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Icariin improves cognitive deficits and activates quiescent neural stem cells in aging rats
Journal of Ethnopharmacology 142 (2012) 746–753
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Icariin improves cognitive deficits and activates quiescent neural stem cells in aging rats Bin Wu a,b,1, Yang Chen a,1, Jianhua Huang a, You Ning a, Qin Bian a, Yimin Shan c, Waijiao Cai a, Xinming Zhang a, Ziyin Shen a,n a
Institute of Integrative Chinese and Western Medicine, Huashan Hospital, Fudan University, No. 12, Wu Lu Mu Qi (Middle) Road, Shanghai 200040, PR China Department of Rheumatology and Geratology, Chongqing Chinese Medicine Hospital, Chongqing 400021, PR China c Department of Health Care Wine Technology, Jing Brand Co. Ltd., Hubei 435100, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 January 2012 Received in revised form 27 March 2012 Accepted 29 May 2012 Available online 9 June 2012
Ethnopharmacological relevance: Icariin represents an important active component in Herba Epimedii, which is a famous Chinese herbal medicine that is widely used to treat some age-related diseases in oriental countries. Objective: The aim of this work was to investigate the effects of icariin on cognitive function in natural aging rats, and then to explore its mechanism by investigating the activation of quiescent neural stem cells (NSCs) in the hippocampus. Materials and methods: Sprague-Dawley rats that were 18 months of age were divided into two groups including treated rats (i.e., icariin was administered from the age of 18 months to 21 months) and control rats (i.e., only saline was administered). The Morris water maze (MWM) tasks were then employed to measure spatial learning and memory. Subsequently, AraC was infused into the brain with osmotic minipumps in order to destroy proliferative stem cells primarily leaving quiescent NSCs. After seven days of recovery, 5-bromodeoxyuridine (BrdU) was co-labeled with markers for NSC to identify NSCs. Results: The results from the MWM indicated that icariin has a beneficial effect on cognitive function in aging rats. In addition, by double-labeling BrdU and glial fibrillary acidic protein (GFAP), our findings indicated that NSC activation is markedly increased in the icariin-treated rats compared to control rats. For example, a much greater increase was produced in BrdU and highly polysialylated neural cell adhesion molecule (PSA-NCAM) and BrdU and Olig2 double-labeled cells following icariin treatment. Conclusion: Our findings suggest that icariin represents a promising candidate for the modulation of aging. Therefore, icariin administration may effectively prevent or delay the onset of age-related cognitive degeneration, and its capability to activate quiescent NSCs may potentially be one of its mechanisms. & 2012 Elsevier Ireland Ltd. All rights reserved.
Keywords: Aging Morris water maze Quiescent neural stem cells Icariin
1. Introduction Aging is a time-dependent process that is progressive and associated with functional impairment. The increase in life expectancy during the 20th century has significantly increased the number of people who suffer from age-related diseases (Vaupel, 2010). In particular, older individuals show a higher deficit in cognitive function and a lower quality of life. Therefore, the age-dependent loss in cognitive functions has attracted extensive attention worldwide (Yankner et al., 2010).
n
Corresponding author. Tel./fax: þ 86 21 62490934. E-mail address: [email protected] (Z. Shen). 1 These authors contributed equally to this work.
0378-8741/$ - see front matter & 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2012.05.056
Previous evidence has established that the hippocampus plays a crucial role in cognitive function (Barnes and Erickson, 2003). In the dentate gyrus of the hippocampus, new neurons are generated from neural stem cells (NSCs) throughout life. As aging progresses, the rate of neurogenesis decreases exponentially, and this decrease in neurogenesis has been implicated in agedependent cognitive decline in both animals and humans (LaFerla et al., 2007; Song et al., 2007). Thereby, the study of the neurobiological mechanism(s) that underlie the age-related reduction in neurogenesis is crucial in order to understand cognitive decline during aging. Recent advances in stem cell research indicate that both quiescent (i.e., out of the cell cycle and in a lower metabolic state) and active (i.e., during the cell cycle and not able to retain DNA labels) stem cell subpopulations may coexist in the hippocampus (Li and
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Clevers, 2010), and the decreased dentate neurogenesis during aging is ascribed to the increase in quiescence of NSCs as well as decrease in active stem cells (Shetty and Hattiangady, 2008). The literature suggests that physical exercise and brain injury can result in the activation of quiescent NSCs (Chen et al., 2009; Taylor et al., 2010); moreover, many researchers are making efforts to explore the mechanism and intervention measures associated with this system (Wang et al., 2011). Therefore, recruiting quiescent cells to enter the cell cycle and transform NSCs from the quiescent state to active growth may improve cognitive decline and have a significant impact on quality of life during aging. Traditional Chinese medicine, with its long history of clinical practice, occupies an important place as an alternative medicine. Herba Epimedii (family Berberidaceae; Ying-Yang-Huo in Chinese) is a Chinese herbal medicine with proven efficacy in treating cardiovascular diseases and osteoporosis, and in improving sexual and neurological functions, which was widely used in oriental countries, such as China, Japan, and Korea (Pei et al., 2008; Sze et al., 2010). Total flavone of Epimedium (TFE) is generally considered as the major active compound found in Herba Epimedii. Previous studies in our laboratory have found that TFE prolongs the lifespan and has strong effects in 2BS cells (Hu et al., 2004), C. elegans (Cai et al., 2008), and Drosophila (Shen et al., 2005). In addition, TFE recovered the metabolic trajectory in aging rats to levels consistent with younger rodents in urine and plasma metabonomic research (Shen et al., 2008, 2009). Icariin represents an important active component in TFE, and exhibits similar life prolonging effects as TFE in C. elegans (Cai et al., 2011) and mice (unpublished). In recent years, several studies have suggested that icariin may improve learning and memory deficits in animal models (Luo et al., 2007; Guo et al., 2010; Li et al., 2010; Urano and Tohda, 2010). However, little is known regarding whether icariin can ameliorate the cognitive decline associated with natural aging. Furthermore, it is necessary to assess icariin’s anti-aging effects associated with enhancing cognitive function and quality of life. In the present study, we evaluated the effect of icariin on neurobehavioral outcomes in natural aging rats, and then explore its mechanism by investigating the activation of quiescent NSCs in the hippocampus.
2. Materials and methods 2.1. Quality control of icariin The dry icariin powder was purchased from Nanjing Zelang Medical Technology Company Ltd. (Nanjing China). A LC/MS system was used to identify its structure and analyze its content. The analysis conditions were as follows. The column was Kromasil C18 (250 4.6 mm, 5.0 mm, Eka chemicals, SW) with a mobile phase consisting of A (water) and B (100% acetonitrile). The gradient elution was 0 min (95% A, 5% B), and 30–50 min (100% B). The flow rate was 0.8 mL min 1, and the column temperature was 25 1C with an injection volume of 20 mL. The MS detection conditions were as follows. The drying gas N2 was 8 L/min, and the temperature was 350 1C. The nebulizer pressure was 35 psi in the negative ionization mode with a capillary voltage of 2500 V and scan range of 100–2200 m/z. The results depicted in Fig. 1 suggest that the purity of icariin is more than 99.95%.
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well as the current version of the China Law on the Protection of Animals. The rats were housed in well-ventilated rooms at 19–23 1C and 45–70% relative humidity with a regular light–dark cycle (12 h of light, 07:00–19:00 h). Food and tap water were provided ad libitum. After 2 weeks of acclimation, the rats were randomly divided into two groups (n¼ 15 rats) including (a) aged rats with orally administrated icariin (0.02 g/kg body weight/day) from the age of 18 months to 21 months and (b) aged controlled rats orally administrated with the same volumes of saline in place of the drugs. The icariin was dissolved in water at 1% concentration before the solution was orally administered to the rats. 2.3. Morris Water Maze Following 3 months of icariin or vehicle administration, the rats’ cognitive function was assessed using the Morris Water Maze (MWM), which is regarded as the standard procedure to evaluate the spatial learning and memory in rodents (Morris, 1984). The MWM apparatus consisted of a circular pool (180 cm in diameter and 50 cm in height) surrounded by a white curtain to ensure that the rats were not affected by the outside environment. The pool was divided into four quadrants and filled with water that was maintained at room temperature (2372 1C). An escape platform was placed in the center of the northeast quadrant, and 1.0 cm below the water surface. Four extra-maze cues were also set on the quadrant walls surrounding the pool. Thereby, the platform provided the only means of escape from the water, and was located in the same quadrant during every trial. A digital video camera was positioned directly above the pool enabling full collection of swim activity in the different quadrants, and was attached to a computer-controlled system (Jiliang Software Company, Shanghai, China). This process of Morris water maze consisted of 5 days of learning-memory training, and a probe trial that was applied on day six. On the day prior to the MWM test, each rat was allowed to swim in the MWM for 120 s in order to acclimate them to the novel environment and to allow them to locate and climb onto the escape platform. On each day, the rats were placed in the MWM facing the wall at the starting points in different quadrants of the tank. The rats were trained for one morning as well as one afternoon block from 08:00 to 12:00 h and 13:00 to 17:00 h, respectively. Each block consisted of two trials, and each trial lasted for 120 s or was completed if the rats reached the submerged platform. If the animal did not find the platform within 120 s, the rat was manually guided to the platform and placed on the platform for 30 s by the experimenter. A 2 min break was provided between trials. The latency (i.e., swimming time from the starting point to the platform) was recorded during the experiment for each rat. Every trial lasted a maximum of 120 s. If the rat failed to find the platform in time, the value for this latency was set at 120 s as a representation of learning performance. The escape latency(s) was calculated by averaging four trial values. The probe trial was performed by removing the platform and allowing each rat to swim freely for 120 s inside the pool. The mean number of animals which crossed the normal position of the platform was also collected. The ratio of time to distance in the target quadrant to that in the whole pool was also calculated.
2.2. Animals
2.4. Implantation osmotic minipumps containing Ara-C or vehicle alone
Thirty male Sprague-Dawley rats (610–720 g; 18 months of age) were purchased from the Scientific Animal and Plant Center, Fudan University (Shanghai, China). The principles of laboratory animal care (NIH publication no. 86–23, revised 1985) were followed as
Before the implantation of osmotic minipumps, three animals in each group were sacrificed to evaluate the neurogenesis state at the end of the icariin treatment. Cytosine-b-d-arabiofuranoside (Ara-C, Sigma) was infused into the other rats at the right lateral
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Fig. 1. Chemical profile of icariin by Agilent 1100 HPLC/MS/MS/UV. (A) Icariin displays a weak response in positive ionization mode. (B), (C) Icariin exhibits strong signals in the negative ionization mode (R.T. 10.9 min) and UV detection (344–348 nm), which indicates high purity of icariin. (D), (E) The MS spectrum of the peak R.T 10.9 min is depicted in positive and negative ionization mode.
ventricle of male Sprague-Dawley rats, as previously described (Pruitt et al., 2004). Aged rats (21 months of age) were anaesthetized by an i.p. injection of a 10% Chloral Hydrate (0.5 mg/kg) solution and positioned in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, USA). The skull was exposed by a skin incision, and a small hole (1 mm) was drilled through the skull. A stainless steel cannula (outer diameter of 0.8 mm, Shanghai Syringe Co.) was implanted stereotaxically into the lateral ventricle of the right side at 1.4 mm posterior to bregma and 2.0 mm lateral with a final depth at 3.5 mm from the skull. AraC (Sigma; 2% in 0.9% saline) or vehicle alone was infused for 7 days into the brain with an osmotic pump (model 2ML; Alzet). The pumps were then removed, and the first day after the last infusion was regarded as day 0. The animals were then sacrificed, and perfused at day 0 and day 7 in two groups (n ¼3). A single i.p. BrdU injection (50 mg/kg body weight) was administered 2 h before the rats were sacrificed. Brdu immunohistochemistry was used to investigate proliferative NSCs (day 7) and to verify the effectiveness of Ara-C treatment in the elimination of actively dividing cells (day 0).
2.5. Tissue preparation and immunhistochemistry For histology, rats were deeply anaesthetized by intraperitoneal injection of pentobarbital (50 mg/kg body weight), and were perfused with 0.9% saline solution followed by 4% paraformaldehyde (PFA) solution in 0.1 M phosphate buffer solution (PBS). Brains were fixed with 4% PFA overnight. Then, the tissue was dehydrated in 30% sucrose with 4% PFA and in 30% sucrose solution in 0.1 M PBS for 24 h. Subsequently, the tissue was embedded and frozen in OCT (TissueTEK). Coronal sections (40 mm) were obtained through the dorsal hippocampus (at levels corresponding to 2.8–5.6 mm posterior to bregma). The sections were selected in multi-well dishes (Corning), and were stored at 20 1C in an antifreeze solution until they were examined. Every sixth section of the hippocampus was selected (six sections per animal), and was washed three times with 0.01 M PBS for 5 min. The tissue was fixed in 10% paraformaldehyde for 30 s, and washed again in PBS. The sections were then incubated in 2 M HCl at 37 1C for 30 min. The tissue was washed again in PBS, and subsequently permeabilized with 0.4% Triton X-100
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diluted in PBS for 30 min at room temperature. Following an additional wash, the tissue was blocked in 10% goat serum at 37 1C for 1 h. BrdU staining was performed using sheep monoclonal antBrdU (1:200 Novas), and BrdU was double-labeled with anti-GFAP (1:400 Millipore), -PSA-NCAM (1:400 Millipore), and -Olig2 (1:400 Millipore) antibodies. FITC-conjugated antisheep IgG (1:500 Millipore) and Cy3-conjugated antimouse IgG antibodies (1:1000 Millipore) were used as the secondary antibodies. For double staining, primary antibodies or secondary antibodies from
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different species were incubated simultaneously. Sections were incubated overnight at 4 1C with the diluted solution of the primary antibody. The sections were washed three times with 0.01 M PBS (15 min each), and then incubated at 37 1C for 1 h with the diluted solutions of the secondary antibodies. After washing the sections three times with PBS (15 min each), the sections were counter-stained with DAPI (1 mg/ml; Sigma) to visualize the nuclei (Fig. 3C). The co-localization with neuronal stem cell markers was analyzed using a fluorescence microscope (Nikon 80i).
Fig. 2. Morris water maze in aging rats. The typical tracking of the swimming path is represented in aging (A) control and (B) icariin rats. (C) The mean swimming speed in two groups. (D) Comparison of escape latencies to find the platform during 5 days of training. (E) The percentage of searching time and distance spent in the target quadrant. (F) The comparison of platform crosses in two groups. Results are expressed as the mean 7 SD (n¼ 15; *Po 0.05).
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2.6. Cell quantification analysis with stereology
3. Results
BrdU-immunopositive cells were detected with an antibody against BrdU in the hilus of dentate gyrus (DG) of the hippocampus. Newborn neuronal stem cells were localized in the subgranular zone (SGZ). Quantitative analysis of the number of the positively stained cells was performed with optimal magnification ( 100). All sections were defined from the Paxinos & Watson’s rat brain atlas (1997) to ensure that corresponding sections were compared between groups. The double-labeled cells in the SGZ (six sections per animal; n ¼3 for each group) were analyzed and counted using the imageanalysis software Image Pro-Plus 6.0 for windows (Copyright 1993, 2003 Media Cybernetics L.P.). A double-stained cell was defined as having the strong yellow intensity with an overlap of the green (FITC) and red (Cy3) fluorescence. The total number of marked cells in the SGZ sections was tallied and multiplied by the section-sampling fraction (Becker et al., 2007).
3.1. Icariin improves cognitive function of aging rats
2.7. Statistical analysis Data were analyzed using Student’s unpaired t-test if they were normally distributed (Kolmogorov–Smirov test); otherwise, the Mann–Whitney U-test was used. Repeated-measures ANOVAS followed by a post hoc test (Tukey B) was used for the analysis of WMM study data, using the SPSS 16.0 program. A two-tailed value of Po0.05 was considered significant.
The MWM is a well-established paradigm for evaluating deficits in hippocampal-dependent memory, and the improvement in learning and memory can be indicated by the escape latencies in hidden platform test, the percentage of time and distance in target quadrant, and the number of platform crosses in the probe test. In the present study, the results from the MWM test are shown in Fig. 2. Fig. 2A and B represents the typical tracking of the swimming path for the control and icariin rats. Fig. 2C indicates that the average swimming speed is not different when comparing the two groups. As indicated in previous literature (Lindner, 1997), the MWM test is not affected by motor or motivational factors. During the 5 day training (Fig. 2D), the rats in two groups show equivalent escape latencies on day 1, and these results suggest that the rats have similar initial performance. The aging rats following icariin administration exhibit a significant decrease in their escape latency on days 2, 3, and 5, as compared to control rats. Although similar high levels of escape latency were detected on the fourth day of training in both groups, these findings still illustrated that the icariin-treated rats show better acquisition of the task than control rats. In the probe test (Fig. 2E), rats following icariin administration spent significantly longer searching time and distance in the target quadrant compared with the control
Fig. 3. The immunofluorescence of coronal sections. (A), (B) The double-labeling of BrdU and GFAP, as arrows point to the yellow dot, to show the activation of NSCs at the end of icariin treatment. Icariin significantly increases the number of NSCs and promotes neurogenesis. (C) DAPI staining was used to visualize the nuclei. (D) BrdU immunostaining revealed very few labeled cells distributed in the SGZ. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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group (P o0.05, Po0.05, respectively), and exhibit an increased number of platform crosses (Po0.05, Fig. 2F). 3.2. Immunohistochemical analysis To evaluate the supply of newly generated NSCs in the SGZ of the hippocampus, double-labeling with immunofluorescence was utilized. BrdU-positive cells were co-labeled with a neuronal marker, BrdU and GFAP, to show NSCs as well as BrdU and PSANCAM to detect neuroblast/neural precursor cells. Additionally, double-labeling of BrdU and Olig2 was employed to measure transit-amplifying cells (Joyner and Ahn, 2005). At the end of the icariin treatment, the neurogenesis was evaluated using double-labeling of BrdU and GFAP to show the activation of neuronal stem cells. The result indicates the neurogenesis of icariin group is more than the control group in Fig. 3A and B (p o0.05; 1.86 70.54, and 5.2171.29 respectively). In both control and treated rats, BrdU immunostaining revealed very few labeled cells distributed in the SGZ at day 0. A representative image is provided in Fig. 3D. After 7 days of natural recovery, icariin administration is associated with a significant increase in the co-labeling of cells in the SGZ (Fig. 4A). The total number of BrdU and GFAP, PSA-NCAM, and Olig2 double-labeled cells increased 1.94, 3.59, 4.06-fold in icariin-treated rats compared to control rats (Po0.05, Po0.01, Po0.05, respectively; Fig. 4B).
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4. Discussion Along with the increasing average life-span of humans over the last decades, cognitive diseases are emerging as one of the greatest health threats of the twenty-first century. It is estimated that in the coming 30 years, 15 to 20 million elderly in America may exhibit some form of cognitive disability (Brookmeyer et al., 2011). The high incidence of age-related diseases in the increasing population of elderly people has stimulated interest in the search for protective agents that have the capability of preventing premature aging and delaying the onset of degenerative disorders (Poeggeler, 2005). Two monomer compounds have demonstrated some efficacy against cognitive deterioration caused by natural aging, including tetrahydroxystilbene glucoside (Li et al., 2007), and Huperzine A (Tang et al., 2000). The findings in the present study indicate that another monomer component icariin displays neuroprotective effects against cognitive impairments during natural ageing. Likewise, icariin is a component of the Chinese herb Herba Epimedii, which is often used to tonify the kidney according to the theory of traditional Chinese medicine. The Morris water maze is commonly used to assess hippocampal-dependent spatial memory in rodents (Anglade et al., 1993). Behavioral studies in animals have demonstrated that hippocampal damage can produce learning-memory impairments. During 5 days of training trials in this task, a shorter latency was found in icariin-treated animals reaching the hidden
Fig. 4. The immunofluorescence of coronal sections and statistical results of NSCs. (A) The double-labeling of cells was defined as yellow due to the overlap of the green and red fluorescence (refer to arrows). The density of dividing cells is dramatically increased in icariin-treated rats. (B) Icariin markedly elevated the amount of NSCs between 1.9- and 4.1-fold. Results are expressed as the mean 7 SD (n ¼3; *P o0.05; **P o0.01). Scale bars represent 50 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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platform when compared to control animals. An obvious tendency suggested that icariin-treated rats show better performance in Morris water maze than control rats. For instance, we consistently found that the searching time, the distance in the target quadrant, and the number of platform crosses were significantly increased by icariin administration in the probe test (Po0.05, Po0.05, Po0.05, respectively). Collectively, these behavioral results suggest that icariin has a beneficial effect on spatial learning and memory in aging rats. Under appropriate conditions, the quiescent cells may be capable of re-entering the mitotic cell cycle, and this mechanism may contribute to the stem cell pool and maintaining selfrenewal characteristics of stem cells (Alexanian and Kurpad, 2005). In the present study, double-labeling was applied to detect NSCs by utilizing Brdu in order to identify proliferative stem cells, glial fibrillary acidic protein (GFAP) to identify NSCs as well as polysialic acid-neural cell adhesion molecule (PSA-NCAM) and Olig2 to identify neuroblast/neural precursor and transit-amplifying cells (Joyner and Ahn, 2005). At the end of the icariin treatment, the neurogenesis or activation of neuronal stem cells was elevated by icariin (Fig. 3A and B). In order to explore the mechanism of the newly generated cells, AraC, which is an antimitotic reagent, was used to kills fast-dividing cells in the SGZ and left quiescent NSCs. AraC was infused into the brain of SD rats for one week using a mini-osmotic pump. At the end of AraC treatment (day 0), Brdu immunoreactivity was quantified in the lateral wall of the lateral ventricles, and our results showed that the fast-dividing precursors/neuroblasts were almost completely depleted, confirming the efficacy of AraC treatment. One week after the AraC osmotic minipumps were removed, icariin was associated with 1.9- to 4.1-fold increase in proliferative NSCs and neurogenesis. Numerous studies have shown that the dramatic decline in neurogenesis with age may contribute to impairments in learning and memory (Lazarov et al., 2010). Therefore, these results provide strong evidence that icariin can activate quiescent NSCs to stimulate neurogenesis, and may ameliorate cognitive decline. The activation of the quiescent stem cells results in increased neurogenesis. This increase in neurogenesis may potentially be one of the mechanisms associated with an improvement in the aging rat’s cognitive function. Of course, other cellular effects of icariin may be acting on the neural subpopulations of dentate gyrus. For instance, icariin could accelerate the maturation of early neural progenitor cells. Therefore, further studies are needed to determine other biological mechanism that may be involved in the improvement of age-related memory impairments. In conclusion, the overall evidence in the present study indicates that icariin treatment alleviates the age-dependent deficit in cognitive function, and promoting the activation of quiescent NSCs may be one of its mechanisms.
Acknowledgments This study was financially supported by the National Science Funds of China (30873319) and the National basic research programs of China (2010CB530402). The authors graciously acknowledge the editorial assistance of International Science Editing. References Alexanian, A.R., Kurpad, S.N., 2005. Quiescent neural cells regain multipotent stem cell characteristics influenced by adult neural stem cells in co-culture. Experimental Neurology 191, 193–197.
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Icariin improves cognitive deficits and activates quiescent neural stem cells in aging rats Bin Wu a,b,1, Yang Chen a,1, Jianhua Huang a, You Ning a, Qin Bian a, Yimin Shan c, Waijiao Cai a, Xinming Zhang a, Ziyin Shen a,n a
Institute of Integrative Chinese and Western Medicine, Huashan Hospital, Fudan University, No. 12, Wu Lu Mu Qi (Middle) Road, Shanghai 200040, PR China Department of Rheumatology and Geratology, Chongqing Chinese Medicine Hospital, Chongqing 400021, PR China c Department of Health Care Wine Technology, Jing Brand Co. Ltd., Hubei 435100, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 January 2012 Received in revised form 27 March 2012 Accepted 29 May 2012 Available online 9 June 2012
Ethnopharmacological relevance: Icariin represents an important active component in Herba Epimedii, which is a famous Chinese herbal medicine that is widely used to treat some age-related diseases in oriental countries. Objective: The aim of this work was to investigate the effects of icariin on cognitive function in natural aging rats, and then to explore its mechanism by investigating the activation of quiescent neural stem cells (NSCs) in the hippocampus. Materials and methods: Sprague-Dawley rats that were 18 months of age were divided into two groups including treated rats (i.e., icariin was administered from the age of 18 months to 21 months) and control rats (i.e., only saline was administered). The Morris water maze (MWM) tasks were then employed to measure spatial learning and memory. Subsequently, AraC was infused into the brain with osmotic minipumps in order to destroy proliferative stem cells primarily leaving quiescent NSCs. After seven days of recovery, 5-bromodeoxyuridine (BrdU) was co-labeled with markers for NSC to identify NSCs. Results: The results from the MWM indicated that icariin has a beneficial effect on cognitive function in aging rats. In addition, by double-labeling BrdU and glial fibrillary acidic protein (GFAP), our findings indicated that NSC activation is markedly increased in the icariin-treated rats compared to control rats. For example, a much greater increase was produced in BrdU and highly polysialylated neural cell adhesion molecule (PSA-NCAM) and BrdU and Olig2 double-labeled cells following icariin treatment. Conclusion: Our findings suggest that icariin represents a promising candidate for the modulation of aging. Therefore, icariin administration may effectively prevent or delay the onset of age-related cognitive degeneration, and its capability to activate quiescent NSCs may potentially be one of its mechanisms. & 2012 Elsevier Ireland Ltd. All rights reserved.
Keywords: Aging Morris water maze Quiescent neural stem cells Icariin
1. Introduction Aging is a time-dependent process that is progressive and associated with functional impairment. The increase in life expectancy during the 20th century has significantly increased the number of people who suffer from age-related diseases (Vaupel, 2010). In particular, older individuals show a higher deficit in cognitive function and a lower quality of life. Therefore, the age-dependent loss in cognitive functions has attracted extensive attention worldwide (Yankner et al., 2010).
n
Corresponding author. Tel./fax: þ 86 21 62490934. E-mail address: [email protected] (Z. Shen). 1 These authors contributed equally to this work.
0378-8741/$ - see front matter & 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jep.2012.05.056
Previous evidence has established that the hippocampus plays a crucial role in cognitive function (Barnes and Erickson, 2003). In the dentate gyrus of the hippocampus, new neurons are generated from neural stem cells (NSCs) throughout life. As aging progresses, the rate of neurogenesis decreases exponentially, and this decrease in neurogenesis has been implicated in agedependent cognitive decline in both animals and humans (LaFerla et al., 2007; Song et al., 2007). Thereby, the study of the neurobiological mechanism(s) that underlie the age-related reduction in neurogenesis is crucial in order to understand cognitive decline during aging. Recent advances in stem cell research indicate that both quiescent (i.e., out of the cell cycle and in a lower metabolic state) and active (i.e., during the cell cycle and not able to retain DNA labels) stem cell subpopulations may coexist in the hippocampus (Li and
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Clevers, 2010), and the decreased dentate neurogenesis during aging is ascribed to the increase in quiescence of NSCs as well as decrease in active stem cells (Shetty and Hattiangady, 2008). The literature suggests that physical exercise and brain injury can result in the activation of quiescent NSCs (Chen et al., 2009; Taylor et al., 2010); moreover, many researchers are making efforts to explore the mechanism and intervention measures associated with this system (Wang et al., 2011). Therefore, recruiting quiescent cells to enter the cell cycle and transform NSCs from the quiescent state to active growth may improve cognitive decline and have a significant impact on quality of life during aging. Traditional Chinese medicine, with its long history of clinical practice, occupies an important place as an alternative medicine. Herba Epimedii (family Berberidaceae; Ying-Yang-Huo in Chinese) is a Chinese herbal medicine with proven efficacy in treating cardiovascular diseases and osteoporosis, and in improving sexual and neurological functions, which was widely used in oriental countries, such as China, Japan, and Korea (Pei et al., 2008; Sze et al., 2010). Total flavone of Epimedium (TFE) is generally considered as the major active compound found in Herba Epimedii. Previous studies in our laboratory have found that TFE prolongs the lifespan and has strong effects in 2BS cells (Hu et al., 2004), C. elegans (Cai et al., 2008), and Drosophila (Shen et al., 2005). In addition, TFE recovered the metabolic trajectory in aging rats to levels consistent with younger rodents in urine and plasma metabonomic research (Shen et al., 2008, 2009). Icariin represents an important active component in TFE, and exhibits similar life prolonging effects as TFE in C. elegans (Cai et al., 2011) and mice (unpublished). In recent years, several studies have suggested that icariin may improve learning and memory deficits in animal models (Luo et al., 2007; Guo et al., 2010; Li et al., 2010; Urano and Tohda, 2010). However, little is known regarding whether icariin can ameliorate the cognitive decline associated with natural aging. Furthermore, it is necessary to assess icariin’s anti-aging effects associated with enhancing cognitive function and quality of life. In the present study, we evaluated the effect of icariin on neurobehavioral outcomes in natural aging rats, and then explore its mechanism by investigating the activation of quiescent NSCs in the hippocampus.
2. Materials and methods 2.1. Quality control of icariin The dry icariin powder was purchased from Nanjing Zelang Medical Technology Company Ltd. (Nanjing China). A LC/MS system was used to identify its structure and analyze its content. The analysis conditions were as follows. The column was Kromasil C18 (250 4.6 mm, 5.0 mm, Eka chemicals, SW) with a mobile phase consisting of A (water) and B (100% acetonitrile). The gradient elution was 0 min (95% A, 5% B), and 30–50 min (100% B). The flow rate was 0.8 mL min 1, and the column temperature was 25 1C with an injection volume of 20 mL. The MS detection conditions were as follows. The drying gas N2 was 8 L/min, and the temperature was 350 1C. The nebulizer pressure was 35 psi in the negative ionization mode with a capillary voltage of 2500 V and scan range of 100–2200 m/z. The results depicted in Fig. 1 suggest that the purity of icariin is more than 99.95%.
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well as the current version of the China Law on the Protection of Animals. The rats were housed in well-ventilated rooms at 19–23 1C and 45–70% relative humidity with a regular light–dark cycle (12 h of light, 07:00–19:00 h). Food and tap water were provided ad libitum. After 2 weeks of acclimation, the rats were randomly divided into two groups (n¼ 15 rats) including (a) aged rats with orally administrated icariin (0.02 g/kg body weight/day) from the age of 18 months to 21 months and (b) aged controlled rats orally administrated with the same volumes of saline in place of the drugs. The icariin was dissolved in water at 1% concentration before the solution was orally administered to the rats. 2.3. Morris Water Maze Following 3 months of icariin or vehicle administration, the rats’ cognitive function was assessed using the Morris Water Maze (MWM), which is regarded as the standard procedure to evaluate the spatial learning and memory in rodents (Morris, 1984). The MWM apparatus consisted of a circular pool (180 cm in diameter and 50 cm in height) surrounded by a white curtain to ensure that the rats were not affected by the outside environment. The pool was divided into four quadrants and filled with water that was maintained at room temperature (2372 1C). An escape platform was placed in the center of the northeast quadrant, and 1.0 cm below the water surface. Four extra-maze cues were also set on the quadrant walls surrounding the pool. Thereby, the platform provided the only means of escape from the water, and was located in the same quadrant during every trial. A digital video camera was positioned directly above the pool enabling full collection of swim activity in the different quadrants, and was attached to a computer-controlled system (Jiliang Software Company, Shanghai, China). This process of Morris water maze consisted of 5 days of learning-memory training, and a probe trial that was applied on day six. On the day prior to the MWM test, each rat was allowed to swim in the MWM for 120 s in order to acclimate them to the novel environment and to allow them to locate and climb onto the escape platform. On each day, the rats were placed in the MWM facing the wall at the starting points in different quadrants of the tank. The rats were trained for one morning as well as one afternoon block from 08:00 to 12:00 h and 13:00 to 17:00 h, respectively. Each block consisted of two trials, and each trial lasted for 120 s or was completed if the rats reached the submerged platform. If the animal did not find the platform within 120 s, the rat was manually guided to the platform and placed on the platform for 30 s by the experimenter. A 2 min break was provided between trials. The latency (i.e., swimming time from the starting point to the platform) was recorded during the experiment for each rat. Every trial lasted a maximum of 120 s. If the rat failed to find the platform in time, the value for this latency was set at 120 s as a representation of learning performance. The escape latency(s) was calculated by averaging four trial values. The probe trial was performed by removing the platform and allowing each rat to swim freely for 120 s inside the pool. The mean number of animals which crossed the normal position of the platform was also collected. The ratio of time to distance in the target quadrant to that in the whole pool was also calculated.
2.2. Animals
2.4. Implantation osmotic minipumps containing Ara-C or vehicle alone
Thirty male Sprague-Dawley rats (610–720 g; 18 months of age) were purchased from the Scientific Animal and Plant Center, Fudan University (Shanghai, China). The principles of laboratory animal care (NIH publication no. 86–23, revised 1985) were followed as
Before the implantation of osmotic minipumps, three animals in each group were sacrificed to evaluate the neurogenesis state at the end of the icariin treatment. Cytosine-b-d-arabiofuranoside (Ara-C, Sigma) was infused into the other rats at the right lateral
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Fig. 1. Chemical profile of icariin by Agilent 1100 HPLC/MS/MS/UV. (A) Icariin displays a weak response in positive ionization mode. (B), (C) Icariin exhibits strong signals in the negative ionization mode (R.T. 10.9 min) and UV detection (344–348 nm), which indicates high purity of icariin. (D), (E) The MS spectrum of the peak R.T 10.9 min is depicted in positive and negative ionization mode.
ventricle of male Sprague-Dawley rats, as previously described (Pruitt et al., 2004). Aged rats (21 months of age) were anaesthetized by an i.p. injection of a 10% Chloral Hydrate (0.5 mg/kg) solution and positioned in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, USA). The skull was exposed by a skin incision, and a small hole (1 mm) was drilled through the skull. A stainless steel cannula (outer diameter of 0.8 mm, Shanghai Syringe Co.) was implanted stereotaxically into the lateral ventricle of the right side at 1.4 mm posterior to bregma and 2.0 mm lateral with a final depth at 3.5 mm from the skull. AraC (Sigma; 2% in 0.9% saline) or vehicle alone was infused for 7 days into the brain with an osmotic pump (model 2ML; Alzet). The pumps were then removed, and the first day after the last infusion was regarded as day 0. The animals were then sacrificed, and perfused at day 0 and day 7 in two groups (n ¼3). A single i.p. BrdU injection (50 mg/kg body weight) was administered 2 h before the rats were sacrificed. Brdu immunohistochemistry was used to investigate proliferative NSCs (day 7) and to verify the effectiveness of Ara-C treatment in the elimination of actively dividing cells (day 0).
2.5. Tissue preparation and immunhistochemistry For histology, rats were deeply anaesthetized by intraperitoneal injection of pentobarbital (50 mg/kg body weight), and were perfused with 0.9% saline solution followed by 4% paraformaldehyde (PFA) solution in 0.1 M phosphate buffer solution (PBS). Brains were fixed with 4% PFA overnight. Then, the tissue was dehydrated in 30% sucrose with 4% PFA and in 30% sucrose solution in 0.1 M PBS for 24 h. Subsequently, the tissue was embedded and frozen in OCT (TissueTEK). Coronal sections (40 mm) were obtained through the dorsal hippocampus (at levels corresponding to 2.8–5.6 mm posterior to bregma). The sections were selected in multi-well dishes (Corning), and were stored at 20 1C in an antifreeze solution until they were examined. Every sixth section of the hippocampus was selected (six sections per animal), and was washed three times with 0.01 M PBS for 5 min. The tissue was fixed in 10% paraformaldehyde for 30 s, and washed again in PBS. The sections were then incubated in 2 M HCl at 37 1C for 30 min. The tissue was washed again in PBS, and subsequently permeabilized with 0.4% Triton X-100
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diluted in PBS for 30 min at room temperature. Following an additional wash, the tissue was blocked in 10% goat serum at 37 1C for 1 h. BrdU staining was performed using sheep monoclonal antBrdU (1:200 Novas), and BrdU was double-labeled with anti-GFAP (1:400 Millipore), -PSA-NCAM (1:400 Millipore), and -Olig2 (1:400 Millipore) antibodies. FITC-conjugated antisheep IgG (1:500 Millipore) and Cy3-conjugated antimouse IgG antibodies (1:1000 Millipore) were used as the secondary antibodies. For double staining, primary antibodies or secondary antibodies from
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different species were incubated simultaneously. Sections were incubated overnight at 4 1C with the diluted solution of the primary antibody. The sections were washed three times with 0.01 M PBS (15 min each), and then incubated at 37 1C for 1 h with the diluted solutions of the secondary antibodies. After washing the sections three times with PBS (15 min each), the sections were counter-stained with DAPI (1 mg/ml; Sigma) to visualize the nuclei (Fig. 3C). The co-localization with neuronal stem cell markers was analyzed using a fluorescence microscope (Nikon 80i).
Fig. 2. Morris water maze in aging rats. The typical tracking of the swimming path is represented in aging (A) control and (B) icariin rats. (C) The mean swimming speed in two groups. (D) Comparison of escape latencies to find the platform during 5 days of training. (E) The percentage of searching time and distance spent in the target quadrant. (F) The comparison of platform crosses in two groups. Results are expressed as the mean 7 SD (n¼ 15; *Po 0.05).
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2.6. Cell quantification analysis with stereology
3. Results
BrdU-immunopositive cells were detected with an antibody against BrdU in the hilus of dentate gyrus (DG) of the hippocampus. Newborn neuronal stem cells were localized in the subgranular zone (SGZ). Quantitative analysis of the number of the positively stained cells was performed with optimal magnification ( 100). All sections were defined from the Paxinos & Watson’s rat brain atlas (1997) to ensure that corresponding sections were compared between groups. The double-labeled cells in the SGZ (six sections per animal; n ¼3 for each group) were analyzed and counted using the imageanalysis software Image Pro-Plus 6.0 for windows (Copyright 1993, 2003 Media Cybernetics L.P.). A double-stained cell was defined as having the strong yellow intensity with an overlap of the green (FITC) and red (Cy3) fluorescence. The total number of marked cells in the SGZ sections was tallied and multiplied by the section-sampling fraction (Becker et al., 2007).
3.1. Icariin improves cognitive function of aging rats
2.7. Statistical analysis Data were analyzed using Student’s unpaired t-test if they were normally distributed (Kolmogorov–Smirov test); otherwise, the Mann–Whitney U-test was used. Repeated-measures ANOVAS followed by a post hoc test (Tukey B) was used for the analysis of WMM study data, using the SPSS 16.0 program. A two-tailed value of Po0.05 was considered significant.
The MWM is a well-established paradigm for evaluating deficits in hippocampal-dependent memory, and the improvement in learning and memory can be indicated by the escape latencies in hidden platform test, the percentage of time and distance in target quadrant, and the number of platform crosses in the probe test. In the present study, the results from the MWM test are shown in Fig. 2. Fig. 2A and B represents the typical tracking of the swimming path for the control and icariin rats. Fig. 2C indicates that the average swimming speed is not different when comparing the two groups. As indicated in previous literature (Lindner, 1997), the MWM test is not affected by motor or motivational factors. During the 5 day training (Fig. 2D), the rats in two groups show equivalent escape latencies on day 1, and these results suggest that the rats have similar initial performance. The aging rats following icariin administration exhibit a significant decrease in their escape latency on days 2, 3, and 5, as compared to control rats. Although similar high levels of escape latency were detected on the fourth day of training in both groups, these findings still illustrated that the icariin-treated rats show better acquisition of the task than control rats. In the probe test (Fig. 2E), rats following icariin administration spent significantly longer searching time and distance in the target quadrant compared with the control
Fig. 3. The immunofluorescence of coronal sections. (A), (B) The double-labeling of BrdU and GFAP, as arrows point to the yellow dot, to show the activation of NSCs at the end of icariin treatment. Icariin significantly increases the number of NSCs and promotes neurogenesis. (C) DAPI staining was used to visualize the nuclei. (D) BrdU immunostaining revealed very few labeled cells distributed in the SGZ. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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group (P o0.05, Po0.05, respectively), and exhibit an increased number of platform crosses (Po0.05, Fig. 2F). 3.2. Immunohistochemical analysis To evaluate the supply of newly generated NSCs in the SGZ of the hippocampus, double-labeling with immunofluorescence was utilized. BrdU-positive cells were co-labeled with a neuronal marker, BrdU and GFAP, to show NSCs as well as BrdU and PSANCAM to detect neuroblast/neural precursor cells. Additionally, double-labeling of BrdU and Olig2 was employed to measure transit-amplifying cells (Joyner and Ahn, 2005). At the end of the icariin treatment, the neurogenesis was evaluated using double-labeling of BrdU and GFAP to show the activation of neuronal stem cells. The result indicates the neurogenesis of icariin group is more than the control group in Fig. 3A and B (p o0.05; 1.86 70.54, and 5.2171.29 respectively). In both control and treated rats, BrdU immunostaining revealed very few labeled cells distributed in the SGZ at day 0. A representative image is provided in Fig. 3D. After 7 days of natural recovery, icariin administration is associated with a significant increase in the co-labeling of cells in the SGZ (Fig. 4A). The total number of BrdU and GFAP, PSA-NCAM, and Olig2 double-labeled cells increased 1.94, 3.59, 4.06-fold in icariin-treated rats compared to control rats (Po0.05, Po0.01, Po0.05, respectively; Fig. 4B).
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4. Discussion Along with the increasing average life-span of humans over the last decades, cognitive diseases are emerging as one of the greatest health threats of the twenty-first century. It is estimated that in the coming 30 years, 15 to 20 million elderly in America may exhibit some form of cognitive disability (Brookmeyer et al., 2011). The high incidence of age-related diseases in the increasing population of elderly people has stimulated interest in the search for protective agents that have the capability of preventing premature aging and delaying the onset of degenerative disorders (Poeggeler, 2005). Two monomer compounds have demonstrated some efficacy against cognitive deterioration caused by natural aging, including tetrahydroxystilbene glucoside (Li et al., 2007), and Huperzine A (Tang et al., 2000). The findings in the present study indicate that another monomer component icariin displays neuroprotective effects against cognitive impairments during natural ageing. Likewise, icariin is a component of the Chinese herb Herba Epimedii, which is often used to tonify the kidney according to the theory of traditional Chinese medicine. The Morris water maze is commonly used to assess hippocampal-dependent spatial memory in rodents (Anglade et al., 1993). Behavioral studies in animals have demonstrated that hippocampal damage can produce learning-memory impairments. During 5 days of training trials in this task, a shorter latency was found in icariin-treated animals reaching the hidden
Fig. 4. The immunofluorescence of coronal sections and statistical results of NSCs. (A) The double-labeling of cells was defined as yellow due to the overlap of the green and red fluorescence (refer to arrows). The density of dividing cells is dramatically increased in icariin-treated rats. (B) Icariin markedly elevated the amount of NSCs between 1.9- and 4.1-fold. Results are expressed as the mean 7 SD (n ¼3; *P o0.05; **P o0.01). Scale bars represent 50 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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platform when compared to control animals. An obvious tendency suggested that icariin-treated rats show better performance in Morris water maze than control rats. For instance, we consistently found that the searching time, the distance in the target quadrant, and the number of platform crosses were significantly increased by icariin administration in the probe test (Po0.05, Po0.05, Po0.05, respectively). Collectively, these behavioral results suggest that icariin has a beneficial effect on spatial learning and memory in aging rats. Under appropriate conditions, the quiescent cells may be capable of re-entering the mitotic cell cycle, and this mechanism may contribute to the stem cell pool and maintaining selfrenewal characteristics of stem cells (Alexanian and Kurpad, 2005). In the present study, double-labeling was applied to detect NSCs by utilizing Brdu in order to identify proliferative stem cells, glial fibrillary acidic protein (GFAP) to identify NSCs as well as polysialic acid-neural cell adhesion molecule (PSA-NCAM) and Olig2 to identify neuroblast/neural precursor and transit-amplifying cells (Joyner and Ahn, 2005). At the end of the icariin treatment, the neurogenesis or activation of neuronal stem cells was elevated by icariin (Fig. 3A and B). In order to explore the mechanism of the newly generated cells, AraC, which is an antimitotic reagent, was used to kills fast-dividing cells in the SGZ and left quiescent NSCs. AraC was infused into the brain of SD rats for one week using a mini-osmotic pump. At the end of AraC treatment (day 0), Brdu immunoreactivity was quantified in the lateral wall of the lateral ventricles, and our results showed that the fast-dividing precursors/neuroblasts were almost completely depleted, confirming the efficacy of AraC treatment. One week after the AraC osmotic minipumps were removed, icariin was associated with 1.9- to 4.1-fold increase in proliferative NSCs and neurogenesis. Numerous studies have shown that the dramatic decline in neurogenesis with age may contribute to impairments in learning and memory (Lazarov et al., 2010). Therefore, these results provide strong evidence that icariin can activate quiescent NSCs to stimulate neurogenesis, and may ameliorate cognitive decline. The activation of the quiescent stem cells results in increased neurogenesis. This increase in neurogenesis may potentially be one of the mechanisms associated with an improvement in the aging rat’s cognitive function. Of course, other cellular effects of icariin may be acting on the neural subpopulations of dentate gyrus. For instance, icariin could accelerate the maturation of early neural progenitor cells. Therefore, further studies are needed to determine other biological mechanism that may be involved in the improvement of age-related memory impairments. In conclusion, the overall evidence in the present study indicates that icariin treatment alleviates the age-dependent deficit in cognitive function, and promoting the activation of quiescent NSCs may be one of its mechanisms.
Acknowledgments This study was financially supported by the National Science Funds of China (30873319) and the National basic research programs of China (2010CB530402). The authors graciously acknowledge the editorial assistance of International Science Editing. References Alexanian, A.R., Kurpad, S.N., 2005. Quiescent neural cells regain multipotent stem cell characteristics influenced by adult neural stem cells in co-culture. Experimental Neurology 191, 193–197.
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