Hormones and Behavior 65 (2014) 505–515

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Dihydrotestosterone treatment delays the conversion from mild cognitive impairment to Alzheimer's disease in SAMP8 mice Lin Kang a,1, Sha Li a,1, Zhaoguo Xing a, Jianzhong Li a, Yuhong Su a, Ping Fan a, Lei Wang a, Huixian Cui a,b,⁎ a b

Department of Human Anatomy, Hebei Medical University, Hebei, PR China Hebei Key Laboratory for Brain Aging and Cognitive Neuroscience, Hebei, PR China

a r t i c l e

i n f o

Article history: Received 10 December 2013 Revised 7 March 2014 Accepted 31 March 2014 Available online 6 April 2014 Keywords: Mild cognitive impairment Alzheimer's disease SAMP8 Testosterone Dihydrotestosterone

a b s t r a c t The senescence-accelerated-prone mouse 8 (SAMP8) has been proposed as a suitable, naturally derived animal model for Alzheimer's disease (AD). In the current study, we focus on the problem whether SAMP8 mice show abnormal behavioral and neuropathological signs before they present the characteristic of AD. Our results demonstrated that given the presence of the senescent, behavioral and neuropathological characteristics, the “middle-aged” SAMP8 mice appear to be a suitable and naturally derived animal model for MCI basic research. There is relatively less evidence that androgen may be involved in the pathogenesis of AD. We determined testosterone (T) levels of SAMR1 and SAMP8 mice and found that the marked age-related decrease in serum androgen levels may be one of the risk factors for Alzheimer's dementia. We also evaluated the interventional effect on MCI phase by dihydrotestosterone (DHT) in male SAMP8 mice and found that timely and appropriate androgen intervention can postpone the onset and improve the symptoms of dementia. © 2014 Elsevier Inc. All rights reserved.

Introduction Mild cognitive impairment (MCI) as a descriptive term, referred to as stage 3 of the Global Deterioration Scale, was introduced into the literature in 1988 by Reisberg et al. (1988). MCI is currently described as a transitional condition with cognitive changes between normal aging and Alzheimer's dementia (DeCarli, 2003; Petersen et al., 1999, 2007). Alzheimer's disease (AD) is the most common type of dementia in the modern aging society (Alzheimer's Association, 2012; Schott et al., 2006; Yaari and Corey-Bloom, 2007). New diagnostic criteria and guidelines for AD were proposed and published by the National Institute on Aging (NIA) and the Alzheimer's Association (AA) in 2011 (Albert et al., 2011; Jack et al., 2011; McKhann et al., 2011; Sperling et al., 2011). They recommend that AD is considered a disease with three stages: (1) preclinical AD, (2) MCI due to AD (all “MCI” that are subsequently mentioned refer to this stage), and (3) dementia due to AD. This reflects the current thinking that AD causes distinct and measurable changes in the brains of affected people years, perhaps decades, before memory and cognitive symptoms are noticeable. MCI is used to refer to the symptomatic pre-dementia phase of AD and can thus be regarded as a condition with a high risk of progression to dementia due to AD. However, the current challenge for AD is that there

⁎ Corresponding author at: Department of Human Anatomy, Hebei Medical University, 361 Zhongshan East Road, Shijiazhuang, Hebei, 050017, PR China. Fax: +86 311 8626 6615. E-mail address: [email protected] (H. Cui). 1 These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.yhbeh.2014.03.017 0018-506X/© 2014 Elsevier Inc. All rights reserved.

is no single, generally accepted way to identify the disease in the earliest presymptomatic stage. The assessment of the conversion from MCI to AD may lead to preventive strategies by controlling risk factors that can prevent or delay the onset of disease. In the basic studies of MCI due to AD, appropriate animal models are necessary in order to understand the pathogenic mechanisms and to test the new therapeutic strategies prior to clinical trials. The senescence-accelerated mouse (SAM) has been established as an inbred mouse model of accelerated aging by Takeda et al. (1981, 1991). According to aging rate and age-associated pathologic phenotypes, the SAM model includes strains of senescence-accelerated-prone mouse (SAMP) and strains of senescence-accelerated-resistant mouse (SAMR). Among the SAMP strains, SAMP8 mice are characterized by an agerelated spontaneous deterioration in learning and memory abilities. This deterioration often accompanies changes in cortical and hippocampal gene expression and various other pathological features that are similar to AD (Butterfield and Poon, 2005; Del Valle et al., 2010; Nomura and Ohkuma, 1999). Nevertheless, even if SAMP8 is a reasonable and naturally derived animal model for investigating the fundamental mechanisms of AD, we focus on the problem whether SAMP8 mice show abnormal behavioral, neuropathological and neurochemistry signs before they present the characteristic of AD, which brings this model to the assessment of incipient AD in MCI subjects. It is widely accepted that changes in the levels of gonadal hormones over the course of life contribute not only to variations in cognitive function (Jonasson, 2005; Linn and Petersen, 1985; Voyer et al., 1995) but also to the incidence of certain types of neurodegenerative disorders. While relatively less attention has been devoted to the effects of

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androgens compared to estrogens, androgens also clearly have positive effects on cognitive performance and may contribute to a protective role against AD (Cherrier et al., 2005b; Rosario and Pike, 2008; Yao et al., 2008). Scholars from different scientific research organizations proposed that an age-related decrease in serum androgen levels has been associated with an increased risk of MCI and the development of AD. Older men with low serum levels of circulating testosterone (T) appear to be at higher risk of developing AD than men with normal levels of this hormone (Moffat et al., 2004). However, the mechanisms responsible for the androgen-induced influence on AD remain poorly understood. If we could determine the risk for developing Alzheimer's dementia in healthy or MCI individuals, we can identify a crucial window of opportunity to intervene with disease-modifying therapy. In addition, these data aid the selection of appropriate populations for the treatment of presymptomatic/preclinical AD. The aim of present research is to extend the utilization of SAMP8 mice and characterize the conversion from presymptomatic stage to obvious cognitive impairment and neuropathological stage in this strain. It also remains unclear whether timely and appropriate androgen intervention can postpone the appearance of Alzheimer's dementia. To test this hypothesis, we evaluate the interventional effect on the MCI phase using DHT in male SAMP8 mice, which may be a treatment option for MCI in the future. Materials and methods Animals Experiments were carried out in male SAMP8 and SAMR1 mice, obtained from our breeding colonies and maintained as an inbred strain and originally provided by Prof. Yew David of The Chinese University of Hong Kong. All of the mice received a standard rodent diet and water ad libitum and were housed under a 12-h light-dark cycle (lights at 6:00 AM) at a room temperature of 21 ± 2 °C. All of the experimental procedures were performed in compliance with the Guidelines for the Care and Use of Mammals in Neuroscience Research and approved by the Committee of Ethics on Animal Experiments at Hebei Medical University. Assessment of the conversion from MCI to Alzheimer's dementia in SAMP8 mice This study consisted of three sub-experiments designed to assess the conversion from MCI to Alzheimer's dementia in SAMP8 mice (Fig. 1 provides the experimental procedure). In this study, SAMP8 and SAMR1 mice (n = 8 animals per strain) were examined for 5 months, from 3-month-old to 7-month-old, to characterize the senescence grading scores and mild cognitive impairment. In addition, SAMP8 and SAMR1 mice from each age group (5 groups in all from 3-month-old to 7-month-old,n = 16 animals per group) were used to assess the

mild changes in pathology, including the loss of neurons, decrease in dendritic spine density and increase in Aβ depositions. The grading score The items to be examined in the grading score system include 11 categories selected from the clinical signs and gross lesions considered to be associated with the aging process. The 11 categories include reactivity, passivity, glossiness, coarseness, hair loss, ulcer, periophthalmic lesions, cataract, corneal ulcer, corneal opacity and lordokyphosis. In general, each category has five grades corresponding to the intensity of the changes. Grade 0 represents no particular changes and grade 4 represents the most severe changes. Each grade in each category is clearly defined (Hosokawa et al., 1984; Takeda et al., 1981). Mild cognitive impairment The Morris water maze (MWM), a behavioral test in which mild cognitive impairments can be detected early on, was utilized to confirm SAMP8 mice as a reasonable model of MCI due to AD. The water maze was a black circular pool (120 cm in diameter and 60 cm in height) filled with opaque water (21 ± 1 °C) by the addition of white, nontoxic liquid washable paint. The maze was located in a room containing many stable external cues and divided geographically into four quadrants. A hidden circular platform (10 cm in diameter) was located in the center of the target quadrant, submerged 1 cm below the surface of the water. A video camera was mounted above the center of the water maze and was linked to a computer to record and store the swimming path data. The test contains two tests: a spatial reference memory (hidden platform) test and a spatial probe test. Before hidden platform training, the mice were familiarized with the task (e.g., locate and climb onto the escape platform), and any non-cognitive impairments (e.g., visual impairments and swimming difficulties) that may impact performance on the task were ruled out. After familiarization, the mice were trained for 5 days to locate the hidden platform by learning multiple spatial relationships. Each animal was allowed to swim until they found the hidden platform for a maximum duration of 120 s. If the mouse failed to locate the platform, it would be gently guided to sit on the platform for a maximum duration of 30 s. The mice were also left to sit on the platform for a maximum of 10 s on successful trial. A tracking system was used to measure the escape latency. After the mice completed the trials, a spatial probe test was performed. In this test, the platform was removed from the pool to check the memory of the animal for the platform location. The mice were reintroduced to the pool and allowed to swim for 120 s. Suspended above the pool was a video camera connected to a video tracking system (WinFast PVR, Jiliang, China). By videotaping the swim paths, the number of platform crossings in the probe trial and the percent time spent in the target quadrant were calculated. Mild pathologic morphology

Fig. 1. The experimental procedure for the assessment of the conversion from MCI to Alzheimer's dementia.

Groups of mice (n = 8) were deeply anesthetized with 6% chloral hydrate (5 mg/kg, i.p. injection) and perfused with 0.9% physiological saline through the left ventricle, followed by a fixative containing 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4. The brains were carefully removed, and tissue samples were obtained from the superior colliculus to the optic chiasma. Samples were postfixed overnight in the same fixative at 4 °C. Next, the tissue block was divided into two halves along the median plane, dehydrated in a graded series of ethanol, cleared in xylene and embedded in paraffin. The paraffin blocks were cut serially on a sliding microtome (Leica-RM2145, Germany) into 5 μm thick coronal sections. Two successive sections were collected every 50 μm and mounted onto polylysine-coated slides for Nissl staining and immunohistochemical staining. (1) Nissl staining. After being deparaffinized and dehydrated, the sections were soaked with 1% cresyl

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for 30 min at 60 °C and rinsed in distilled water. The sections were then differentiated in 90% ethanol, dehydrated with 100% ethanol, cleared in xylene, and coverslipped using a resinous mounting medium. Nisslpositive neuronal cell counts within the hippocampal CA1 region were manually performed without knowledge of the experimental conditions. (2) Immunohistochemical staining. After being deparaffinized and dehydrated, the sections were subjected to antigen retrieval using a microwave for 30 min. The sections were immersed in 3% hydrogen peroxide in methanol for 30 min to abolish endogenous peroxidase activity. Then the sections were incubated with 5% normal goat serum to block nonspecific binding, followed by an overnight incubation with rabbit anti-β-amyloid (Aβ) antibody (1:100, product code: bs0107R, Biosynthesis Biotechnology Corp., Beijing, China) at 4 °C. After washing, the sections were incubated with biotinylated goat antirabbit IgG for 1 h. The computer image analysis system Image-Pro Plus 6.0 was used to determine the average values of the optical density (OD) of Aβ in the hippocampal CA1 region. (3) Golgi staining. Groups of mice (n = 8) were deeply anesthetized with 6% chloral hydrate and rapidly decapitated. The brains were removed, and tissue samples were immediately dissected from the superior colliculus to the optic chiasma. The tissue blocks were processed for Golgi–Cox staining using a Rapid Golgi Stain Kit (FD Neuro-Technologies, Inc., USA). Golgi staining was carried out as previously reported (Li et al., 2013). Secondary and tertiary apical dendrites were selected for quantitative analysis in which 10 μm segments were magnified 1000 × in the digitized images. The dendritic spine density values were expressed as the number of thorns/10 μm of dendrite.

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presented in the results. For experiments grading score, repeated measure analysis of variance (ANOVA) was used to analyze the main effects of age (3, 4, 5, 6 and 7 months) and strain (SAMR1 and SAMP8) on the grading score. For experiments hidden platform test, analysis of repeated measure ANOVA was used to analyze the main effects of age and day (1, 2, 3, 4 and 5 day) on the escape latency. For experiments spatial probe test, pathologic morphology and serum T levels, a two-way ANOVA (2wANOVA) was used to analyze the main effects of age and strain on the conversion from MCI to Alzheimer's dementia and serum T levels. A “single-strain” ANOVA (ssANOVA) and multiple comparisons were performed for significant differences using Fisher's least significant difference (LSD) post-hoc test (experiment mild cognitive impairment and mild pathologic morphology) and Student–Newman–Keuls (SNK)q post-hoc test (experiment serum T levels) to reveal a significant main effect of age. Similarly, a “single-age” ANOVA (saANOVA) was used to reveal the main effect of strain. Eta-squared (η2) was used for standardized effect-size estimates. For experiment effects of castration and DHT intervention, analysis of repeated measure ANOVA was used to analyze the main effects of group and day (1, 2, 3, 4 and 5 day) on the escape latency in SAMP8 mice. We performed the other results for normality (Kolmogorov–Smirnov test) and equal variance (Levene's test) for all of the data. If both normal distribution and homogeneity of variance were found, then comparisons were analyzed using one-way ANOVA, and Cohen's d test were used for pair-wise comparisons. All of the statistical analyses were performed using SPSS 13.0 (SPSS Inc., Chicago, IL, USA). The results were expressed as the mean ± standard deviation (SD). The criterion for statistical significance was set at P b 0.05.

Assessment of strain and age-related differences in serum T levels Results SAMP8 and SAMR1 mice (5 groups/strain from 3-month-old to 7-month-old, n = 8 animals per group) were used to assess the strain- and age-related differences in serum T. Animals were anesthetized with 10% chloral hydrate, and 0.5 ml of blood was drawn from the tail vein into a heparinized syringe. The sample was transferred to a clean microcentrifuge tube on ice and centrifuged at 5000 × g for 20 min at 4 °C. The supernatant was then transferred to a clean microcentrifuge tube and stored at −20 °C until assayed. Serum T levels were assessed using a competitive binding radioimmunoassay kit (Jiudin Corp., Tianjin, China) as directed by the manufacturer. This kit had an analytical sensitivity of 0.02 ng/ml. Effects of castration and DHT intervention on the conversion from MCI to Alzheimer's dementia in SAMP8 mice Male 5-month-old SAMP8 mice (n = 24) were randomly divided into a sham-operated control group (P8-sham group, n = 8) and a castration group (Cast group, n = 8) and castration plus DHT group (DHT group, n = 8). Bilateral testes were removed from the Cast and DHT groups at 5 months old. Three days after castration, the DHT group was administered standard dose of DHT (product code: 2500981, International Laboratory, CA, USA) for 2 months by subcutaneous injection (1 mg/kg per day administered between 5:00 PM and 6:00 PM). Other groups were injected aseptically with equal doses of medical maize oil. Groups of mice at 7 months of age were used to determine the senescence grading scores, cognitive impairment and pathologic morphology. All of the referenced indicators were examined as described in the above experimental procedures. Statistical analysis For the Nissl and immunohistochemical staining, 10 sections were analyzed for each mouse and the average number of pyramidal cells and OD value were presented for each mouse in the results. For the Golgi staining, 9 randomly chosen, representative neurons from 3 sections were analyzed and the average value for each animal was

The grading score For the mice tested at all five time points (3, 4, 5, 6 and 7 months), the total score began to increase at 3 months of age and continuously increased with advancing age in both SAMR1 and SAMP8 mice. Analysis of repeated measure indicated that both the age [F(4,11) = 1155.37, P b 0.001; η2 = 0.08] and the strain [F(1,14) = 1120.81, P b 0.001; η2 = 0.15] significantly affected the senescence grading scores. The interaction of age × strain also had a significant effect on the score of the SAM mice (P b 0.001). The total score of the SAMP8 mice was clearly higher than that of the age-matched SAMR1 mice. The SAMR1 mice showed a normal aging pattern and only a slight increase in the total senescence score was observed from 3-month-old to 7-month-old. However, SAMP8 mice showed remarkable age-associated increases in the senescence grading scores. The post hoc analysis indicated that age significantly impacted on the total score (4 months vs. 5 months, P b 0.001; 5 months vs. 6 months, P b 0.001; 6 months vs. 7 months, P b 0.001) (Fig. 2). Mild cognitive impairment All of the SAMR1 and SAMP8 mice tested at all five time points (3, 4, 5, 6 and 7 months) showed decreases in escape latency over the 5 days of the spatial reference memory (hidden platform) trials in the MWM, indicating that the mice learned the location of the platform. The analysis indicated that both the age [F(4,75) = 875.81, P b 0.001; η2 = 0.09] and the strain [F(1,78) = 641.45, P b 0.001; η2 = 0.18] significantly affected the performance of the SAM mice. The interaction of age × strain also had a significant effect on the performance of the SAM mice (P b 0.001) (Fig. 3A, B). We observed that the SAMR1 groups exhibited linear improvement in performance across training, thus demonstrating good spatial memory (Fig. 3A). In contrast, the SAMP8 groups were slower in their rates of improvement, indicating an age-associated cognitive deficit. The severity of the impairment gradually increased with age. In the SAMP8 mice, post hoc analysis

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Fig. 2. Assessment of the conversion from MCI to Alzheimer's dementia through senescence grading scores. Both age and strain had significant effect on the performance. The grading score of the SAMP8 mice was clearly higher than that of the age-matched SAMR1 mice. The SAMR1 mice showed a slight increase in the total senescence score. However, SAMP8 mice showed remarkable age-associated increases in the senescence grading scores. The results were expressed as the mean ± SD. Statistical analysis was performed using repeated measure ANOVA with LSD post-hoc test. 4 months vs. 5 months, P b 0.001; 5 months vs. 6 months, P b 0.001; 6 months vs. 7 months, P b 0.001.

of the escape latency for consecutive days showed no statistically significant impairment in spatial reference memory at 5, 6 and 7 months of age (P N 0.05) (Fig. 3B). After completion of the training trials, the spatial probe trial was performed, and both the number of platform crossings and the percent time spent in the target quadrant were analyzed. All treatment groups spent more time swimming in the target quadrant. The 2wANOVA indicated that both the age [probe platform crossings, F(4,75) = 32.65, P b 0.001; η2 = 0.07. percent time in target quadrant, F(4,75) = 171.68, P b 0.001; η2 = 0.12] and the strain [probe platform crossings, F(1,78) = 178.83, P b 0.001; η2 = 0.20% time in target quadrant, F(1,78) = 1963.80, P b 0.001; η2 = 0.23] significantly affected the performance of the SAM mice. The saANOVA revealed a strain effect at all five time points (3, 4, 5, 6 and 7 months, P b 0.05). The SAMR1 mice performed significantly better than SAMP8 mice in crossing the exact platform location significantly more times (Fig. 3C). Likewise, the SAMR1 mice spent a higher percentage of total time in target quadrant than the SAMP8 mice (Fig. 3D). These results again indicated that SAMP8 mice showed an age-associated cognitive deficit. Post-hoc analyses among the groups of 3-, 4-, 5-, 6- and 7-month-old SAMP8 mice demonstrated a progressive and relatively rapid memory decline in the probe trial (P b 0.05). It is worth noting that the mild cognitive impairment could be detected beginning at 5 months of age and was severe at 7 months [the number of platform crossings, 4 months vs. 5 months, P b 0.001; 6 months vs. 7 months, P b 0.001. the percent time spent in target quadrant, 4 months vs. 5 months, P b 0.001; 6 months vs. 7 months, P b 0.001] (Fig. 3C, D). Mild pathologic morphology The 2wANOVA indicated that both the age [F(4,75) = 289.66, P b 0.001; η2 = 0.10] and the strain [F(1,78) = 2389.53, P b 0.001; η2 = 0.24] significantly affected the number of pyramidal cells in CA1 of the SAM mice. The saANOVA showed that the neuronal number was significantly different between the two strains and a significant decrease in SAMP8 mice compared to age-matched SAMR1 mice (P b 0.001). The ssANOVA showed that there was an age effect in SAMP8 mice [F(4,35) = 168.24, P b 0.001], but not in the SAMR1 mice [F(4,35) = 2.32, P N 0.05]. Most notably, 5-month-old SAMP8 mice show significant neuron loss compared to 4-month-old mice [F(1,14) = 13.47, P b 0.05]. The severe hippocampal neuron loss was

found in the 6- and 7-month-old SAMP8 mice [5 months vs. 6 months, P b 0.001; 6 months vs. 7 months, P b 0.001] (Fig. 4A1–A10, B). Immunohistochemical staining for Aβ demonstrate brownish yellow granules are located in the membrane and cytoplasm of pyramidal cells. The 2wANOVA indicated that both the age [F(4,75) = 87.01, P b 0.001; η 2 = 0.11] and the strain [F(1,78) = 336.07, P b 0.001; η2 = 0.15] significantly affected the deposit of the SAM mice. The saANOVA showed that SAMP8 mice had a significant stronger staining in SAMP8 mice compared to age-matched SAMR1 mice (P b 0.001). Most importantly, the LSD post-hoc test revealed that in the groups of young (3 and 4 months) SAMP8 mice, there were few Aβ deposits. With aging, they presented mild Aβ deposits beginning from the age of 5 months [4 months vs. 5 months, P b 0.001], and presented extensive Aβ deposits at 7 months of age [5 months vs. 6 months, P b 0.001; 6 months vs. 7 months, P b 0.001]. The results reflected the likelihood of the progression for an MCI animal (Fig. 4C1–C10, D). In the coronal section, neurons impregnated with the Golgi–Cox solution clearly display dendritic spines. The secondary and tertiary apical dendrites in the CA1 region of the hippocampus were observed in the digitized images. The 2wANOVA indicated that both age [F(4,75) = 67.81, P b 0.001; η2 = 0.17] and strain [F(1,78) = 147.51, P b 0.001; η2 = 0.25] significantly affected the dendritic spine density of the SAM mice. The dendritic spines in the hippocampal CA1 region of the SAMR1 mice were regular and intense, revealing a significant difference compared to age-matched SAMP8 mice (P b 0.001). The ssANOVA showed that the fluctuating trends of the dendritic spine density in SAMP8 mice were similar to that of Nissl staining results. With aging, the mild decrease at 5 months [3 months vs. 4 months, P b 0.05; 4 months vs. 5 months, P b 0.001] subsequently progressed to a more severe stage at 7 months [5 months vs. 6 months, P b 0.001; 6 months vs. 7 months, P b 0.001] (Fig. 4E1–E10, F).

Assessment of strain and age-related differences in serum T levels The 2wANOVA indicated that both age [F(4,75) = 175.04, P b 0.001; η2 = 0.12] and strain [F(1,78) = 817.26, P b 0.001; η2 = 0.27] significantly affected the serum T levels. The ssANOVA showed that there was an age effect in SAMP8 mice [F(4,35) = 86.45, P b 0.001] but not in the SAMR1 mice [F(4,35) = 1.67, P N 0.05]. SNK-q test revealed that after the development and maturation process, the SAMP8 mice showed decreases in T beginning at 5 months of age [3 months vs. 4 months, P N 0.05; 4 months vs. 5 months, P b 0.001]. They subsequently underwent an age-related decline in serum T levels [5 months vs. 6 months, P b 0.001; 6 months vs. 7 months, P b 0.001]. The mean T levels of 5- and 7-month-old SAMP8 mice were significantly lower than that at 3 months [5 months vs. 3 months, F(1,14) = 36.89, P b 0.001; 7 months vs. 3 months, F(1,14) = 28.06, P b 0.001]. The serum T levels of 5- and 7-month-old SAMP8 mice were 10.31% and 30.98% lower than that in 3-month-old mice (Fig. 5).

Effects of castration and DHT intervention on the conversion from MCI to Alzheimer's dementia in SAMP8 mice The effects of castration and DHT intervention on the conversion from MCI to Alzheimer's dementia in SAMP8 mice are shown in Fig. 6. We analyzed the senescence grading scores (Fig. 6A), mild cognitive impairment (Fig. 6B–D) and pathologic morphology (Fig. 6E–J) using Cohen's d test. The results showed that all of the parameters in Cast group were significantly different between P8-sham and DHT group (P b 0.001). Androgen deficiency after castration hastened senescence, cognitive impairment and changes of pathology morphology by comparing gonad-intact and DHT-intervened male SAMP8 mice. Androgen replacement therapy reversed the rapid conversion from MCI to AD.

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Fig. 3. Assessment of the conversion from MCI to Alzheimer's dementia through mild cognitive impairment detected in MWM. Both age and strain had significant effect on the performance. During 5 days of spatial reference memory (hidden platform) test (A, B), all of the SAMR1 and SAMP8 mice tested at all five time points (3, 4, 5, 6 and 7 months) showed decreases in escape latency. SAMR1 groups exhibited good spatial memory (A). In contrast, SAMP8 groups were slower in their rates of improvement, indicating an age-associated cognitive deficit (B). During the spatial probe test, both the number of platform crossings (C) and the percent time spent in the target quadrant (D) were analyzed. All treatment groups spent more time swimming in the target quadrant. SAMP8 mice demonstrated a progressive and relatively rapid memory decline in the MWM. It is worth noting that the mild cognitive impairment could be detected beginning at 5 months of age and was severe at 7 months. The results were expressed as the mean ± SD. Statistical analysis was performed using repeated measure ANOVA (hidden platform test) a two-way ANOVA with LSD post-hoc test (spatial probe test). * P b 0.05, ** P b 0.001.

Discussion According to the NIA/AA diagnostic criteria for AD, it is difficult to identify or develop an animal model that reproduces all or most of the

features of human MCI. To begin with, formal cognitive testing for an animal is not feasible, and it is difficult to assess whether its daily life is affected. Second, it is important to emphasize that standardization of these biomarkers is currently limited and it is difficult to define

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Fig. 4. Assessment of the conversion from MCI to Alzheimer's dementia through mild pathologic morphology. Photomicrographs showing the Nissl staining (A1–A10), immunohistochemical staining (C1–C10) and Golgi staining (E1–E10). Photomicrographs (A1–A5, C1–C5 and E1–E5) and photomicrographs (A6–A10, C6–C10 and E6–E10) respectively showed SAMR1group and SANP8 group. Scale bar = 100 μm (A1–A10 and C1–C10), Scale bar = 50 μm (E1–E10). Statistical graphs showing the statistical analysis results of the pathologic morphology (B, D and F). Both age and strain had significant effect on the pathologic morphology. These results indicated that SAMP8 mice showed an age-associated pathologic morphology. With aging, SAMP8 mice presented mild pathologic morphology beginning from the age of 5 months and subsequently progressed to a more severe stage at 7 months. The results were expressed as the mean ± SD. Statistical analysis was performed using a two-way ANOVA with LSD post-hoc test. * P b 0.05, ** P b 0.001.

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Fig. 5. Assessment of strain and age-related differences in serum T levels. Serum testosterone levels decreased significantly as a function of age among SAMP8 but not SAMR1 mice. Post-hoc analyses revealed that after the development and maturation process, the SAMP8 mice showed decreases in T beginning at 5 months of age and subsequently underwent an age-related decline. The serum T levels of 5- and 7-month-old SAMP8 mice were 10.31% and 30.98% lower than that the levels in 3-month-old mice. The results were expressed as the mean ± SD. Statistical analysis was performed using a two-way ANOVA with SNK-q post-hoc test. * P b 0.05, ** P b 0.001.

biomarkers in the diagnosis and pathophysiological processes of MCI. Additionally, to identify MCI animal models, the problem of how to distinguish them from AD animal models needs to be considered. This problem parallels the situation facing the clinician, who must distinguish between MCI and dementia due to AD versus normal aging (Grundman et al., 2004). There is currently no uniform standard to identify MCI animal models. However, based on the clinical description of MCI and other considerations, we selected several indicators as reference criteria in the discrimination and assessment of incipient AD in MCI subjects. Studies are needed to clarify the extent to which aging can be considered to research the MCI phase. In addition, more research is required to identify the animal model “diagnosis” of MCI due to AD. The grading score system is a unique, useful and convenient method for evaluating the degree of senescence in mice based on changes in their behavior and appearance (Hosokawa et al., 1984). There was a significant correlation between the total grading score and the degree of senescence. Using the grading score, we observed SAMR1 mice showed a normal aging pattern, while SAMP8 mice showed remarkable age-associated increases in the senescence grading scores. It is worth noting that during the 5 months observation period, the increase in total scores was significant at 5 and 7 months of age. The increasing scores indicated that the SAMP8 mice showed an irreversible advancement of senescence at 5 months of age and a serious senescence deficit at 7 months of age. The grading score system is thought to accurately represent the degree of senescence of individual animals; the novel findings presented here may answer to what extent aging can be considered to research MCI phase. The cognitive ability test is optimal for objectively assessing the degree of cognitive impairment. Behavioral studies with SAMP8 mice show age-related deficits in the acquisition and retention of passive avoidance training (Flood and Morley, 1998). Available data indicate that this deficiency can be detected with different behavioral tests at different time points. However, in most studies using SAMP8 mice to study cognitive decline, only two or three representative ages, young and old, are compared (Chen et al., 2007; Flood et al., 1995b). In the present study, we investigate the earliest age that the decline can be detected, mimicking MCI in humans. In this study, we are the first to report the decline of spatial learning and memory during the processes of maturation and senescence, which occurs from 3 to 7 months of age. At 3 months of age, the SAMP8 and SAMR1 mice do not differ in their performance of the tasks. A mild impairment, the characteristic feature of

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the MCI animal model, could be measured as early as 5 months of age. SAMP8 mice subsequently show a clear age-related decline. At 7 months of age, SAMP8 mice develop severe deficits in learning and memory. Thus, it is noted that the observed impairment is becoming progressively worse with age. The 7-month-old SAMP8 mice are relatively “old” in their average life span of 12 months, and at this age they become a valid animal model of Alzheimer's dementia. Our results indicated that their performance in the MWM provides a mechanism to assess the mild cognitive impairment in the MCI animal model. Numerous observational studies find that SAMP8 mice develop deficits in learning and memory relatively early in there lifespan (Flood and Morley, 1992; Miyamoto et al., 1986; Yagi et al., 1988). Here, we investigate the mild changes in cognition across several months. To our knowledge, this is the only study to identify a progressive impairment of learning and memory, with the initial deficit beginning at 5 months of age. The 2011 criteria and guidelines propose that biomarker tests are essential to identify individuals in the preclinical and MCI stages of AD. Biomarkers may help address the likelihood of disease progression from MCI to a more severe stage of MCI or to dementia (Albert et al., 2011). We selected two classes of biomarkers: (1) markers that directly reflect the pathology of AD by providing evidence of the presence of key proteins deposited in the brain during the course of AD (e.g., Aβ); (2) markers that less directly or nonspecifically reflect the pathology of AD by tracking indices of neuronal injury (e.g., neuron loss and synaptic damage). The neuropathological hallmarks of AD are Aβ deposits as well as neuronal and synaptic loss (Coleman and Yao, 2003; DeKosky et al., 1996; Terry et al., 1991). These pathologic changes accumulate gradually over decades before becoming clinically evident (Morris, 2005). SAMP8 mice are characterized by an age-related spontaneous deterioration in learning and memory abilities, preceded or accompanied by pathological features that are similar to AD (Butterfield and Poon, 2005; Del Valle et al., 2010; Nomura and Ohkuma, 1999). In the present study, we compared the neuropathological changes in the hippocampus and demonstrated progressive neuropathology by comparing different age points in SAMP8 mice. Up to now, the neuron loss was not included in the diagnostic criteria, but was considered an important pathological component of AD. Studies have demonstrated that the loss of hippocampal neurons in AD brains could explain the cognitive impairment, even in the earliest stage of AD (Apostolova et al., 2010; Kril et al., 2004; Rossler et al., 2002; Simić et al., 1997; van de Pol et al., 2006; West et al., 1994, 2004). In this study, we found a significant reduction of neurons in SAMP8 mice at 5 months of age. At this time, they developed cognitive deficits as examined by MWM. Our result is consistent with many studies which have associated hippocampal neuron loss with cognitive deficits or progression to dementia in MCI individuals. Thus, the change in neuron number may be helpful over a relatively long period of observation. The SAMP8 mice at 7 months of age show a significant neuron loss as seen in AD patients. Biomarkers can detect and quantify the Aβ protein that accumulates in the brain, a hallmark feature of AD (Albert et al., 2011). The level of Aβ accumulation is incorporated in research criteria and provides information about the stage or severity of disease. Previous studies demonstrate that SAMP8 mice, but not SAMR1 mice, show a significant age-related increase in Aβ depositions (Del Valle et al., 2010; Morley et al., 2000, 2002; Kumar et al., 2000). Our present study confirms that SAMP8 mice are considered a model of AD, as they present with extensive Aβ deposits. It is worth noting that SAMP8 mice presented mild Aβ deposits beginning at the age of 5 months. Because MCI is considered a transitional state between normal aging and dementia, the phase before extensive deposit formation are worthy of our attention. Synapses are the site of memory storage in the nervous system and through which information is transferred between neurons. Accumulating evidence demonstrates that a decrease in dendritic spine density correlates strongly with cognitive decline (Coleman and Yao, 2003; Martin et al., 2000; Neves et al., 2008; Selkoe, 2002; Terry et al., 1991).

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Fig. 6. Effects of castration and DHT intervention on the conversion from MCI to Alzheimer's dementia in SAMP8 mice. Statistical graph showing the effects of castration and DHT intervention on the conversion from MCI to Alzheimer's dementia through senescence grading scores (A) and cognitive impairment detected in MWM (B, C and D). Photomicrographs showing the Nissl staining (E1–E3), immunohistochemical staining (G1–G3) and Golgi staining (I1–I3). Scale bar = 100 μm (E1–E3 and G1–G3), Scale bar = 50 μm (I1–I3). Statistical graph showing the effects of castration and DHT intervention on the conversion from MCI to Alzheimer's dementia through pathologic morphology (F, H and J). Castration hastened the conversion from MCI to AD by comparing gonad-intact and DHT male SAMP8 mice. The results were expressed as the mean ± SD. Statistical analysis was performed using repeated measure ANOVA (hidden platform test) and one-way ANOVA with Cohen's d test (the other results). ** P b 0.001.

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Therefore, dendritic spine loss can be used to assess the conversion from MCI to AD in the SAMP8 mice. Our results demonstrate a fluctuating trend in dendritic spine density, similar to the results we find for Aβ deposits. At approximately 5 months of age, the SAMP8 mice show mild synaptic damage. Combining the positive Aβ biomarker and the neuronal injury, the research of age-related cognitive and neurobiological changes in SAMP8 would be useful to identify the disease in the earliest presymptomatic stage. We could not relate the more behavioral tasks reflecting cognitive impairment and the more biomarkers reflecting the pathologic alterations, but to a certain extent, the presence of the senescent, behavioral and neuropathological signs in 5-month-old male SAMP8 mice can character an animal model to research presymptomatic stage. Given such characteristics, the “middle-aged” SAMP8 mice appear to be a suitable and naturally derived animal model for MCI basic research. Both laboratory work and clinical trials aim to improve early detection and risk evaluation; earlier therapeutic intervention is more likely to achieve disease modification. There is strong evidence from the basic sciences and epidemiological studies that androgens play a protective role in neurodegeneration (Geerlings et al., 2006; Moffat et al., 2004; Paoletti et al., 2004). This demonstration addresses the interaction of androgen with the brain, focusing on men with androgen decline in aging males (ADAM) syndrome or andropause (Morley and Perry, 1999). The subject undergoes gradual androgen depletion with aging, which increases the risk of disease in androgen-responsive tissues throughout the body, including the brain (Giannoulis et al., 2012; Morris and Channer, 2012; Seidman, 2007; Sheffield-Moore and Urban, 2004; Yeap, 2009a, 2009b). Profitable studies demonstrate that androgen influence learning and memory (Holland et al., 2011; Maggio et al., 2012) and may contribute to a protective role against AD. Furthermore, these data built the foundation for the hypothesis that timely and appropriate androgen intervention may postpone the onset or improve the symptoms of AD. In fact, androgen levels are often low long before AD manifests (Kenny et al., 2004; Tan et al., 2004). Individuals with low levels of androgen may be at increased risk for developing cognitive impairment, and low androgen may be associated with the MCI that often forebodes AD (Moffat et al., 2004). In this study, we determined the T levels in the blood serum of SAMR1 and SAMP8 mice from 3 months to 7 months. The data demonstrated that after the development and maturation process, SAMP8 mice showed a decrease in T starting at 5 months. They subsequently presented with an age-related decrease in serum T levels. The 7month-old SAMP8 mice had T levels 30.98% lower than 3-month-old mice. Nomura et al. (Nomura et al., 1989) found that SAMP8 mice had significantly lower levels of serum T than SAMR1 mice at 11–12 months of age. Flood et al. (1995a) reported a 71% age-related decrease in serum T levels between 4- and 12-month-old SAMP8 mice, but only a 26% decrease was found between age-matched SAMR1 mice. A surprising finding was that SAMP8 mice had significant higher T levels than SAMR1 mice until 7 months of age. However, the observation of pathology in the testes and neuroendocrine dysfunction haven't been determined and whether it affects cognition remains unexplored. For now, we can only say that the SAMP8 mice have the higher serum T levels than the SAMR1 mice at the age of 3–6 months and this situation does not contradict the SAMP8 mice perform worse on MWM compared with the SAMR1 mice. The reasonable interpretation may be the marked agerelated decrease in serum T levels is one of the risk factors for the development Alzheimer's dementia. The strengths of this study was the continuous and monitoring of T levels in the blood serum. We cannot claim that low T per se was responsible for the cognitive impairment seen in the SAMP8 mice. AD, like other common chronic diseases, develops as a result of multiple factors rather than a single cause. So one of the complexities in understanding the effects of androgen is that the impaired cognitive function and various pathological features similar to AD of the SAMP8 is due to an interaction of aging and reduced androgen levels.

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It should also be noted that the decreased levels of T imply the possibility that the beneficial neural effects of androgens may be compromised during aging. Thus, reduced T levels may be responsible, in part, for the age-related cognitive impairment and the development of Alzheimer's dementia associated with the SAMP8 strain. However, no studies to data confirm the role of androgens in these processes of AD. It is unknown if androgen treatment has therapeutic implications for MCI and dementia. Generally, castration results in impairment of the reproductive tract and endogenous androgens are rapidly depleted. Baulieu and Robel (1998) demonstrate that T in the brain completely disappears within 1 day after castration. Therefore, sustained castration can identify the involvement of androgens in the processes of MCI and conversion to Alzheimer's dementia. We confirmed that castration hastened the conversion from MCI to AD by comparing gonad-intact and DHT male SAMP8 mice. All of the indicators reflected that androgen depletion in male SAMP8 mice is a risk factor for the development of AD. Although the majority of studies have identified a relationship between low levels of androgen and increased AD risk, most were unable to determine whether low T contributes to the disease process or is merely a result of it. However, it is worth noting that in the present study, we observed that SAMP8 mice do experience an age-related androgen decrease, which was somewhat similar to the process of ADAM. From these results, we speculated that low serum T levels occurs prior to or in the early stages of AD pathogenesis and thus likely act a risk factor. By improving early detection and evaluation of risk for AD, we can test potential therapies and eventually prescribe them for people at increased risk. Our study focuses on the hypothesis that androgen replacement therapy may prevent or delay the progression of symptoms and conversion to AD. Indeed, there is some evidence for this hypothesis from androgen therapy in subjects with AD (Cherrier et al., 2003, 2005b; Tan and Pu, 2003). However, not all studies confirm these results. Lu et al. report that T replacement therapy improves overall quality of life in patients with AD but has minimal effects on cognition (Lu et al., 2006). There are likely several factors that contribute to differences between the studies on the androgen in AD, including cognitive domains, treatment type and duration, age and other characteristics (Pike et al., 2009). Based on the hypothesis that androgen deficiency or deprivation is a risk for the development of Alzheimer's dementia, we observed whether treatment with androgen in castrated SAMP8 mice can delay the onset of disease. DHT, the most potent naturally-occurring androgen, is produced from testosterone through the action of cholestenone 5a-reductase. Because T may be partially converted into estradiol by endogenous aromatase (Cherrier et al., 2005a; Hojo et al., 2004; Ishii et al., 2007), we used DHT, a non-aromatizable androgen, in our studies. Recently, we reported that 1 mg/kg dose of DHT replacement therapy played an important role in sustaining and regulating structural synaptic plasticity in the hippocampus of male SAMP8 mice, whereas androgen deficiency may contribute to the etiology of age-related cognitive impairment, corresponding to the decline in endogenous androgen production (Li et al., 2013). This study provides a valuable theoretical basis for understanding the function of androgen and for therapeutic targets of androgen in AD. However, whether timely androgen intervention can postpone the onset or improve the symptoms of AD is a key question of our study. In an attempt to define the role of androgens in these processes, we investigated the effect of androgen deficiency in male SAMP8 mice after castration and DHT administration. In this study, the castrated 5-month-old male SAMP8 mice were administered 1 mg/kg of DHT for 2 months for investigating the effect of prevention of dementia. It is not the case that the dose must be the most optimal dosage. So here, we can only conclude that under the current experimental conditions, the 1 mg/kg dose of DHT replacement therapy is effective. We found that DTH-treated SAMP8 mice demonstrated improvement across the referenced indicators. Thus, DHT supplementation may be

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an effective disease-modifying therapy during the MCI phase under the current experimental conditions. This report is an extension of our prior study. We identify features of age-related cognitive and neurobiological changes in SAMP8 mice to research the conversion from MCI to Alzheimer's dementia, including changes in senescence, mild cognitive impairment and mild neuropathological changes. To a certain extent, the above indicators characterize the “middle-aged” SAMP8 mice, and identify them as a suitable and naturally derived animal model for MCI research. In the present study, the marked age-related decrease in serum androgen levels may be a risk factor for the development of Alzheimer's dementia. Timely and appropriate androgen intervention can postpone the onset and improve the symptoms of Alzheimer's dementia. Taken together, by improving early detection and risk evaluation, we hope bring the field closer to earlier detection, and ultimately lead to potential therapies for people at increased risk, once they are developed. Acknowledgments This work was supported by the National Natural Science Foundation of China Grants (31271191) and the Natural Science Foundation of Hebei Province Grants (C2009001081 and C2012206128). References Albert, M.S., DeKosky, S.T., Dickson, D., Dubois, B., Feldman, H.H., Fox, N.C., Gamst, A., Holtzman, D.M., Jagust, W.J., Petersen, R.C., Snyder, P.J., Carrillo, M.C., Thies, B., Phelps, C.H., 2011. The diagnosis of mild cognitive impairment due to Alzheimer's disease: recommendations from the National Institute on Aging–Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease. Alzheimers Dement. 7, 270–279. Alzheimer's Association, 2012. Alzheimer's disease facts and figures. Alzheimers Dement. 8, 131–168. Apostolova, L.G., Mosconi, L., Thompson, P.M., Green, A.E., Hwang, K.S., Ramirez, A., Mistur, R., Tsui, W.H., de Leon, M.J., 2010. Subregional hippocampal atrophy predicts Alzheimer's dementia in the cognitively normal. Neurobiol. Aging 31, 1077–1088. Baulieu, E.E., Robel, P., 1998. Dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) as neuroactive neurosteroids. Proc. Natl. Acad. Sci. U. S. A. 95, 4089–4091. Butterfield, D.A., Poon, H.F., 2005. The senescence-accelerated prone mouse (SAMP8): a model of age-related cognitive decline with relevance to alterations of the gene expression and protein abnormalities in Alzheimer's disease. Exp. Neurol. 40, 774–783. Chen, G.H., Wang, Y.J., Qin, S., Yang, Q.G., Zhou, J.N., Liu, R.Y., 2007. Age-related spatial cognitive impairment is correlated with increase of synaptotagmin 1 in dorsal hippocampus in SAMP8 mice. Neurobiol. Aging 28, 611–618. Cherrier, M.M., Craft, S., Matsumoto, A.H., 2003. Cognitive changes associated with supplementation of testosterone or dihydrotestosterone in mildly hypogonadal men: a preliminary report. J. Androl. 24, 568–576. Cherrier, M.M., Matsumoto, A.M., Amory, J.K., Ahmed, S., Bremner, W., Peskind, E.R., Raskind, M.A., Johnson, M., Craft, S., 2005a. The role of aromatization in testosterone supplementation: effects on cognition in older men. Neurology 64, 290–296. Cherrier, M.M., Matsumoto, A.M., Amory, J.K., Asthana, S., Bremner, W., Peskind, E.R., Raskind, M.A., Craft, S., 2005b. Testosterone improves spatial memory in men with Alzheimer disease and mild cognitive impairment. Neurology 64, 2063–2068. Coleman, P.D., Yao, P.J., 2003. Synaptic slaughter in Alzheimer's disease. Neurobiol. Aging 24, 1023–1027. DeCarli, C., 2003. Mild cognitive impairment: prevalence, prognosis, aetiology, and treatment. Lancet Neurol. 2, 15–21. DeKosky, S.T., Scheff, S.W., Styren, S.D., 1996. Structural correlates of cognition in dementia: quantification and assessment of synapse change. Neurodegeneration 5, 417–421. Del Valle, J., Duran-Vilaregut, J., Manich, G., Casadesús, G., Smith, M.A., Camins, A., Pallàs, M., Pelegrí, C., Vilaplana, J., 2010. Early amyloid accumulation in the hippocampus of SAMP8 mice. J. Alzheimers Dis. 19, 1303–1315. Flood, J.F., Morley, J.E., 1992. Early onset of age-related impairment of aversive and appetitive learning in the SAM-P/8 mouse. J. Gerontol. Biol. Sci. 47, B52–B59. Flood, J.F., Morley, J.E., 1998. Learning and memory in the SAMP8 mouse. Neurosci. Biobehav. Rev. 22, 1–20. Flood, J.F., Farr, S., Kaiser, F.E., La Regina, M., Morley, J.E., 1995a. Age-related decrease of plasma testosterone in SAMP8 mice: replacement improves age-related impairment of learning and memory. Physiol. Behav. 57, 669–673. Flood, J.F., Morley, P.M., Morley, J.E., 1995b. Age-related changes in learning, memory, and lipofuscin as a function of the percentage of SAMP8 genes. Physiol. Behav. 58, 819–822. Geerlings, M.I., Strozyk, D., Masaki, K., Remaley, A.T., Petrovitch, H., Ross, G.W., White, L.R., Launer, L.J., 2006. Endogenous sex hormones, cognitive decline, and future dementia in old men. Ann. Neurol. 60, 346–355.

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