Comparison of Neurogenesis in the Dentate Gyrus Between the Adult and Aged Gerbil Following Transient Global Cerebral Ischemia

In the present study, we compared differences in cell proliferation, neuroblast differentiation and neuronal maturation in the hippocampal dentate gyrus (DG) between the adult and aged gerbil induced by 5 min of transient global cerebral ischemia using Ki-67 and BrdU (markers for cell proliferation), doublecortin (DCX, a marker for neuroblast differentiation) and neuronal nuclei (NeuN, a marker for mature neuron). The number of Ki-67-immunoreactive (⁺) cells in the DG of both the groups peaked 7 days after ischemia/reperfusion (I/R). However, the number in the aged DG was 40.6 ± 1.8% of that in the adult DG. Thereafter, the number decreased with time. After ischemic damage, DCX immunoreactivity and its protein level in the adult and aged DG peaked at 10 and 15 days post-ischemia, respectively. However, DCX immunoreactivity and its protein levels in the aged DG were much lower than those in the adult. DCX immunoreactivity and its protein level in the aged DG were 11.1 ± 0.6% and 34.4 ± 2.1% of the adult DG, respectively. In addition, the number of Ki-67⁺ cells and DCX immunoreactivity in both groups were similar to those in the sham at 60 days postischemia. At 30 days post-ischemia, the number of BrdU⁺ cells and BrdU⁺/NeuN⁺ cells in the adult-group were much higher (281.2 ± 23.4% and 126.4 ± 7.4%, respectively) than the aged-group (35.6 ± 6.8% and 79.5 ± 6.1%, respectively). These results suggest that the ability of neurogenesis in the ischemic aged DG is much lower than that in the ischemic adult DG.     

Effects of <Emphasis Type="Italic">Melissa officinalis</Emphasis> L. (Lemon Balm) Extract on Neurogenesis Associated with Serum Corticosterone and GABA in the Mouse Dentate Gyrus

Lemon balm, leaves of Melissa officinalis L., has been used for anti-anxiety and spasmolytics. We observed the extract of Melissa officinalis L. (MOE) on cell proliferation and neuroblast differentiation in the hippocampal dentate gyrus (DG) of middle-aged mice (12 months of age) using Ki67 and doublecortin (DCX), respectively. We also observed changes in corticosterone, GAD67 and GABA-transaminase (GABA-T) to check their possible mechanisms related to neurogenesis. We administered 50 or 200 mg/kg MOE to the animals once a day for 3 weeks. For labeling of newly generated cells, we also administered 5-bromodeoxyuridine (BrdU) twice a day for 3 days from the day of the first MOE treatment. Administration of 50 or 200 mg/kg MOE dose-dependently increased Ki67 positive nuclei to 244.1 and 763.9% of the vehicle-treated group, respectively. In addition, 50 or 200 mg/kg MOE significantly increased DCX positive neuroblasts with well-developed (tertiary) dendrites. Furthermore, MOE administration significantly increased BrdU/calbindin D-28 k double labeled cells (integrated neurons into granule cells in the DG) to 245.2% of the vehicle-treated group. On the other hand, administration of MOE reduced corticosterone levels in serum and decreased GABA-T levels in the DG homogenates. These results suggest that MOE increases cell proliferation, neuroblast differentiation and integration into granule cells by decreasing serum corticosterone levels as well as by increasing GABA levels in the mouse DG.     

Comparison of Newly Generated Doublecortin-immunoreactive Neuronal Progenitors in the Main Olfactory Bulb among Variously Aged Gerbils

In the present study, we investigated age-related differences in neuronal progenitors in the gerbil main olfactory bulb (MOB) using doublecortin (DCX), a marker for neuronal progenitors which differentiate into neurons in the brain. No difference in the number of neuronal nuclei (NeuN)-immunoreactive neurons was found in the MOB at variously aged gerbils. At postnatal month (PM) 1, DCX immunoreaction was detected in all layers of the MOB except for the olfactory nerve layer. At this time point, DCX-immunoreactive cells (neuronal progenitors) were very abundant; however, they did not have fully developed-processes. From PM 3, the number of DCX-immunoreactive neuronal progenitors was decreased with age. At PM 6, DCX-immunoreactive cells showed very well-developed processes. In western blot analysis, DCX protein level in the MOB was highest at PM 1. Thereafter, levels of DCX protein were decreased with age. In the subventricular zone of the lateral ventricle, the number of Ki-67-immunoractive cells (proliferating cells) was also significantly decreased with age. In addition, increases of α-synuclein-immunoreactive structures were observed in the MOB with age. These results suggest that decrease in DCX-immunoreactive neuronal progenitors and its protein levels in the MOB with age may be associated with reduction of cell proliferation in the SVZ and with an increase in α-synuclein in the MOB.     

Aripiprazole, An Atypical Antipsychotic Drug, Improves Maturation and Complexity of Neuroblast Dendrites in the Mouse Dentate Gyrus Via Increasing Superoxide Dismutases

Apripiprazole (APZ) is well known as an atypical antipsychotic and antidepressant. In the present study, we investigated effects of APZ on cell proliferation and neuronal differentiation in the dentate gyrus (DG) of the adolescent mouse using BruU, Ki-67 and doublecortin (DCX) immunohistochemistry. BruU, Ki-67 and DCX-positive (+) cells were easily detected in the subgranular zone of the DG in the vehicle- and APZ-treated group. We found that in the 8 mg/kg APZ-treated group numbers of Ki-67⁺, DCX⁺ and BrdU⁺/DCX⁺ cells were significantly increased compared with those in the vehicle-treated group. We also found that maturation and complexity of DCX⁺ dendrites in the 8 mg/kg APZ-treated group was well improved compared with those in the vehicle-treated group. In addition, markedly decreased lipid peroxidation and increased superoxide dismutase 2 (SOD2) level were observed in the DG of the 8 mg/kg APZ-treated group. Our present findings indicate that APZ can enhance cell proliferation and neuroblast differentiation, particularly maturation and complexity of neuroblast dendrites, in the DG via decreasing lipid peroxidation and increasing SOD2 level.     

Metformin Normalizes Type 2 Diabetes-Induced Decrease in Cell Proliferation and Neuroblast Differentiation in the Rat Dentate Gyrus

In this study, we observed the effects of metformin, one of the most widely prescribed drugs for the treatment of type 2 diabetes, on cell proliferation and neuroblast differentiation in the subgranular zone of the hippocampal dentate gyrus (SZDG) in Zucker diabetic fatty (ZDF) rats, which are a model for type 2 diabetes. For this, metformin was administered orally once a day to 14-week-old ZDF rats for 2 weeks and the animals were sacrificed at 16 weeks of age. During this period, blood glucose levels were higher in the vehicle-treated ZDF rats than in the Zucker lean control (ZLC) rats. Metformin treatment significantly decreased the blood glucose levels from 15.5 weeks of age. In the SZDG, Ki67 (a marker for cell proliferation)- and doublecortin (DCX, a marker for differentiated neuroblasts)-immunoreactive cells were much lower in the vehicle-treated ZDF rats than in the ZLC rats. In the metformin-treated ZDF group, Ki67- and DCX-immunoreactive cells were significantly increased in the SZDG compared to those in the vehicle-treated ZDF group. These results suggest that diabetes significantly reduces cell proliferation and neuroblast differentiation in the SZDG and that metformin treatment normalizes the reduction of cell proliferation and neuroblast differentiation in the SZDG in diabetic rats.     

Effects of Treadmill Exercise on Cell Proliferation and Differentiation in the Subgranular Zone of the Dentate Gyrus in a Rat Model of Type II Diabetes

In the present study, we investigated the effects of a treadmill exercise on serum glucose levels and Ki67 and doublecortin (DCX) immunoreactivity, which is a marker of cell proliferation expressed during cell cycles except G0 and early G1 and a marker of progenitors differentiating into neurons, respectively, in the subgranular zone of the dentate gyrus (SZDG) using a type II diabetic model. At 6 weeks of age, Zucker lean control (ZLC) and Zucker diabetic fatty (ZDF) rats were put on a treadmill with or without running for 1 h/day/5 consecutive days at 22 m/min for 5 weeks. Body weight was significantly increased in the control (without running)-ZDF rats compared to that in the other groups. In the control groups blood glucose levels were increased by 392.7 mg/dl in the control-ZDF rats and by 143.3 mg/dl in the control-ZLC rats. However, in the exercise groups, blood glucose levels were similar between the exercise-ZLC and ZDF rats: The blood glucose levels were 110.0 and 118.2 mg/dl, respectively. Ki67 positive nuclei were detected in the SZDG in control and exercise groups. The number of Ki67 positive nuclei was significantly high in exercise groups compared to that in the control groups. In addition, Ki67 positive cells were abundant in ZLC groups compared to those in ZDF groups. DCX-immunoreactive structures in the control-ZDF rats were lower than that in the control-ZLC rats. In the exercise groups, DCX-immunoreactive structures (somata and processes with tertiary dendrites) and DCX protein levels were markedly increased in both the exercise-ZLC and ZDF rats compared to that in the control groups. These results suggest that a treadmill exercise reduces blood glucose levels in ZDF rats and increases cell proliferation and differentiation in the SZDG in ZLC and ZDF rats compared to those in control groups.     

Reduced cell proliferation and neuroblast differentiation in the dentate gyrus of high fat diet-fed mice are ameliorated by metformin and glimepiride treatment

We investigated the effects of a high-fat diet (HFD) and the subsequent treatment of metformin (met) and glimepiride (glim), which are widely prescribed for type 2 diabetes, on cell proliferation and neuroblast differentiation using Ki67 and doublecortin (DCX) immunohistochemistry, respectively. Animals were fed low-fat diet (LFD) or HFD for 8 weeks. After 5 weeks of the HFD treatment, met alone or met + glim was administered orally once a day for 3 weeks. Body weight and food intake were much higher in the HFD + vehicle-treated group than the LFD-treated group. The administration of met or met + glim to the HFD-treated group resulted in a decrease in weight gain and food intake. Ki67-immunoreactive (⁺) nuclei, DCX⁺ neuroblasts and brain-derived neurotrophic factor (BDNF) protein levels were markedly decreased in the dentate gyrus (DG) of the HFD + vehicle-treated group compared to the LFD-treated group. The administration of met or met + glim to the HFD-treated group prevented the reduction of Ki67⁺ nuclei, DCX⁺ neuroblasts, BDNF levels in the DG. The intraventricular injection of K252a (a BDNF receptor blocker) to the HFD-treated group treated met or met + glim distinctively lowered the reduction of cell proliferation and neuroblast differentiation induced by HFD. These results suggest that a HFD significantly reduces cell proliferation and neuroblast differentiation by reducing BDNF levels and these effects are ameliorated by treatment with met or met + glim.     

Tanshinone I Enhances Neurogenesis in the Mouse Hippocampal Dentate Gyrus via Increasing Wnt-3, Phosphorylated Glycogen Synthase Kinase-3β and β-Catenin Immunoreactivities

Tanshinone I (TsI), a lipophilic diterpene extracted from Danshan (Radix Salvia miltiorrhizae), exerts neuroprotection in cerebrovascular diseases including transient ischemic attack. In this study, we examined effects of TsI on cell proliferation and neuronal differentiation in the subgranular zone (SGZ) of the mouse dentate gyrus (DG) using Ki-67, BrdU and doublecortin (DCX) immunohistochemistry. Mice were treated with 1 and 2 mg/kg TsI for 28 days. In the 1 mg/kg TsI-treated-group, distribution patterns of BrdU, Ki-67 and DCX positive (⁺) cells in the SGZ were similar to those in the vehicle-treated-group. However, in the 2 mg/kg TsI-treated-group, double labeled BrdU⁺/NeuN⁺ cells, which are mature neurons, as well as Ki-67⁺, DCX⁺ and BrdU⁺ cells were significantly increased compared with those in the vehicle-treated-group. On the other hand, immunoreactivities and protein levels of Wnt-3, β-catenin and serine-9-glycogen synthase kinase-3β (p-GSK-3β), which are related with morphogenesis, were significantly increased in the granule cell layer of the DG only in the 2 mg/kg TsI-treated-group. Therefore, these findings indicate that TsI can promote neurogenesis in the mouse DG and that the neurogenesis is related with increases of Wnt-3, p-GSK-3β and β-catenin immunoreactivities.     

Effects of a new synthetic butyrylcholinesterase inhibitor, HBU-39, on cell proliferation and neuroblast differentiation in the hippocampal dentate gyrus in a scopolamine-induced amnesia animal model

In this study, we synthesized [1-(4-(benzo[d][1,3]dioxol-5-ylmethyl)piperazin-1-yl)-5-(1,2-dithiolan-3-yl)pentan-1-one, HBU-39], a (α)-lipoic acid derivative, and found this compound strongly inhibited butyrylcholinesterase (BuChE) in an in vitro experiment. We also examined the effects of HBU-39 on cell proliferation and neuroblast differentiation using the specific markers Ki67 and doublecortin (DCX), respectively, in the hippocampal dentate gyrus of a rat model of scopolamine-induced amnesia. For this, scopolamine was subcutaneously administered for 28 days by an ALzet osmotic minipump (44 mg/mL delivered at 2.5 μL/h). HBU-39 (1 mg/kg per day) and galantamine (an acetylcholinesterase inhibitor used as a control; 5 mg/kg per day) were intraperitoneally administered for 28 days. The administration of scopolamine significantly decreased the mean number of Ki67- and DCX-immunoreactive cells in the dentate gyrus. However, treatment with both HBU-39 and galantamine significantly ameliorated the reductions in cell proliferation and neuroblast differentiation. In particular, the mean number of Ki67- and DCX-immunoreactive cells was prominently abundant in the HBU-treated group compared to that in the galantamine-treated group. These results suggest that the BuChE inhibitor, HBU-39, can ameliorate the scopolamine-induced reductions of cell proliferation and neuroblast differentiation, and HBU-39 may be applicable to amnesia patients to promote memory functions.     

Topiramate Improves Neuroblast Differentiation of Hippocampal Dentate Gyrus in the <Emphasis Type="SmallCaps">d</Emphasis>-Galactose-Induced Aging Mice via Its Antioxidant Effects

Some anticonvulsant drugs are associated with cognitive ability in patients; Topiramate (TPM) is well known as an effective anticonvulsant agent applied in clinical settings. However, the effect of TPM on the cognitive function is rarely studied. In this study, we aimed to observe the effects of TPM on cell proliferation and neuronal differentiation in the dentate gyrus (DG) of the d-galactose-induced aging mice by Ki-67 and doublecortin (DCX) immunohistochemistry. The study is divided into four groups including control, d-galactose-treated group, 25 and 50 mg/kg TPM-treated plus d-galactose-treated groups. We found, 50 mg/kg (not 25 mg/kg) TPM treatment significantly increased the numbers of Ki-67⁺ cells and DCX immunoreactivity, and improved neuroblast injury induced by d-galactose treatment. In addition, we also found that decreased immunoreactivities and protein levels of antioxidants including superoxide dismutase and catalase induced by d-galactose treatment were significantly recovered by 50 mg/kg TPM treatment in the mice hippocampal DG (P < 0.05). In conclusion, our present results indicate that TPM can ameliorate neuroblast damage and promote cell proliferation and neuroblast differentiation in the hippocampal DG via increasing SODs and catalase levels in the d-galactose mice.     

Zinc chelation reduces traumatic brain injury-induced neurogenesis in the subgranular zone of the hippocampal dentate gyrus

Numerous studies have demonstrated that traumatic brain injury (TBI) increases hippocampal neurogenesis in the rodent brain. However, the mechanisms underlying increased neurogenesis after TBI remain unknown. Continuous neurogenesis occurs in the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG) in the adult brain. The mechanism that maintains active neurogenesis in the hippocampal area is not known. A high level of vesicular zinc is localized in the presynaptic terminals of the SGZ (mossy fiber). The mossy fiber of dentate granular cells contains high levels of chelatable zinc in their terminal vesicles, which can be released into the extracellular space during neuronal activity. Previously, our lab presented findings indicating that a possible correlation may exist between synaptic zinc localization and high rates of neurogenesis in this area after hypoglycemia or epilepsy. Using a weight drop animal model to mimic human TBI, we tested our hypothesis that zinc plays a key role in modulating hippocampal neurogenesis after TBI. Thus, we injected a zinc chelator, clioquinol (CQ, 30 mg/kg), into the intraperitoneal space to reduce brain zinc availability twice per day for 1 week. Neuronal death was evaluated with Fluoro Jade-B and NeuN staining to determine whether CQ has neuroprotective effects after TBI. The number of degenerating neurons (FJB (+)) and live neurons (NeuN (+)) was similar in vehicle and in CQ-treated rats at 1 week after TBI. Neurogenesis was evaluated using BrdU, Ki67 and doublecortin (DCX) immunostaining 1 week after TBI. The number of BrdU, Ki67 and DCX positive cell was increased after TBI. However, the number of BrdU, Ki67 and DCX positive cells was significantly decreased by CQ treatment. The present study shows that zinc chelation did not prevent neurodegeneration but did reduce TBI-induced progenitor cell proliferation and neurogenesis. Therefore, this study suggests that zinc has an essential role for modulating hippocampal neurogenesis after TBI.     

Effects of Sensitive to Apoptosis Gene Protein on Cell Proliferation, Neuroblast Differentiation, and Oxidative Stress in the Mouse Dentate Gyrus

Sensitive to apoptosis gene (SAG) protein is a redox-inducible protein that protects cells against apoptosis induced by redox agents. In this study, we observed effects of SAG on cell proliferation and neuroblast differentiation in the mouse hippocampal dentate gyrus (DG) using Ki67 and doublecortin (DCX), respectively. For easy penetration into neurons, Tat-SAG expression vector was constructed by ligation with SAG and expression vector, Tat, in-frame with six histidine open-reading frames to generate the expression vector, and cloned into E. coli DH5α cells. One or 5?mg/kg Tat-SAG fusion protein (Tat-SAG) was intraperitoneally administered to mice once a day for 3?weeks. The administration of Tat-SAG significantly increased the number of 5-bromodeoxyuridine positive cells, Ki67 positive cells and DCX immunoreactive neuroblast in the mouse DG: Especially, in the 5?mg/kg Tat-SAG-treated mice, DCX positive neuroblasts showed a well-developed arborization of tertiary dendrites in the DG. On the other hand, we examined that the administration of Tat-SAG significantly reduced the DNA damage and lipid peroxidation judging from 8-hydroxy-2'-deoxyguanosine and 4-hydroxynonenal immunohistochemistry: The decrease was much more distinct in the 5?mg/kg Tat-SAG-treated mice than 1?mg/kg Tat-SAG-treated mice. This result suggests that SAG significantly increases cell proliferation, neuroblast differentiation and oxidative stress in normal states.     

Stress-Induced Anxiety- and Depressive-Like Phenotype Associated with Transient Reduction in Neurogenesis in Adult Nestin-CreERT2/Diphtheria Toxin Fragment A Transgenic Mice

Depression and anxiety involve hippocampal dysfunction, but the specific relationship between these mood disorders and adult hippocampal dentate gyrus neurogenesis remains unclear. In both humans with MDD and rodent models of depression, administration of antidepressants increases DG progenitor and granule cell number, yet rodents with induced ablation of DG neurogenesis typically do not demonstrate depressive- or anxiety-like behaviors. The conflicting data may be explained by the varied duration and degree to which adult neurogenesis is reduced in different rodent neurogenesis ablation models. In order to test this hypothesis we examined how a transient–rather than permanent–inducible reduction in neurogenesis would alter depressive- and anxiety-like behaviors. Transgenic Nestin-CreER^T2/floxed diphtheria toxin fragment A (DTA) mice (Cre+DTA+) and littermates (Cre+DTA-; control) were given tamoxifen (TAM) to induce recombination and decrease nestin-expressing stem cells and their progeny. The decreased neurogenesis was transient: 12 days post-TAM Cre+DTA+ mice had fewer DG proliferating Ki67+ cells and fewer DCX+ neuroblasts/immature neurons relative to control, but 30 days post-TAM Cre+DTA+ mice had the same DCX+ cell number as control. This ability of DG neurogenesis to recover after partial ablation also correlated with changes in behavior. Relative to control, Cre+DTA+ mice tested between 12–30 days post-TAM displayed indices of a stress-induced anxiety phenotype–longer latency to consume highly palatable food in the unfamiliar cage in the novelty-induced hypophagia test, and a depression phenotype–longer time of immobility in the tail suspension test, but Cre+DTA+ mice tested after 30 days post-TAM did not. These findings suggest a functional association between adult neurogenesis and stress induced anxiety- and depressive-like behaviors, where induced reduction in DCX+ cells at the time of behavioral testing is coupled with stress-induced anxiety and a depressive phenotype, and recovery of DCX+ cell number corresponds to normalization of these behaviors.     

PEP-1-frataxin significantly increases cell proliferation and neuroblast differentiation by reducing lipid peroxidation in the mouse dentate gyrus

Frataxin plays important roles in the mitochondrial respiratory chain and in the differentiation of neurons during early development. In this study, we observed the effects of frataxin on cell proliferation and neuroblast differentiation in the mouse hippocampal dentate gyrus. For this, we constructed an expression vector, PEP-1, that was fused with frataxin to create a PEP-1-frataxin fusion protein that easily penetrated frataxin into the blood-brain barrier. Three mg/kg PEP-1-frataxin was intraperitoneally administered to mice once a day for 2 weeks. The administration of PEP-1 alone did not result in any significant changes in the number of Ki67-positive cells and doublecortin (DCX)-immunoreactive neuroblasts in the mouse dentate gyrus. However, the administration of PEP-1-frataxin significantly increased the number of Ki67-positive cells and DCX-immunoreactive neuroblasts in the mouse dentate gyrus. In addition, PEP-1-frataxin significantly reduced 4-hydroxynonenal protein levels and malondialdehyde formation, while Cu, Zn-superoxide dismutase protein levels were maintained. These results suggest that frataxin effectively increased cell proliferation and neuroblast differentiation by decreasing lipid peroxidation in the dentate gyrus.     

Cyclosporine A Reduces Dendritic Outgrowth of Neuroblasts in the Subgranular Zone of the Dentate Gyrus in C57BL/6 Mice

In the present study, we observed the effects of cyclosporine A (CsA), an efficient immunosuppressant, on cell proliferation and neuroblast differentiation in the subgranular zone of the dentate gyrus (SZDG) in normal C57BL/6 mice using Ki67 and doublecortin (DCX) immunohistochemical staining, respectively. At 8 weeks of age, vehicle (physiological saline) or CsA was daily administered (40 mg/kg, i.p.) for 1 week. Animals were sacrificed at 2 weeks after last administration. CsA treatment did not show any influences in neurons, astrocytes and microglia based on immunohistochemistry for its markers, respectively. However, in the CsA-treated group, Fluoro-Jade B, a marker for neurodegeneration, positive cells were found in the SZDG, not in the vehicle-treated group. In the vehicle-treated group, Ki67 immunoreactive (⁺) nuclei were clustered in the SZDG, whereas in the CsA-treated group Ki67⁺ nuclei were scattered in the SZDG, showing no difference in cell numbers. Numbers of DCX⁺ neuroblasts with well-developed processes (tertiary dendrites) were much lower in the CsA-treated group than those in the vehicle-treated group; however, numbers of DCX⁺ neuroblasts with secondary dendrites were similar in both the groups. These results suggest that CsA significantly reduces dendritic outgrowth and complexity from neuroblasts in the SZDG without any affecting in neurons, astrocytes and microglia in normal mice.     

Reduced Hippocampal Cell Differentiation in the Subgranular Zone of the Dentate Gyrus in a Rat Model of Type II Diabetes

It has recently been reported that diabetes mellitus is strongly associated with neurodegenerative and functional disorders of the central nervous system. In the present study, we investigated the changes in proliferating neurons in the dentate gyrus of type II diabetic rats using doublecortin (DCX), a marker of progenitors differentiating into neurons. At 4 weeks after birth, there were no differences in the blood glucose levels of Zucker diabetic fatty (ZDF) rats or Zucker lean control (ZLC) rats. DCX-immunoreactive neurons were detectable in the subgranular zone of the dentate gyrus in both the ZDF and ZLC rats; however, DCX immunoreactivity was higher in the ZLC rats than in the ZDF rats. At 12 weeks after birth, the blood glucose level was significantly increased by 400 mg/dl in the ZDF rats, but the blood glucose level in the ZLC rats was only slightly increased by 152.3 mg/dl. DCX immunoreactivity was significantly decreased in 12-week-old rats in comparison to 4-week-old rats. Some DCX-immunoreactive neurons were detectable in the subgranular zone of the dentate gyrus in the ZLC rats. However, only a few DCX-immunoreactive neurons were observed in the ZDF rats, and the DCX-immunoreactive neurons in the ZDF rats did not show fully developed processes. These results suggest that DCX-immunoreactive neurons were significantly decreased in an age-dependent manner and that DCX-immunoreactive neurons were also reduced in diabetic rats. In addition, the reduction in DCX-immunoreactive neurons in age matched rats may be associated with type II diabetes.     

Effects of Treadmill Exercise Combined with MK 801 Treatment on Neuroblast Differentiation in the Dentate Gyrus in Rats

N-methyl-d-aspartate receptor (NR) is involved in activity-dependent synaptic plasticity, such as associative long-term potentiation, and in related central functions, such as learning and memory. In this study, we observed effects of treadmill exercise on NR1 and doublecortin (DCX, a marker for neuroblast differentiation) in the subgranular zone of the dentate gyrus (DG). At 6 weeks of age, rats were put on a treadmill with or without running for 1 h/day for 5 consecutive days at 22 m/min for 5 weeks. Exercise increased NR1 immunoreactivity and protein level in the hippocampus. To identify the correlations between NR and neuroblasts, we intraperitoneally administered a NR antagonist, MK-801, to the exercised rats. MK-801 treatment reduced NR1 protein level in the hippocampus of the exercised rats. In addition, in the MK-801-treated group, the number of DCX cells was significantly decreased in the subgranular zone of the DG. These results suggest that NR may be one of the important factors that modulate neuroblast differentiation during exercise in rats.     

Systemic Inflammation Impairs Proliferation of Hippocampal Type 2 Intermediate Precursor Cells

Neurogenesis is a plastic event modulated by external cues. Systemic inflammation decreases neurogenesis in the dentate gyrus (DG) in part through the proliferative restrain of neural precursor cells (NPCs). To evaluate if inflammation affects the cell cycle progression of particular populations of NPCs, we treated young-adult mice with a single i.p. injection of saline or 1 mg/kg LPS. After 7 days, we analysed proliferation of new BrdU+/DCX+ cells through immunohistochemistry. We extracted the hippocampus and performed a neurosphere assay and a flow cytometric analysis to evaluate proliferation and to identify the phase of the cell cycle in specific populations of DG-derived NPCs. We show that the number of BrdU+/DCX+ cells diminishes in the LPS-treated group and that the number of primary neurospheres derived from LPS-injected animals is significantly reduced compared to the saline-injected group. Flow cytometry revealed that inflammation does not affect the total number of Type 1 BLBP+/TBR2? cells, while the total number of Type 2 intermediate precursor cells (IPCs) (TBR2+) from the LPS-treated group was increased. Cell cycle analysis shows a decrease in the total rate of NPCs in phases S, G2 and M in the LPS-treated group. The percentage of Type 1 BLBP+/TBR2? cells in each cell cycle phase was not different between groups, while there was a fewer number of Type 2 TBR2+ cells in S/G2/M phase. These results show that inflammation alters the appropriate cell cycle progression of Type 2 IPCs, which may contribute to the decrease in the birth rate of DG neurons.     

Simvastatin augments the efficacy of therapeutic angiogenesis induced by bone marrow-derived mesenchymal stem cells in a murine model of hindlimb ischemia

Many studies showed beneficial effects of either statin or bone marrow-derived mesenchymal stem cells (MSC) treatment in ischemic disease. In an attempt to further improve postischemic tissue repair, we investigated the effect of a local administration of MSC, in the presence or not of low-dose simvastatin, on angiogenesis and functional recovery in a mouse model of hindlimb ischemia. In vitro, the proliferation, migration, apoptosis, and tube formation of bone marrow MSC derived from transgenic mice expressing green fluorescent protein (GFP) were detected in the presence or not of 0.01 μmol/l simvastatin, respectively. In vivo, immediately after hindlimb ischemia, the mice were divided into four groups, namely control, MSC, statin, and statin-MSC, and received a single local injection of MSC (2 × 10⁶ cells) and/or a repeated gavages’ administration of simvastatin (0.2 mg/kg) for 21 days. The blood flow was measured by laser Doppler imaging, the capillary density was detected by alkaline phosphatase staining and, the MSC differentiation was assessed by immunofluorescent staining at 21 days after the ischemia. In vitro, the MSC proliferation rate, migration ability and tube formation number were increased significantly in simvastatin group relative to control group. Whereas, the H₂O₂ induced-apoptosis was inhibited significantly in simvastatin group relative to control group. In vivo, hindlimb blood reperfusion was significantly improved (MSC 0.55 ± 0.08, statin 0.57 ± 0.05, vs. control 0.47 ± 0.07, P < 0.05) and capillary density was obviously higher at day 21 post-ischemia by Laser Doppler Imaging in the MSC group and the Statin group when compared with control group. The combined use of statin and MSC further improved revascularization (perfusion ratio of 0.70 ± 0.09; P < 0.001 verse other groups) and resulted in the highest capillary density (P < 0.05 vs. all other groups). GFP-labeled transplanted cells were more frequently observed in the Statin-MSC group than in the MSC group (6.8 ± 0.5–3.1 ± 0.7, P < 0.05). Low-dose simvastatin could act in a synergistic way with MSC to potentiate the functional neovascularization in a mouse model of hind limb ischemia.     

Epileptogenesis following Kainic Acid-Induced Status Epilepticus in Cyclin D2 Knock-Out Mice with Diminished Adult Neurogenesis

The goal of this study was to determine whether a substantial decrease in adult neurogenesis influences epileptogenesis evoked by the intra-amygdala injection of kainic acid (KA). Cyclin D2 knockout (cD2 KO) mice, which lack adult neurogenesis almost entirely, were used as a model. First, we examined whether status epilepticus (SE) evoked by an intra-amygdala injection of KA induces cell proliferation in cD2 KO mice. On the day after SE, we injected BrdU into mice for 5 days and evaluated the number of DCX- and DCX/BrdU-immunopositive cells 3 days later. In cD2 KO control animals, only a small number of DCX+ cells was observed. The number of DCX+ and DCX/BrdU+ cells/mm of subgranular layer in cD2 KO mice increased significantly following SE (p<0.05). However, the number of newly born cells was very low and was significantly lower than in KA-treated wild type (wt) mice. To evaluate the impact of diminished neurogenesis on epileptogenesis and early epilepsy, we performed video-EEG monitoring of wt and cD2 KO mice for 16 days following SE. The number of animals with seizures did not differ between wt (11 out of 15) and cD2 KO (9 out of 12) mice. The median latency to the first spontaneous seizure was 4 days (range 2 – 10 days) in wt mice and 8 days (range 2 – 16 days) in cD2 KO mice and did not differ significantly between groups. Similarly, no differences were observed in median seizure frequency (wt: 1.23, range 0.1 – 3.4; cD2 KO: 0.57, range 0.1 – 2.0 seizures/day) or median seizure duration (wt: 51 s, range 23 – 103; cD2 KO: 51 s, range 23 – 103). Our results indicate that SE-induced epileptogenesis is not disrupted in mice with markedly reduced adult neurogenesis. However, we cannot exclude the contribution of reduced neurogenesis to the chronic epileptic state.