Epigenetic aging in adult neurogenesis

Neural stem cells (NSCs) in the hippocampus generate new neurons throughout life, which functionally contribute to cognitive flexibility and mood regulation. Yet adult hippocampal neurogenesis substantially declines with age and age-related impairments in NSC activity underlie this reduction. Particularly, increased NSC quiescence and consequently reduced NSC proliferation are considered to be major drivers of the low neurogenesis levels in the aged brain. Epigenetic regulators control the gene expression programs underlying NSC quiescence, proliferation and differentiation and are hence critical to the regulation of adult neurogenesis. Epigenetic alterations have also emerged as central hallmarks of aging, and recent studies suggest the deterioration of the NSC-specific epigenetic landscape as a driver of the age-dependent decline in adult neurogenesis. In this review, we summarize the recently accumulating evidence for a role of epigenetic dysregulation in NSC aging and propose perspectives for future research directions.     

Derivation of neural stem cells from an animal model of psychiatric disease

Several psychiatric and neurological diseases are associated with altered hippocampal neurogenesis, suggesting differing neural stem cell (NSC) function may play a critical role in these diseases. To investigate the role of resident NSCs in a murine model of psychiatric disease, we sought to isolate and characterize NSCs from alpha-calcium-/calmodulin-dependent protein kinase II heterozygous knockout (CaMK2α-hKO) mice, a model of schizophrenia/bipolar disorder. These mice display altered neurogenesis, impaired neuronal development and are part of a larger family possessing phenotypic and behavioral correlates of schizophrenia/bipolar disorder and a shared pathology referred to as the immature dentate gyrus (iDG). The extent to which NSCs contribute to iDG pathophysiology remains unclear. To address this, we established heterogeneous cultures of NSCs isolated from the hippocampal neuropoietic niche. When induced to differentiate, CaMK2α-hKO-derived NSCs recapitulate organotypic hippocampal neurogenesis, but generate larger numbers of immature neurons than wild-type (WT) littermates. Furthermore, mutant neurons fail to assume mature phenotypes (including morphology and MAP2/calbindin expression) at the same rate observed in WT counterparts. The increased production of immature neurons which fail to mature indicates that this reductionist model retains key animal- and iDG-specific maturational deficits observed in animal models and human patients. This is doubly significant, as these stem cells lack several developmental inputs present in vivo. Interestingly, NSCs were isolated from animals prior to the emergence of overt iDG pathophysiology, suggesting mutant NSCs may possess lasting intrinsic alterations and that altered NSC function may contribute to iDG pathophysiology in adult animals.     

NSCs promote hippocampal neurogenesis,metabolic changes and synaptogenesis in APP/PS1 transgenic mice

Adult neurogenesis and synaptic remodeling persist as a unique form of structural and functional plasticity in the hippocampal dentate gyrus (DG) and subventricular zone (SVZ) of the lateral ventricles due to the existence of neural stem cells (NSCs). Transplantation of NSCs may represent a promising approach for the recovery of neural circuits. Here, we aimed to examine effects of highly neuronal differentiation of NSCs transplantation on hippocampal neurogenesis, metabolic changes and synaptic formation in APP/PS1 mice. 12‐month‐old APP/PS1 mice were used for behavioral tests, immunohistochemistry, western blot, transmission electron microscopy and proton magnetic resonance spectroscopy (1H‐MRS). The results showed that N‐acetylaspartate (NAA) and Glutamate (Glu) levels were increased in the Tg‐NSC mice compared with the Tg‐PBS and Tg‐AD mice 10 weeks after NSCs transplantation. NSC‐induced an increase in expression of synaptophysin and postsynaptic protein‐95, and the number of neurons with normal synapses was significantly increased in Tg‐NSC mice. More doublecortin‐, BrdU/NeuN‐ and Nestin‐positive neurons were observed in the hippocampal DG and SVZ of the Tg‐NSC mice. This is the first demonstration that engrafted NSCs with a high differentiation rate to neurons can enhance neurogenesis in a mouse model of AD and can be detected by 1H‐MRS in vivo. It is suggested that engraft of NSCs can restore memory and promote endogenous neurogenesis and synaptic remodeling, moreover, 1H‐MRS can detect metabolite changes in AD mice in vivo. The observed changes in NAA/creatine (Cr) and glutamate (Glu)/Cr may be correlated with newborn neurons and new synapse formation.     

Spatiotemporal changes to the subventricular zone stem cell pool through aging

Through adulthood, the rodent subventricular zone (SVZ) stem cell niche generates new olfactory bulb interneurons. We had previously reported that the number of new neurons produced in the SVZ declines through aging; however, age-related changes attributable specifically to the SVZ neural stem cell (NSC) population have not been fully characterized. Here, we conducted a spatiotemporal evaluation of adult SVZ NSCs. We assessed ventricle-contacting NSCs, which together with ependymal cells form regenerative units (pinwheels) along the lateral wall of the lateral ventricle. Based on their apical GFAP-expressing process, individual NSCs were identified across the ventricle surface using serial reconstruction of the SVZ. We observed an 86% decline in total NSCs/mm2 of intact ependyma in 2-year old versus 3-month-old mice, with fewer NSC processes within each aged pinwheel. This resulted in an associated 78% decline in total pinwheel units/mm2. Regional analysis along the lateral ventricle surface revealed that the age-dependent decline of NSCs and pinwheels is spatially uniform and ultimately maintains the conserved ratio of olfactory bulb interneuron subtypes generated in young mice. However, the overall neurogenic output of the aged SVZ is reduced. Surprisingly, we found no significant change in the number of actively proliferating NSCs per mm2 of ventricle surface. Instead, our data reveal that, although the total NSC number, pinwheel units and NSCs per pinwheel decline with age, the percentage of actively, mitotic NSCs increases, indicating that age-related declines in SVZ-mediated olfactory bulb neurogenesis occur downstream of NSC proliferation.     

Activation of GPR55 induces neuroprotection of hippocampal neurogenesis and immune responses of neural stem cells following chronic,systemic inflammation

New neurons are continuously produced by neural stem cells (NSCs) within the adult hippocampus. Numerous diseases, including major depressive disorder and HIV-1 associated neurocognitive disorder, are associated with decreased rates of adult neurogenesis. A hallmark of these conditions is a chronic release of neuroinflammatory mediators by activated resident glia. Recent studies have shown a neuroprotective role on NSCs of cannabinoid receptor activation. Yet, little is known about the effects of GPR55, a candidate cannabinoid receptor, activation on reductions of neurogenesis in response to inflammatory insult. In the present study, we examined NSCs exposed to IL-1β in vitro to assess inflammation-caused effects on NSC differentiation and the ability of GPR55 agonists to attenuate NSC injury. NSC differentiation and neurogenesis was determined via immunofluorescence and flow cytometric analysis of NSC markers (Nestin, Sox2, DCX, S100β, βIII Tubulin, GFAP). GPR55 agonist treatment protected against IL-1β induced reductions in neurogenesis rates. Moreover, inflammatory cytokine receptor mRNA expression was down regulated by GPR55 activation in a neuroprotective manner. To determine inflammatory responses in vivo, we treated C57BL/6 and GPR55^−/− mice with LPS (0.2 mg/kg/day) continuously for 14 days via osmotic mini-pump. Reductions in NSC survival (as determined by BrdU incorporation), immature neurons, and neuroblast formation due to LPS were attenuated by concurrent direct intrahippocampal administration of the GPR55 agonist, O-1602 (4 µg/kg/day). Molecular analysis of the hippocampal region showed a suppressed ability to regulate immune responses by GPR55^−/− animals manifesting in a prolonged inflammatory response (IL-1β, IL-6, TNFα) after chronic, systemic inflammation as compared to C57BL/6 animals. Taken together, these results suggest a neuroprotective role of GPR55 activation on NSCs in vitro and in vivo and that GPR55 provides a novel therapeutic target against negative regulation of hippocampal neurogenesis by inflammatory insult.     

CXCR4 prevents dispersion of granule neuron precursors in the adult dentate gyrus

Neurogenesis in the adult dentate gyrus (DG) generates new granule neurons that differentiate in the inner one‐third of the granule cell layer (GCL). The migrating precursors of these neurons arise from neural stem cells (NSCs) in the subgranular zone (SGZ). Although it is established that pathological conditions, including epilepsy and stroke, cause dispersion of granule neuron precursors, little is known about the factors that regulate their normal placement. Based on the high expression of the chemokine CXCL12 in the adult GCL and its role in guiding neuronal migration in development, we addressed the function of the CXCL12 receptor CXCR4 in adult neurogenesis. Using transgenic reporter mice, we detected Cxcr4‐GFP expression in NSCs, neuronal‐committed progenitors, and immature neurons of adult and aged mice. Analyses of hippocampal NSC cultures and hippocampal tissue by immunoblot and immunohistochemistry provided evidence for CXCL12‐promoted phosphorylation/activation of CXCR4 receptors in NSCs in vivo and in vitro. Cxcr4 deletion in NSCs of the postnatal or mature DG using Cre technology reduced neurogenesis. Fifty days after Cxcr4 ablation in the mature DG, the SGZ showed a severe reduction of Sox2‐positive neural stem/early progenitor cells, NeuroD‐positive neuronal‐committed progenitors, and DCX‐positive immature neurons. Many immature neurons were ectopically placed in the hilus and inner molecular layer, and some developed an aberrant dendritic morphology. Only few misplaced cells survived permanently as ectopic neurons. Thus, CXCR4 signaling maintains the NSC pool in the DG and specifies the inner one‐third of the GCL as differentiation area for immature granule neurons. © 2013 Wiley Periodicals, Inc.     

Early‐life stress diminishes the increase in neurogenesis after exercise in adult female mice

Exposure to early‐life stress (ES) has long‐lasting consequences for later cognition and hippocampal plasticity, including adult hippocampal neurogenesis (AHN), i.e., the generation of new neurons from stem/progenitor cells in the adult hippocampal dentate gyrus. We had previously demonstrated a sex‐specific vulnerability to ES exposure; female mice exposed to ES from P2‐P9 exhibited only very mild cognitive changes and no reductions in AHN as adult, whereas ES‐exposed male mice showed impaired cognition closely associated with reductions in AHN. Given the apparent resilience of AHN to ES in females, we here questioned whether ES has also altered the capacity to respond to positive stimuli for neurogenesis. We therefore investigated whether exercise, known for its strong pro‐neurogenic effects, can still stimulate AHN in adult female mice that had been earlier exposed to ES. We confirm a strong pro‐neurogenic effect of exercise in the dorsal hippocampus of 8‐month‐old control female mice, but this positive neurogenic response is less apparent in female ES mice. These data provide novel insights in the lasting consequences of ES on hippocampal plasticity in females and also indicate that ES might lastingly reduce the responsiveness of the hippocampal stem cell pool, to exercise, in female mice.     

Nerve growth factor promotes survival of new neurons in the adult hippocampus

Exogenously provided NGF enhances cognitive performance in impaired rodents and humans and is currently a promising compound for the treatment of dementia. To investigate whether NGF-dependent cognitive improvement may be due in part to increased hippocampal neurogenesis, adult and aged male rats were treated with NGF or vehicle intracerebroventricularly for 6 or 20 days followed by evaluation of cholinergic parameters and hippocampal neurogenesis. We show that NGF increases hippocampal cholinergic activity as rapidly as 3 days after initiation of treatment. NGF treatment for 6 days did not affect proliferation of progenitor cells in the dentate gyrus granule cell layer (GCL). However, continuous NGF infusion enhanced survival of new neurons in the GCL of young adult, but not aged rats. Taken together, these findings suggest that NGF, likely mediated through increased cholinergic tone, promotes neurogenesis in the adult hippocampus, which may relate to the nootropic action of NGF.     

Practical issues in stem cell therapy for Alzheimer's disease

We have demonstrated that aged animals show significant improvements in cognitive function and neurogenesis after brain transplantation of human neural stem cells or of human adult mesenchymal stem cells that have been dedifferentiated by transfection of the embryonic stem cell gene. We have also demonstrated that peripheral administration of a pyrimidine derivative increased cognition, endogenous brain stem cell proliferation and neurogenesis. These results indicate a bright future for stem cell therapies in Alzheimer's disease (AD). Before this is realized, however, we need to consider the affect of AD pathology on stem cell biology to establish an effective stem cell therapy for this disease. Although amyloid-beta (Abeta) deposition is a hallmark of AD, an absence of a phenotype in the beta-amyloid precursor protein (APP) knockout mouse, might lead one to underestimate the potential physiological functions of APP and suggest that it is unessential or can be compensated for. We have found, however, that APP is needed for differentiation of neural stem cells (NSCs) in vitro, and that NSCs transplanted into a APP-knockout mouse did not migrate or differentiate -- indicating that APP plays an important role in differentiation or migration process of NSCs in the brain. Then again, treatment with high a concentration of APP or its over-expression increased glial differentiation of NSCs. Human NSCs transplanted into APP-transgenic mouse brain exhibited less neurogenesis and active gliosis around the plaque like formations. Treatment of such animals with the compound, (+)-phenserine, that is known to reduce APP protein levels, increased neurogenesis and suppressed gliosis. These results suggest APP levels can regulate NSC biology in the adult brain, that altered APP metabolism in Down syndrome or AD may have implications for the pathophysiology of these diseases, and that a combination of stem cell therapy and regulation of APP levels could provide a treatment strategy for these disorders.     

Transgenic inactivation of acetylcholinesterase impairs homeostasis in mouse hippocampal granule cells

In the adult murine hippocampus, dentate gyrus (DG), neurogenesis and neural cell death are thought to affect learning and memory in incompletely understood mechanism(s). Because cholinergic neurotransmission influences both of these functions, we hypothesized that cholinergic signaling, affected by acetylcholinesterase (AChE) activity, expression level, and alternative splicing, may provide a link between these processes. To challenge this hypothesis, we compared DG neurogenesis in transgenic mice overexpressing engineered "synaptic" AChE-S, incapable of acetylcholine (ACh) hydrolysis (TgSin) with strain-matched controls. In control mice, we observed increasing AChE gene expression with progressing neurogenesis. This involved dividing DG neurons expressing proliferating cell nuclear antigen (PCNA) and Tuj1-positive committed neurons compared with neighboring cells. However, TgSin hippocampi with lower hydrolytic AChE activity showed more PCNA-labeled cells than controls. In contrast, TgS mice overexpressing catalytically active AChE-S, with higher than control levels of AChE hydrolytic activity, presented elevated cell labeling by both bromodeoxyuridine and caspase-3, reflecting facilitated survival of newly born neurons as well as increased neural apoptosis. In comparison, overexpression of the stress-induced "readthrough" AChE-R variant in TgR mice resulted in higher hydrolytic activities but unchanged neurogenesis and apoptosis parameters, while all strains presented similar granule cell layer areas, cell density, and neuron numbers. Importantly, this homeostasis was maintained at a cognitive cost: in the hippocampal-dependent socially transmitted food preference task, TgS and TgSin mice showed impaired acquisition and retention, respectively. Our findings suggest that replacement of AChE-S with AChE-R serves to maintain DG homeostasis and associated cognitive tasks, highlighting the role of cholinergic signaling in adult hippocampal neurogenesis and functioning.     

Mechanisms underlying the effect of voluntary running on adult hippocampal neurogenesis

Adult hippocampal neurogenesis is important for preserving learning and memory-related cognitive functions. Physical exercise, especially voluntary running, is one of the strongest stimuli to promote neurogenesis and has beneficial effects on cognitive functions. Voluntary running promotes exit of neural stem cells (NSCs) from the quiescent stage, proliferation of NSCs and progenitors, survival of newborn cells, morphological development of immature neuron, and integration of new neurons into the hippocampal circuitry. However, the detailed mechanisms driving these changes remain unclear. In this review, we will summarize current knowledge with respect to molecular mechanisms underlying voluntary running-induced neurogenesis, highlighting recent genome-wide gene expression analyses. In addition, we will discuss new approaches and future directions for dissecting the complex cellular mechanisms driving change in adult-born new neurons in response to physical exercise.     

Environmental enrichment enhances cellular plasticity in transgenic mice with Alzheimer-like pathology

Alzheimer's disease (AD) is accompanied by hippocampal neuronal loss and abnormal neurogenesis, both of which probably contributing to AD-related cognitive deficits. Mounting evidence indicates that cognitive and physical stimulation provided by environmental enrichment improves neurogenesis in healthy animals and counteracts beta-amyloid pathology in mouse models of AD. Here, we hypothesized that environmental enrichment has also an impact on hippocampal neurogenesis in mice with AD-like pathology. Therefore, TgCRND8 mice and wild type littermates were either housed under standard conditions or in an enriched environment for 4 months. Standard housed TgCRND8 mice revealed diminished hippocampal cell proliferation and reduced number of mature newborn neurons compared to wild type littermates under the same housing condition. However, environmental enrichment reversed this genotype effect. Here, we show that cognitive and physical stimulation is capable of increasing the number of newborn mature hippocampal neurons in transgenic mice to wild type levels. Moreover, the expression of various plasticity associated molecules was enhanced in transgenic mice due to enriched housing. This study identifies that environmental enrichment improves diminished cellular plasticity in AD brain, probably enhancing the brain capacity to better compensate for neurodegeneration.     

Pituitary adenylate cyclase-activating polypeptide regulates forebrain neural stem cells and neurogenesis in vitro and in vivo

Recent studies suggest that adult neurogenesis can contribute significantly to recovery from brain damage. As a result, there is strong interest in the field in identifying potentially therapeutic factors capable of promoting increased expansion of endogenous neural stem cell (NSC) populations and increased neurogenesis. In the present study, we have investigated the effects of PACAP on the NSC populations of the embryonic and adult forebrain. Our results demonstrate that the PACAP receptor, PAC1-R, is expressed by both embryonic and adult NSCs. The activation of PACAP signaling in vitro enhanced NSC proliferation/survival through a protein kinase A (PKA)-independent mechanism. In contrast, PACAP promoted NSC self-renewal and neurogenesis through a mechanism dependent on PKA activation. Finally, we determined that the intracerebroventricular infusion of PACAP into the adult forebrain was sufficient to increase neurogenesis significantly in both the hippocampus and the subventricular zone. These results demonstrate PACAP is unique in that it is capable of promoting NSC proliferation/survival, self-renewal, and neurogenesis and, therefore, may be ideal for promoting the endogenous regeneration of damaged brain tissue.     

Increased neurogenesis in young transgenic mice overexpressing human APP(Sw, Ind)

APP overexpressing mice have been widely used in the study of Alzheimer's disease (AD), focusing mainly at older ages, with higher accumulation of amyloid-beta peptide (Abeta). A decrease in hippocampal adult neurogenesis has been described in these models and proposed to be a consequence of Abeta accumulation. Only one study demonstrates increased neurogenesis in the hippocampus of APP-overexpressing J20 mice, and suggests it is a compensatory effect due to a subtle Abeta-induced damage. We have previously reported that a specific aggregation of Abeta has neurogenic potential on neural stem cells (NSC) in vitro. In order to clarify the contradicting data reported in vivo, we investigated NSC proliferation and neuronal differentiation in the hippocampi of J20 mice at a broader range of ages. Using immunohistochemistry, we show increased proliferation and neuronal differentiation in the hippocampi of 3 month-old J20 mice that reverted when animals became older. The increase in neurogenesis correlated with detectable levels of oligomeric Abeta, measured by ELISA and western blot. We suggest that oligomeric Abeta directly induces neurogenesis in vivo as has been demonstrated in vitro. Understanding the mechanisms underlying these changes could lead to treatments to control the neuronal differentiation of endogenous precursors through the progress of AD.     

β1‐integrin restricts astrocytic differentiation of adult hippocampal neural stem cells

Integrins are transmembrane receptors that mediate cell‐extracellular matrix and cell–cell interactions. The β1‐integrin subunit is highly expressed by embryonic neural stem cells (NSCs) and is critical for NSC maintenance in the developing nervous system, but its role in the adult hippocampal niche remains unexplored. We show that β1‐integrin expression in the adult mouse dentate gyrus (DG) is localized to radial NSCs and early progenitors, but is lost in more mature progeny. Although NSCs in the hippocampal subgranular zone (SGZ) normally only infrequently differentiate into astrocytes, deletion of β1‐integrin significantly enhanced astrocyte differentiation. Ablation of β1‐integrin also led to reduced neurogenesis as well as depletion of the radial NSC population. Activation of integrin‐linked kinase (ILK) in cultured adult NSCs from β1‐integrin knockout mice reduced astrocyte differentiation, suggesting that at least some of the inhibitory effects of β1‐integrin on astrocytic differentiation are mediated through ILK. In addition, β1‐integrin conditional knockout also resulted in extensive cellular disorganization of the SGZ as well as non‐neurogenic regions of the DG. The effects of β1‐integrin ablation on DG structure and astrogliogenesis show sex‐specific differences, with the effects following a substantially slower time‐course in males. β1‐integrin thus plays a dual role in maintaining the adult hippocampal NSC population by supporting the structural integrity of the NSC niche and by inhibiting astrocytic lineage commitment. GLIA 2016;64:1235–1251     

Neural stem cell protects aged rat brain from ischemia–reperfusion injury through neurogenesis and angiogenesis

Neural stem cells (NSCs) show therapeutic potential for ischemia in young-adult animals. However, the effect of aging on NSC therapy is largely unknown. In this work, NSCs were transplanted into aged (24-month-old) and young-adult (3-month-old) rats at 1 day after stroke. Infarct volume and neurobehavioral outcomes were examined. The number of differentiated NSCs was compared in aged and young-adult ischemic rats and angiogenesis and neurogenesis were also determined. We found that aged rats developed larger infarcts than young-adult rats after ischemia (P<0.05). The neurobehavioral outcome was also worse for aged rats comparing with young-adult rats. Brain infarction and neurologic deficits were attenuated after NSC transplantation in both aged and young-adult rats. The number of survived NSCs in aged rats was similar to that of the young-adult rats (P>0.05) and most of them were differentiated into glial fibrillary acidic protein⁺ (GFAP⁺) cells. More importantly, angiogenesis and neurogenesis were greatly enhanced in both aged and young-adult rats after transplantation compared with phosphate-buffered saline (PBS) control (P<0.05), accompanied by increased expression of vascular endothelial growth factor (VEGF). Our results showed that NSC therapy reduced ischemic brain injury, along with increased angiogenesis and neurogenesis in aged rats, suggesting that aging-related microenvironment does not preclude a beneficial response to NSCs transplantation during cerebral ischemia.     

Extremely low‐frequency electromagnetic fields enhance the survival of newborn neurons in the mouse hippocampus

In recent years, much effort has been devoted to identifying stimuli capable of enhancing adult neurogenesis, a process that generates new neurons throughout life, and that appears to be dysfunctional in the senescent brain and in several neuropsychiatric and neurodegenerative diseases. We previously reported that in vivo exposure to extremely low‐frequency electromagnetic fields (ELFEFs) promotes the proliferation and neuronal differentiation of hippocampal neural stem cells (NSCs) that functionally integrate in the dentate gyrus. Here, we extended our studies to specifically assess the influence of ELFEFs on hippocampal newborn cell survival, which is a very critical issue in adult neurogenesis regulation. Mice were injected with 5‐bromo‐2′‐deoxyuridine (BrdU) to label newborn cells, and were exposed to ELFEFs 9 days later, when the most dramatic decrease in the number of newly generated neurons occurs. The results showed that ELFEF exposure (3.5 h/day for 6 days) enhanced newborn neuron survival as documented by double staining for BrdU and doublecortin, to identify immature neurons, or NeuN labeling of mature neurons. The effects of ELFEFs were associated with enhanced spatial learning and memory. In an in vitro model of hippocampal NSCs, ELFEFs exerted their pro‐survival action by rescuing differentiating neurons from apoptotic cell death. Western immunoblot assay revealed reduced expression of the pro‐apoptotic protein Bax, and increased levels of the anti‐apoptotic protein Bcl‐2, in the hippocampi of ELFEF‐exposed mice as well as in ELFEF‐exposed NSC cultures, as compared with their sham‐exposed counterparts. Our results may have clinical implications for the treatment of impaired neurogenesis associated with brain aging and neurodegenerative diseases.     

Exercise increases neural stem cell proliferation surrounding the area of damage following rat traumatic brain injury

Exercise enhances neuronal stem cell (NSC) proliferation and neurogenesis. However, the effect of exercise on NSC proliferation surrounding the area of damage after traumatic brain injury (TBI) is unknown. Here, we investigate the effect of running on NSC proliferation following TBI in the rat. Wistar rats received TBI and were randomly divided into two groups: (1) non-exercise group and (2) exercise group. The exercise group ran on a treadmill for 30 min/day at 22 m/min for 7 consecutive days. Immunohistochemistry was used to monitor NSC proliferation around the damaged area, and ex vivo techniques were used to isolate NSCs from the damaged region in both groups. The number of nestin- and Ki67-positive cells observed at 3 and 7 days after TBI was significantly greater in the exercise group than in the non-exercise group (P < 0.01). Furthermore, most nestin-positive cells in the exercise group co-localized with Ki67-positive cells. In ex vivo studies, spheres could be isolated from injured brain tissue from the exercise group at 3 and 7 days following TBI, but at only 3 days in the non-exercise group. The number of spheres isolated from injured brain tissue was greater in the exercise group than in the non-exercise group. Spheres were immunopositive for nestin and comprised NSCs that could differentiate into neurons and glia. Exercise increases the proliferation of NSCs around the damaged area following TBI. Therefore, exercise therapy (rehabilitation) in the early phase following TBI is important for recuperation from cerebral dysfunction induced by TBI.