treatment can signicantly improve cognition and emotion in patients (Bauer et al., 2008; Bunevicius, 2009). These results imply that there may be a role for TH to increase the ability of learning and memory of AD. Although laboratory studies and clinical reports support a close link between TH and Alzheimer's pathophysiology (Ceresini et al., 2009; Rivas and Naranjo, 2007; van den Beld et al., 2005; Yoshimasu et al., 1991), studies examining the relationship between TH and AD have yielded contradictory results. Low TH level is associated with AD in some studies (Breteler et al., 1991; Ganguli et al., 1996), but not in others (Yoshimasu et al., 1991). Conversely, some studies show that hyper- rather than hypo-thyroidism is proposed as risk factors for AD (van Osch et al., 2004; Kalmijn et al., 2000). In the Rotterdam Scan Study, no signicant correlation is found between free T4 levels and global cognition in euthyroid patients with AD (de Jong et al., 2006). While examining the association between TH and AD and investigating the mechanism by which TH might inuence memory performance are important for applying TH to prevent AD, to date, there are few relative prospective studies. Therefore, in the present study, we used AD mouse model induced by aggregated Ab to explore the possibility that TH might be applied for therapy of AD.

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We found that TH prevented cognitive decit and improved the neurological function in AD model mice by regulating cholinergic function, inhibiting the damage of free radicals, and increasing the number of neurons in the brains of AD mice.
2. Materials and methods 2.1. Materials L-T4 and Ab (1e42) were purchased from Sigma. L-T4 was dissolved in sodium hydroxide, and hydrochloric acid was added to regulate the pH of the solution to 7.4. Ab (1e42) was dissolved in 0.01 M PBS (pH 7.4) at a concentration of 10 mM, sealed and incubated for 3 d at 37  C to form the aggregated Ab. All other reagents were of chemical grade. 2.2. Animals and housing conditions Healthy mice, Kunming species, male, weighing 25e30 g at the beginning of the experiments, provided by the Animal Breeding Center Afliated to Chongqing Medical University, China, were used throughout the study. Animals were housed under conditions of natural illumination with food and water available ad libitum. Animal experiments were performed in accordance with the Chinese Guides for the Care and Use of Laboratory Animals. 2.3. AD model mouse and L-T4 treatment The model of AD mice induced by Ab was made according to the previous reports (Stephan et al., 2001; Yan et al., 2001). Animals were anesthetized with sodium pentobarbital (40 mg/kg), and the injection of aggregated Ab was made bilaterally into the CA1 region of hippocampus (from Bregma 2.3 mm caudally, 2.1 mm laterally and from skull surface 1.8 mm ventrally) (Paxinos and Franklin, 2001) in sterotaxic apparatus using a 26-gauge needle connected to a 5 mL Hamilton microsyringe by polyethylene tubing. For each side of the hippocampus, a volume of 1.0 mL was administered over a period of 2 min (0.5 mL/min) followed by an additional 2 min waiting time before the injection needle was removed. The control animals were infused with PBS accordingly. After administration, the animal was returned to its home cage. The mice were determined as the AD model by behavior tests and immunostaining 7 d after injection of the aggregated Ab. For the AD model, mice were intraperitoneally injected with 2.5 mg/kg of L-T4 once a day for 4 consecutive days as described previously (Meaney et al., 1987; Smith et al., 2002). The control animals were injected with PBS in parallel. The detail is described in Fig. 1. 2.4. Water maze test The Morris water maze apparatus (Chengdu Technology & Market Co. LTD, China) was used to test the spatial learning and memory as previously described (Tsai et al., 2007). The water in the maze was opaque so that the platform, once submerged, was not visible. For navigation test, there were four trials per session and two sessions per day, with one session given in the morning and the other in the afternoon. For a complete test, a total of six sessions over 3 d were given. In each of the four trials, the animals were placed randomly at four different starting positions at the junction between two adjacent quadrants (the east, north, west or south poles of the maze). The animals were allowed 120 s to nd the platform. If an animal could not nd the platform in 120 s, it was guided to it. After mounting the platform, the animals were allowed to stay there for 30 s. The time that an individual mouse spent to reach the platform was recorded as the escape latency. Probe test was performed 24 h after the navigation test completed. The platform was removed from the pool, and the mice began from a unique starting location directly opposite the platform. During the probe trial, mice remained in the pool for the entire 90 s. All trials were recorded with a digital camera using the computer

software of Water Maze. During the navigation test, the escape latencies to platform of all trials were recorded. The spatial learning ability was evaluated by average escape latency to platform in every session. Additionally, time spent in the target quadrant was recorded during the probe trial. The ratio of time spent in the target quadrant in 90 s was used to evaluate the spatial memory ability. 2.5. Biochemistry assay Biochemical parameters were measured after learning and memory tests. The assay methods were described by previous report (Fu et al., 2006). Briey, mice were euthanized by decapitation, and the cortex and hippocampus were dissected and kept at 70  C ready for use. Ten percent (1:10, w/v) homogenate in cold saline were prepared (5000 rpm, 5 s for twice with 30 s interval) in ice bath. Superoxide dismutase (SOD) activity was assayed by the xanthine oxidase method (Sousa et al., 2008). Glutathione (GSH) was determined by the spectrophotometric method based on the use of Ellman's reagent (Beutler, 1979). Choline acetyltransferase (ChAT) activity was determined spectrophotometrically according to Wolfgram (1972), and acetylcholine (ACh) level was examined by using the method of Hestrin (Vincent et al., 1958). Catalase (CAT) activity was measured by the spectrophotometrical method (Johansson and Borg, 1988), and glutathione peroxidase (GSH-Px) activity was assayed according to the method of Beutler (1979). The content of ATP was measured by bioLuminescence method (Fukuda et al., 1983). Protein concentration was determined according to Lowry et al. (1951). All biochemical parameters were normalized to total homogenate protein. 2.6. Immunouorescence staining Immunouorescence staining was performed after animal behavioral tests. The mice were anesthetized and xed by intracardical perfusion of 4% paraformaldehyde in PBS (0.01 M, pH 7.4). After the brains were cryoprotected in sucrose at 4  C, the coronal sections were cut on a cryostat microtome. The slices of the hippocampus (30 mm; Bregma 1.6 to 2.8) were incubated in 10% normal goat serum diluted in PBS at 4  C overnight. For the Ab recognition, we used a mouse monoclonal antibody (Santa Cruz; 1:1000) and for neuron specic enolase (NSE) a rabbit polyclonal antiNSE (Santa Cruz; 1:1000). FITC-labeled goat anti-mouse IgG (Ab) and FITC-labeled goat anti-rabbit IgG (NSE; Beijing Boaoshen Biotechnology Company, China) were used as secondary antibodies. The PBS was used to wash the slices before each addition. The slices were air dried and placed on coverslips using a uorescent mounting medium. All Immunostaining sections were analyzed with a uorescence microscope (Olympus Optical Co., Ltd., Japan). The average number of positive cells from the sections of CA1 region of hippocampus (Bregma 1.6 to 2.8), located according to the atlas of Paxinos and Franklin (2001), was a calculated average of three sequential brain slices throughout the area of interest and measured both in the left and right hemisphere. The counting of positive cells was performed by an individual blind to the treatment conditions, using the same magnication and identical color scale setting as a correction for background staining. 2.7. TUNEL staining The mice were euthanized after behavioral tests. The brains were rapidly dissected, frozen and sectioned (30 mm) on a cryostat microtome, then the sections were processed for TUNEL staining (TUNEL uorescence FITC Kit, Nanjing Kaiji Biotech. Co. Ltd., China). The sections (30 mm thickness) were observed under a uorescence microscope and the TUNEL-positive cells in the CA1 region of hippocampus (Bregma 1.6 to 2.8) were counted. 2.8. Statistical analysis Data were showed as mean S.E.M. The data were analyzed with computer program by one-way analysis of variance (ANOVA), followed by Dunnett's Multiple Range Test, with SPSS 10.0 software. Differences with p < 0.05 were considered statistically signicant.

A injection

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3. Results 3.1. Preparation of AD model mice In this study, we set up the AD mouse model induced by aggregated Ab according to previous reported, and examined the model by immunouorescence staining and animal behavior tests. The results of immunostaining with anti-Ab showed that Ab aggregation was deposited in the cerebral cortex and hippocampus (Fig. 2A). In the animal behavior tests, the Ab-treated mice showed a longer swimming time in water maze performance as compared with PBS-treated mice (Fig. 2B). These results indicated that AD

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Fig. 1. Experimental design for TH treatment in aggregated Ab-induced AD mouse model.

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3.4. L-T4 inhibited the damage of free radicals To assess the effects of L-T4 on free radicals, we examined the activity of the important enzymes against oxidative stress, SOD, CAT, GSH-Px, and the level of GSH (the rst line of defense against oxidative stress in the form of reactive oxygen species) in the cortex and hippocampus of AD model mice. The results showed that the level of GSH and the activity of SOD, CAT and GSH-Px was lower in the AD mice as compared with the control, whereas L-T4 treatment increased the GSH level [F(5,4) 8.78, p < 0.05] and the activity of SOD [F(5,4) 6.39; p < 0.05], CAT [F(5,4) 6.81, p < 0.05], GSH-Px [F(5,4) 5.31, p < 0.05] in the brains of Ab-treated mice (Fig. 5). The results suggested that L-T4 could diminish the damage of free radical in neurons induced by Ab through increasing the activity of antioxidant enzymes. 3.5. L-T4 elevated ATP content in the cortex and hippocampus of AD model mice Total ATP content in the cortex and hippocampus of AD model mice was signicantly decreased as compared with that of the control mice (Fig. 6), while this reduction was reversed after L-T4 treatment [F(5,4) 13.7, p < 0.01]. This suggested that L-T4 could improve cerebral energy production in AD model mice caused by aggregated Ab. 3.6. The anti-apoptotic effects of L-T4 TUNEL staining was used for testing neuronal apoptosis in the CA1 region of the hippocampus, and NSE staining for determination neuronal population. The results showed that there were signicant TUNEL-positive cells in the AD model group (Fig. 7), and NSEpositive cells were markedly lower than that of control group (Fig. 8). However, the number of apoptotic cells and NSE-positive cells were close to that of the control group after L-T4 treatment. The results indicated that L-T4 could effectively inhibit neuron apoptosis and protect neurons against the damage caused by Ab. 4. Discussion 3.2. L-T4 improved learning and memory ability in AD model mice To evaluate whether L-T4 could prevent the neurological defects of the Ab-induced AD mice, the water maze task was used. In navigation test, the escape latency of L-T4-treated AD mice was signicantly less than that of the PBS-treated AD mice from the forth session (Fig. 3A), and there was no difference in swimming speed between the two groups (Fig. 3B), which indicated that the cognitive ability of mice was improved rather than motor function after L-T4 treatment. In probe test, the time spent in target quadrant of L-T4-treated AD mice was markedly longer than that of AD mice [F(9,11) 14.45, p < 0.01] (Fig. 3C). These data indicated that administration of L-T4 signicantly restored learning and memory function of mice with induced AD features. 3.3. L-T4 increased the ChAT activity and ACh level of AD model mice To identify the effects of L-T4 on cholinergic function, we analyzed the activity of ChAT (ACh biosynthetic enzyme and a marker for cholinergic neurons) and the level of ACh. The results were shown in Fig. 4. Aggregated Ab impaired the cholinergic neurons, and L-T4 treatment signicantly increased the activity of ChAT [F(4,5) 18.09, p < 0.01] and the level of ACh [F(4,5) 10.99, p < 0.01] as compared with the Ab-treated mice. The present study demonstrated that L-T4 treatment signicantly enhanced the ability of aggregated Ab-induced AD model mice to learn in a spatial learning and memory task. The mechanisms of L-T4 treating AD might be associated with regulating cholinergic function, protecting the neurons against the damage from free radicals, and preventing neuronal apoptosis in AD model mice. 4.1. L-T4 improved the neurological behavior of an induced AD mouse model The aggregated Ab-induced AD mouse model is a frequently used animal model for AD type amnesia and for identifying potential drugs which have an ability to treat AD (Maurice et al., 1996; Phinney et al., 2003). In this study, we used this animal model to explore the possibility that L-T4 might be applied for therapy of AD. There is a close correlation between aggregated Ab and the neurodegenerative process of AD. Ab is a 39e43 amino acid peptide that is formed by proteolytic processing of a much larger transmembrane protein, the amyloid precursor protein, by b- and g-secretase (Marks and Berg, 2008). Extracellular Ab peptides are highly cytotoxic to neuronal cells by activating a variety of cell signaling pathways. Incubation of Ab peptides in water for several days produces a conformational transformation from random coil to b-sheet coinciding with an increase in peptide

Fig. 2. Preparation of AD model mice by using aggregated Ab. (A) Representative photographs of Ab immunostaining in CA1 region of hippocampus of AD mouse brain 7 d after the Ab injection. Bar, 100 mm. (B) Morris water maze test was used to examine the impairment of learning and memory ability 7 d after Ab administration. The spatial learning ability was evaluated by escape latencies in six consecutive sessions of the test (n 10 for each group). **p < 0.01 compared to control group.

model mice had been successfully reproduced by intra-hippocampal injection of aggregated Ab.

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Fig. 3. L-T4 improved the spatial learning and memory in AD model mice. (A) L-T4 remedied the spatial learning disability of mice induced by Ab in navigation test (n 12 for PBS group, n 10 for Ab group, n 12 for Ab L-T4 group). **p < 0.01 compared to PBS control; #p < 0.05, ##p < 0.01 compared to Ab group. (B) The comparative of swimming speed among groups. (C) The time spent in target quadrant in probe test. **p < 0.01 compared to PBS control; ##p < 0.01 compared to Ab group. (D) The representative swimming route of animals in probe test.

neurotoxic potency. A single acute administration of aggregated Ab fragment in hippocampus or intracerebroventricle signicantly induces neuronal loss and amyloid deposits in brain and causes marked amnesic effects in mice, evidenced as deciencies in learning and memory (Yatin et al., 1999; Yamaguchi and Kawashima, 2001; Tsai et al., 2007). Ab-mediated neurotoxicity has been suggested through various pathways (Hardy and Selkoe, 2002), such as free radical damage, oxidative stress and mitochondrial dysfunction of neurons, and ultimately, apoptotic cell death was induced. In this study, Ab was incubated in 37  C for 3 days to form aggregated Ab, then the aggregated Ab was injected into the hippocampus of mouse brains. Another 7 d were allowed for the

injection to develop AD-like features according to previous reports. As indicated by immunouorescence staining with anti-Ab, which identied the locations of the Ab aggregation indeed formed in the hippocampus and cerebral cortex. Furthermore, behavior tests using the Morris water maze task showed that mice receiving Ab injection were impaired in their learning and memory abilities. However, after L-T4 administration, the neurological defects of the Ab-induced AD mice were prevented. The escape latency of L-T4-treated AD mice was signicant shorter than that of AD mice in navigation test. In probe test, the time spent in target quadrant of L-T4-treated AD mice was markedly longer than that of AD mice. These data implied that L-T4 might be used to prevent AD from the impairment of learning and memory function.

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Fig. 4. Inuence of L-T4 on ChAT activity and ACh level of AD model mice. The cortex and hippocampus of mice were dissected after learning and memory tests, and ChAT activity and ACh level were assayed respectively. Compared with Ab-induced AD mice, L-T4 treatment signicantly increased the activity of ChAT and the level of ACh. **p < 0.01 compared to PBS control; ##p < 0.01 compared to Ab group.

4.2. L-T4 enhanced cholinergic function in AD model mice The cholinergic system in the central nerve system (CNS) is also known to participate in various cognitive and memory functions. Specic lesions of these cholinergic neurons have been shown to interfere with the abilities of learning and memory. It has been well known that Ab can affect cholinergic neurons in vivo and in vitro (Kar et al., 2004; Watanabe et al., 2009). Several Ab fragments, including Ab (1e42), can inhibit ACh release from rat hippocampal slices. Moreover, intraventricular or hippocampal injection of Ab into the rat brain decreases ChAT activity and ACh release in the cortex and hippocampus and impairs memory (Fu et al., 2006; Maurice et al., 1996; Stephan et al., 2001; Tsai et al., 2007). In the present study, the cholinergic function, characterized by ChAT activity and ACh level, is impaired after injection Ab, which is consistent with previous studies. There exists a very close association between TH and cholinergic function. TH appears to support both the development and

maintenance of cholinergic function, especially in basal forebrain and hippocampus (Gould and Butcher, 1989; Rami et al., 1986; Smith et al., 2002). It has been well documented that the development of the cholinergic system has been markedly retarded and the ability of cognitive has been prevented following perinatal thyroid deciency (Ahmed et al., 2008; Smith et al., 2002). In our study, the ChAT activity and ACh level signicantly increased in the cortex and hippocampus of L-T4-treated AD model mice. Our data supported the view that TH enhanced cholinergic function, and that the augmentative effects on cognitive performance may be mediated through this increased cholinergic activity.

4.3. L-T4 prevented the brain against the damage of free radicals According to the oxidative-stress-hypothesis of AD, Ab inserts into the neuronal membrane bilayer and generates oxygendependent free radicals that then cause protein oxidation. Loss of

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Fig. 5. Effects of L-T4 on the activity of antioxidant enzymes SOD, CAT and GSH-Px and the level of GSH in AD model mice. The reduced SOD, CAT, GSH-Px activity (A, B, C) and GSH level (D) by aggregated Ab were recovered after L-T4 treatment. *p < 0.05, **p < 0.01 compared to PBS control; #p < 0.05 compared to Ab group.

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to have the ability of bilateral regulation and can keep a balance of oxidanteantioxidant status. 4.4. L-T4 restored ATP content in AD model mice

ATP (nmol/mgPr)

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Fig. 6. L-T4 restored ATP content in AD model mice. **p < 0.01 compared to PBS control; ##p < 0.01 compared to Ab group.

Several lines of evidence have suggested that mitochondrial dysfunction impacts on the pathogenesis of AD (Mancuso et al., 2009). Neuronal Ab accumulation may be an important factor in mitochondrial dysfunction. Loss of mitochondrial function leads to depletion of ATP reserve, and then neuronal functions are impaired in AD model mice. TH is considered a major regulator of mitochondrial activity. It's reported that TH induces mitochondrial biogenesis and enhances ATP generation within cells (Sterling et al., 1977; Menzies et al., 2009). Consistent with these reports, the ATP content in the cortex and hippocampus was restored in the AD model mice after L-T4 treatment. 4.5. L-T4 inhibited neuronal apoptosis in AD model mice

membrane integrity leads to cellular dysfunction. Neuronal death is the consequence of these cellular dysfunctions (Yatin et al., 1999). TH is also related with antioxidant defense systems. Hypothyroidism in both 1.5-month and 12-month old rat is accompanied by the oxidative stress in the brain (Ali and Davydov, 2007), and hyperthyroidism results in a marked increase in intracellular antioxidant enzymes i.e., catalase and GPx activities as compared to the control (Komosinka-Vassev et al., 2000). In the present study, the AD model mice administered 2.5 mg/kg TH can diminish the radical damage in neurons induced by Ab through increasing the level of GSH and the activity of antioxidant enzymes SOD, CAT and GSH-Px. The possible mechanism of action of TH on antioxidant enzymes is that TH might act on the transcriptional and translational processes of antioxidant enzymes biosynthesis (Oommen et al., 2006). However, some studies have reported that enhanced oxidative stress also existed in hyperthyroidism. Administration of L-T4 of dose 100 mg/kg of body mass to rabbits for 21 days which induces hyperthyroidism causes damage of the oxidanteantioxidant system (Kowalczyk et al., 2003). These results imply that TH seems

Apoptosis in vitro models and animal models of AD has been largely documented, and evidence for DNA fragmentation in tissue sections of brains from AD patients, using TUNEL, has been demonstrated by several groups (Shimohama, 2000). Stimuli for apoptosis in AD include increased oxidative stress, dysregulation of ion homeostasis, growth factor deprivation, accumulation of Ab, metabolic impairment, reduced clearance of toxin, mitochondrial dysfunction and DNA damage (Calissano et al., 2009; Wei et al., 2008). TH is an important factor for stimulating neuronal proliferation and survival (Darras et al., 2009). Rats with perinatal hypo-thyroidism shows a signicant increase number of apoptotic neurons in the hippocampus, which is closely related to the learning and memory decits (Huang et al., 2008). In our study, the neuron apoptosis was prevented and the number of hippocampal neurons was increased after L-T4 treatment. Our data here suggest the feasibility of using L-T4 as a therapeutic strategy for the treatment of AD. In human aging, TH is required for retaining optimal cognition, and dementia appears to

Fig. 7. L-T4 rescued the hippocampal neuron from apoptosis. (A) Representative photomicrographs of TUNEL staining. The arrow points to the TUNEL-positive cells. Bar, 100 mm. (B) Quantitative analyses of TUNEL-positive cells. The number of brains used for uorescence imaging were n 6 for PBS group, n 9 for Ab group, n 6 for Ab L-T4 group. **p < 0.01 compared to PBS control; ##p < 0.01 compared to Ab group.

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Fig. 8. L-T4 increased the number of hippocampal neurons. (A) Representative photomicrographs of NSE immunostaining. The arrows point to an area of the decrease in number of neurons. (B) Quantication of the changes of the number of NSE-positive cells in the granule cell layer of the CA1 region of hippocampus. The number of brains used for imaging were n 6 for PBS group, n 9 for Ab group, n 6 for Ab L-T4 group. **p < 0.01 compared to PBS control; ##p < 0.01 compared to Ab group.

be one of the dominant characteristic in the elderly hypothyroid patients (Loosen, 1992). Moreover, the incidence of hypo-thyroidism increases with age, and CNS-specic hypo-thyroidism has been reported in some patients with AD (Sampaolo et al., 2005). L-T4 treatment increases the free T4 in blood and brain (Kassem et al., 2006), and then improves the cognitive functions in animals. Therefore, administration low dose L-T4 would be a perspective way for AD treatment. References
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