Administration of Human-Derived Mesenchymal Stem Cells Activates Locally Stimulated Endogenous Neural Progenitors and Reduces Neurological Dysfunction in Mice after Ischemic Stroke.

Increasing evidence shows that the administration of mesenchymal stem cells (MSCs) is a promising option for various brain diseases, including ischemic stroke. Studies have demonstrated that MSC transplantation after ischemic stroke provides beneficial effects, such as neural regeneration, partially by activating endogenous neural stem/progenitor cells (NSPCs) in conventional neurogenic zones, such as the subventricular and subgranular zones. However, whether MSC transplantation regulates the fate of injury-induced NSPCs (iNSPCs) regionally activated at injured regions after ischemic stroke remains unclear. Therefore, mice were subjected to ischemic stroke, and mCherry-labeled human MSCs (h-MSCs) were transplanted around the injured sites of nestin-GFP transgenic mice. Immunohistochemistry of brain sections revealed that many GFP+ cells were observed around the grafted sites rather than in the regions in the subventricular zone, suggesting that transplanted mCherry+ h-MSCs stimulated GFP+ locally activated endogenous iNSPCs. In support of these findings, coculture studies have shown that h-MSCs promoted the proliferation and neural differentiation of iNSPCs extracted from ischemic areas. Furthermore, pathway analysis and gene ontology analysis using microarray data showed that the expression patterns of various genes related to self-renewal, neural differentiation, and synapse formation were changed in iNSPCs cocultured with h-MSCs. We also transplanted h-MSCs (5.0 × 104 cells/µL) transcranially into post-stroke mouse brains 6 weeks after middle cerebral artery occlusion. Compared with phosphate-buffered saline-injected controls, h-MSC transplantation displayed significantly improved neurological functions. These results suggest that h-MSC transplantation improves neurological function after ischemic stroke in part by regulating the fate of iNSPCs.

Transplantation of Human Brain-Derived Ischemia-Induced Multipotent Stem Cells Ameliorates Neurological Dysfunction in Mice After Stroke.

We recently demonstrated that injury/ischemia-induced multipotent stem cells (iSCs) develop within post-stroke human brains. Because iSCs are stem cells induced under pathological conditions, such as ischemic stroke, the use of human brain-derived iSCs (h-iSCs) may represent a novel therapy for stroke patients. We performed a preclinical study by transplanting h-iSCs transcranially into post-stroke mouse brains 6 weeks after middle cerebral artery occlusion (MCAO). Compared with PBS-treated controls, h-iSC transplantation significantly improved neurological function. To identify the underlying mechanism, green fluorescent protein (GFP)-labeled h-iSCs were transplanted into post-stroke mouse brains. Immunohistochemistry revealed that GFP+ h-iSCs survived around the ischemic areas and some differentiated into mature neuronal cells. To determine the effect on endogenous neural stem/progenitor cells (NSPCs) by h-iSC transplantation, mCherry-labeled h-iSCs were administered to Nestin-GFP transgenic mice which were subjected to MCAO. As a result, many GFP+ NSPCs were observed around the injured sites compared with controls, indicating that mCherry+ h-iSCs activate GFP+ endogenous NSPCs. In support of these findings, coculture studies revealed that the presence of h-iSCs promotes the proliferation of endogenous NSPCs and increases neurogenesis. In addition, coculture experiments indicated neuronal network formation between h-iSC- and NSPC-derived neurons. These results suggest that h-iSCs exert positive effects on neural regeneration through not only neural replacement by grafted cells but also neurogenesis by activated endogenous NSPCs. Thus, h-iSCs have the potential to be a novel source of cell therapy for stroke patients.

Enhanced angiogenic properties of umbilical cord blood primed by OP9 stromal cells ameliorates neurological deficits in cerebral infarction mouse model.

Umbilical cord blood (UCB) transplantation shows proangiogenic effects and contributes to symptom amelioration in animal models of cerebral infarction. However, the effect of specific cell types within a heterogeneous UCB population are still controversial. OP9 is a stromal cell line used as feeder cells to promote the hematoendothelial differentiation of embryonic stem cells. Hence, we investigated the changes in angiogenic properties, underlying mechanisms, and impact on behavioral deficiencies caused by cerebral infarction in UCB co-cultured with OP9 for up to 24 h. In the network formation assay, only OP9 pre-conditioned UCB formed network structures. Single-cell RNA sequencing and flow cytometry analysis showed a prominent phenotypic shift toward M2 in the monocytic fraction of OP9 pre-conditioned UCB. Further, OP9 pre-conditioned UCB transplantation in mice models of cerebral infarction facilitated angiogenesis in the peri-infarct lesions and ameliorated the associated symptoms. In this study, we developed a strong, fast, and feasible method to augment the M2, tissue-protecting, pro-angiogenic features of UCB using OP9. The ameliorative effect of OP9-pre-conditioned UCB in vivo could be partly due to promotion of innate angiogenesis in peri-infarct lesions.

Acute stress induces severe neural inflammation and overactivation of glucocorticoid signaling in interleukin-18-deficient mice.

Interleukin-18 (IL18) is an inflammatory cytokine that is related to psychiatric disorders such as depression and cognitive impairment. We previously found that IL18 deficiency may cause hippocampal impairment, resulting in depression-like behavioral changes. However, the potential role of IL18 in stressful conditions remains uncertain. In the present study, we examined the effect of IL18 on neural inflammation and stress tolerance during acute stress. Littermate Il18+/+ and Il18-/- mice were exposed to a single restraint stress for 6 h, and all assessments were performed 18 h after the mice were released from the restraint. In Il18-/- mice exposed to acute stress, the immobility times in both the forced swim test and tail suspension test were decreased, although no difference was observed in Il18+/+ mice. Il1ß, Il6, and Tnfα expression levels in the hippocampus of stressed Il18-/- mice were significantly higher than those in the other groups. Moreover, the numbers of astrocytes and microglia, including those in the active form, were also increased compared with those in other groups. Regarding the molecular mechanism, the HSF5 and TTR genes were specifically expressed in stressed Il18-/- mice. As a potential treatment, intracerebral administration of IL18 to Il18-/- mice resulted in partial recovery of changes in behavioral assessments. Our results revealed that IL18-deficient mice were more sensitive and had a longer response to acute stress than that in normal mice. In addition, neural inflammation and augmentation of glucocorticoid signals caused by stress were more intense and remained longer in Il18-/- mice, resulting in behavioral changes. In conclusion, IL18 might be an indispensable factor that modulates the stress response and maintains balance between neural inflammation and glucocorticoid signaling.

Early-phase administration of human amnion-derived stem cells ameliorates neurobehavioral deficits of intracerebral hemorrhage by suppressing local inflammation and apoptosis.

BACKGROUND: Intracerebral hemorrhage (ICH) is a significant cause of death and disabilities. Recently, cell therapies using mesenchymal stem cells have been shown to improve ICH-induced neurobehavioral deficits. Based on these findings, we designed this study to evaluate the therapeutic efficacy and underlying mechanisms by which human amnion-derived stem cells (hAMSCs) would ameliorate neurobehavioral deficits of ICH-bearing hosts. METHODS: hAMSCs were induced from amnia obtained by cesarean section and administered intravenously to ICH-bearing mice during the acute phase. The mice were then subject to multitask neurobehavioral tests at the subacute phase. We attempted to optimize the dosage and timing of the hAMSC administrations. In parallel with the hAMSCs, a tenfold higher dose of human adipose-derived stem cells (ADSCs) were used as an experimental control. Specimens were obtained from the ICH lesions to conduct immunostaining, flow cytometry, and Western blotting to elucidate the underlying mechanisms of the hAMSC treatment. RESULTS: The intravenous administration of hAMSCs to the ICH-bearing mice effectively improved their neurobehavioral deficits, particularly when the treatment was initiated at Day 1 after the ICH induction. Of note, the hAMSCs promoted clinical efficacy equivalent to or better than that of hADSCs at 1/10 the cell number. The systemically administered hAMSCs were found in the ICH lesions along with the local accumulation of macrophages/microglia. In detail, the hAMSC treatment decreased the number of CD11b+CD45+ and Ly6G+ cells in the ICH lesions, while splenocytes were not affected. Moreover, the hAMSC treatment decreased the number of apoptotic cells in the ICH lesions. These results were associated with suppression of the protein expression levels of macrophage-related factors iNOS and TNFα. CONCLUSIONS: Intravenous hAMSC administration during the acute phase would improve ICH-induced neurobehavioral disorders. The underlying mechanism was suggested to be the suppression of subacute inflammation and apoptosis by suppressing macrophage/microglia cell numbers and macrophage functions (such as TNFα and iNOS). From a clinical point of view, hAMSC-based treatment may be a novel strategy for the treatment of ICH.

Voluntary running exercise after focal cerebral ischemia ameliorates dendritic spine loss and promotes functional recovery.

Cerebral infarction causes motor, sensory, and cognitive impairments. Although rehabilitation enhances recovery of activities of daily living after cerebral infarction, its mechanism remains elusive due to the lack of reproducibility and low survival rate of brain ischemic model animals. Here, to investigate the relationship between rehabilitative intervention, motor function, and pathophysiological remodeling of the tissue in the ipsilateral hemisphere after cerebral infarction, we took advantage of a highly reproducible model of cerebral infarction using C.B-17/Icr-+/+Jcl mice. In this model, we confirmed that voluntary running exercise improved functional recovery after ischemia. Exercise did not alter the volume of infarction or survived cortex, or the number of NeuN-labeled cells in the peri-infarct cortex. In mice who did not exercise, the number of basal dendritic spines of layer 5 pyramidal cells decreased in the peri-infarct motor cortex, whereas in mice who exercised it remained at the normal level. The voluntary exercise intervention maintained basal dendritic spine density within the peri-infarct area, which may reflect an adaptive remodeling of the surviving neural circuitry that might contribute to promoting the recovery of activities of daily living.

Intravenous Administration of Human Amniotic Mesenchymal Stem Cells in the Subacute Phase of Cerebral Infarction in a Mouse Model Ameliorates Neurological Disturbance by Suppressing Blood Brain Barrier Disruption and Apoptosis via Immunomodulation.

Neuro-inflammation plays a key role in the pathophysiology of brain infarction. Cell therapy offers a novel therapeutic option due to its effect on immunomodulatory effects. Amniotic stem cells, in particular, show promise owing to their low immunogenicity, tumorigenicity, and easy availability from amniotic membranes discarded following birth. We have successfully isolated and expanded human amniotic mesenchymal stem cells (hAMSCs). Herein, we evaluated the therapeutic effect of hAMSCs on neurological deficits after brain infarction as well as their immunomodulatory effects in a mouse model in order to understand their mechanisms of action. One day after permanent occlusion of the middle cerebral artery (MCAO), hAMSCs were intravenously administered. RT-qPCR for TNFα, iNOS, MMP2, and MMP9, immunofluorescence staining for iNOS and CD11b/c, and a TUNEL assay were performed 8 days following MCAO. An Evans Blue assay and behavioral tests were performed 2 days and several months following MCAO, respectively. The results suggest that the neurological deficits caused by cerebral infarction are improved in dose-dependent manner by the administration of hAMSCs. The mechanism appears to be through a reduction in disruption of the blood brain barrier and apoptosis in the peri-infarct region through the suppression of pro-inflammatory cytokines and the M2-to-M1 phenotype shift.

Early Reperfusion Following Ischemic Stroke Provides Beneficial Effects, Even After Lethal Ischemia with Mature Neural Cell Death.

Ischemic stroke is a critical disease caused by cerebral artery occlusion in the central nervous system (CNS). Recent therapeutic advances, such as neuroendovascular intervention and thrombolytic therapy, have allowed recanalization of occluded brain arteries in an increasing number of stroke patients. Although previous studies have focused on rescuing neural cells that still survive despite decreased blood flow, expanding the therapeutic time window may allow more patients to undergo reperfusion in the near future, even after lethal ischemia, which is characterized by death of mature neural cells, such as neurons and glia. However, it remains unclear whether early reperfusion following lethal ischemia results in positive outcomes. The present study used two ischemic mouse models-90-min transient middle cerebral artery occlusion (t-MCAO) paired with reperfusion to induce lethal ischemia and permanent middle cerebral artery occlusion (p-MCAO)-to investigate the effect of early reperfusion up to 8 w following MCAO. Although early reperfusion following 90-min t-MCAO did not rescue mature neural cells, it preserved the vascular cells within the ischemic areas at 1 d following 90-min t-MCAO compared to that following p-MCAO. In addition, early reperfusion facilitated the healing processes, including not only vascular but also neural repair, during acute and chronic periods and improved recovery. Furthermore, compared with p-MCAO, early reperfusion after t-MCAO prevented behavioral symptoms of neurological deficits without increasing negative complications, including hemorrhagic transformation and mortality. These results indicate that early reperfusion provides beneficial effects presumably via cytoprotective and regenerative mechanisms in the CNS, suggesting that it may be useful for stroke patients that experienced lethal ischemia.

Interleukin-18-deficient mice develop hippocampal abnormalities related to possible depressive-like behaviors.

Interleukin-18 (IL-18) is an inflammatory cytokine linked to major depressive disorder (MDD). MDD is closely related to metabolic disorders, such as diabetes mellitus (DM) and obesity. Moreover, DM is associated with cognitive impairment and promotes apoptosis of hippocampal cells by activating pro-apoptotic and inhibiting anti-apoptotic factors. IL-18-deficient (Il18-/-) mice are obese and have DM. Therefore, we hypothesized a close relationship between IL-18 and death of hippocampal cells, affecting neurogenesis related to behavioral changes such as MDD. Il18-/- male mice were generated on the C57Bl/6 background and Il18+/+ mice were used as controls. Behavioral, histopathological, and molecular responses, as well as responses to intracerebral recombinant IL-18 administration, were examined. Compared with Il18+/+ mice, Il18-/- mice had impaired learning and memory and exhibited lower motivation. In the Il18-/- mice, degenerated mitochondria were detected in synaptic terminals in the molecular layer, the polymorphic layer, and in mossy fibers in the dentate gyrus, suggesting mitochondrial abnormalities. Because of the degeneration of mitochondria in the dentate gyrus, in which pro-apoptotic molecules were upregulated and anti-apoptotic factors were decreased, apoptosis inducers were not cleaved, indicating inhibition of apoptosis. In addition, neurogenesis in the dentate gyrus and the maturity of neuronal cells were decreased in the Il18-/- mice, while intracerebral administration of recombinant IL-18 promoted significant recovery of neurogenesis. Our findings suggested that IL-18 was indispensable for mitochondrial homeostasis, sustaining clearance of degenerative neural cells, and supporting neurogenesis, normal neuronal maturation and hippocampal function.

Adipose-derived stem cell therapy inhibits the deterioration of cerebral infarction by altering macrophage kinetics.

INTRODUCTION: We previously established a method to isolate and culture human adipose-derived stem cells (hADSCs) using fetal bovine serum and showed the therapeutic impact on cerebral infarction. Recently, we modified the culture method with the use of serum-free media for future clinical applications. This study aims to evaluate whether intravenous administration of hADSCs induced by the serum-free culture method would improve neurobehavioral deficits in mice with cerebral infarction. RESULTS: Induced hADSCs possessed the characteristics of mesenchymal stem cells and withstood a freeze-thaw process. hADSC administration improved neurobehavioral deficits in MCAO-treated mice and suppressed brain atrophy at the chronic phase. Although hADSC administration did not affect serum cytokine profiles, it decreased the number of CD11b+ monocytes in the spleen. Concomitantly, hADSC administration increased the local accumulation of CD11b+CD163+ M2 macrophages into the border zone of the cerebral infarction at 4 days post-MCAO (the acute phase). DISCUSSION: Our data indicate that the systemic administration of hADSCs can improve the neurobehavioral deficits that occur after cerebral infarction by modulating the acute immune response mediated by CD11b+CD163+ M2 macrophages in infarcted lesions.

Intravenous administration of human adipose-derived stem cells ameliorates motor and cognitive function for intracerebral hemorrhage mouse model.

Even today, intracerebral hemorrhage (ICH) is a major cause of death and disabilities. Rehabilitation is preferentially applied for functional recovery although its effect is limited. Recent studies have suggested that intravenous administration of mesenchymal stem cells would improve the post-ICH neurological deficits. Human adipose-derived stem cells (hADSCs) have been established in our laboratory. We aimed to evaluate the therapeutic efficacy of the hADSCs on the post-ICH neurological deficits using a clinical-relevant ICH mouse model. We also evaluated immune responses to clarify the underlying mechanisms. The hADSCs expressed MSC markers at high levels. The hADSCs administration into the ICH-bearing mice improved the neurological deficits during the subacute phases, which was shown by neurobehavioral experiments. Besides, the hADSC administration decreased the number of CD11+CD45+ cells and increased the proportion of CD86+ and Ly6C+ cells in the ICH lesions. In summary, intravenous administration of hADSCs during the acute phase improved ICH-induced neurological deficits during the subacute phase because of the suppression of acute inflammation mediated by CD11+CD45+ subpopulations. Our data suggest that hADSCs can be served as a novel strategy for ICH treatment.

Molecular analysis of the mouse brain exposed to chronic mild stress: The influence of hepatocyte nuclear factor 4α on physiological homeostasis.

Major depressive disorder (MDD) is a prevalent disorder that causes considerable disability in social functioning and is a risk factor for physical diseases. Recent clinical reports have demonstrated a marked association between MDD and physiological dyshomeostasis induced by metabolic disorders, including diabetes, hormone abnormalities and autoimmune diseases. The authors of the present study have previously analyzed comparative gene expression profiles in the prefrontal cortex (PFC) of a chronic mild stress (CMS) animal model of MDD. Hepatocyte nuclear factor 4α (Hnf4α) was identified as a central regulator that exerted significant influence on genes associated with physiological homeostasis. The aim of the present study was to investigate: i) the molecular mechanism of the depressive state in the PFC, and ii) the involvement of genes extracted from the comparative gene expression profiles, particularly those applicable to MDD in clinical practice. Core analysis of the previous PFC microarray results was performed using Ingenuity Pathway Analysis (IPA). Subsequently, IPA was used to search for molecules that are regulated by Hnf4α, and exist in the PFC and serum. From the core analysis, 5 genes that are associated with cell death and are expressed in the cortex were selected. Four of the extracted genes, insulin­like growth factor 1, transthyretin, serpin family A member 3 and plasminogen, were markedly affected by Hnf4α. S100 calcium­binding protein A9 (S100a9) and α2-HS-glycoprotein (Ahsg) were also chosen as they exist in serum and are also affected by Hnf4α. A significant group difference in the expression of these two genes was detected in the PFC, thalamus and hippocampus. The protein levels of AHSG and S100A9 in the PFC and hippocampus of the CMS group increased significantly when compared with the control group. These findings support the close association of Hnf4α (through genes such as S100a9 and Ahsg) with the development of various diseases induced by deregulation of physiological homeostasis during the progression of MDD.

CAPS1 RNA Editing Promotes Dense Core Vesicle Exocytosis.

Calcium-dependent activator protein for secretion 1 (CAPS1) plays a distinct role in the priming step of dense core vesicle (DCV) exocytosis. CAPS1 pre-mRNA is known to undergo adenosine-to-inosine RNA editing in its coding region, which results in a glutamate-to-glycine conversion at a site in its C-terminal region. However, the physiological significance of CAPS1 RNA editing remains elusive. Here, we created mutant mice in which edited CAPS1 was solely expressed. These mice were lean due to increased energy expenditure caused by physical hyperactivity. Electrophysiological and biochemical analyses demonstrated that the exocytosis of DCVs was upregulated in the chromaffin cells and neurons of these mice. Furthermore, we showed that edited CAPS1 bound preferentially to the activated form of syntaxin-1A, a component of the exocytotic fusion complex. These findings suggest that RNA editing regulates DCV exocytosis in vivo, affecting physical activity.

Effect of Sema4D on microglial function in middle cerebral artery occlusion mice.

Cerebral ischemia evokes neuroinflammatory response. Inflammatory stimulation induces microglial activation, such as changes of their morphology from ramified to ameboid, expression of iNOS and cytokines, and the elevation of proliferative activity. Activated microglia play important roles in pathogenesis of cerebral ischemia. A previous study indicated that Sema4D promoted iNOS expression in cultured microglia; however, roles of Sema4D on microglial activation in ischemic injury remains unclear. We investigated the effect of Sema4D-deficiency on microglial activation by using permanent middle cerebral artery occlusion (MCAO) in mice. In this study, ischemia-induced activated microglia were classified into activated-ramified microglia and ameboid microglia based on their morphology. We demonstrated that the rate of iNOS expression in activated-ramified microglia was lower than that in ameboid microglia, while the most proliferating microglia were activated-ramified microglia but not ameboid microglia after cerebral ischemia. Sema4D-deficiency decreased the number of ameboid microglia and iNOS-expressing activated-ramified microglia in the peri-ischemic cortex. These changes by Sema4D-deficiency contributed to the reduction of NO production that was estimated by nitrite concentration in ischemic cortex. On the other hand, Sema4D-deficiency promoted proliferation of microglia in the peri-ischemic cortex. Importantly, ischemia-induced apoptosis and postischemic behavioral abnormality were moderated in Sema4D(-/-) mice. These findings suggest that Sema4D promotes cytotoxic activation of microglia and inhibits functional recovery after cerebral ischemia.

Hepatocyte nuclear factor 4 alpha is a key factor related to depression and physiological homeostasis in the mouse brain.

Major depressive disorder (MDD) is a common psychiatric disorder that involves marked disabilities in global functioning, anorexia, and severe medical comorbidities. MDD is associated with not only psychological and sociocultural problems, but also pervasive physical dysfunctions such as metabolic, neurobiological and immunological abnormalities. Nevertheless, the mechanisms underlying the interactions between these factors have yet to be determined in detail. The aim of the present study was to identify the molecular mechanisms responsible for the interactions between MDD and dysregulation of physiological homeostasis, including immunological function as well as lipid metabolism, coagulation, and hormonal activity in the brain. We generated depression-like behavior in mice using chronic mild stress (CMS) as a model of depression. We compared the gene expression profiles in the prefrontal cortex (PFC) of CMS and control mice using microarrays. We subsequently categorized genes using two web-based bioinformatics applications: Ingenuity Pathway Analysis and The Database for Annotation, Visualization, and Integrated Discovery. We then confirmed significant group-differences by analyzing mRNA and protein expression levels not only in the PFC, but also in the thalamus and hippocampus. These web tools revealed that hepatocyte nuclear factor 4 alpha (Hnf4a) may exert direct effects on various genes specifically associated with amine synthesis, such as genes involved in serotonin metabolism and related immunological functions. Moreover, these genes may influence lipid metabolism, coagulation, and hormonal activity. We also confirmed the significant effects of Hnf4a on both mRNA and protein expression levels in the brain. These results suggest that Hnf4a may have a critical influence on physiological homeostasis under depressive states, and may be associated with the mechanisms responsible for the interactions between MDD and the dysregulation of physiological homeostasis in humans.

A DKP cyclo(L-Phe-L-Phe) found in chicken essence is a dual inhibitor of the serotonin transporter and acetylcholinesterase.

Diketopiperazines (DKPs) are naturally-occurring cyclic dipeptides with a small structure and are found in many organisms and in large amounts in some foods and beverages. We found that a chicken essence beverage, which is popular among Southeast Asians as a traditional remedy and a rich source of DKPs, inhibited the serotonin transporter (SERT) and suppressed serotonin uptake from rat brain synaptosomes, which prompted us to isolate and identify the active substance(s). We purified a SERT inhibitor from the chicken essence beverage and identified it as the DKP cyclo(L-Phe-L-Phe). Interestingly, it was a naturally occurring dual inhibitor that inhibited both SERT and acetylcholinesterase (AChE) in vitro. The DKP increased extracellular levels of the cerebral monoamines serotonin, norepinephrine, and dopamine in the medial prefrontal cortex and acetylcholine in the ventral hippocampus of freely moving rats when administered orally. Moreover, cyclo(L-Phe-L-Phe) significantly shortened escape latency in the water maze test in depressed mice previously subjected to a repeated open-space swimming task, which induces a depression-like state. Cyclo(L-Phe-L-Phe) also significantly improved accuracy rates in a radial maze test in rats and increased step-through latencies in a passive avoidance test in mice with scopolamine-induced amnesia. These animal test results suggest that cyclo(L-Phe-L-Phe), which is present abundantly in some foods such as chicken essence, may abrogate the onset of depression and, thus, contribute to preventing the development of Alzheimer's disease and other dementia, because senile depression is a risk factor for dementia.

Hepatocyte growth factor overexpression in the nervous system enhances learning and memory performance in mice.

Hepatocyte growth factor (HGF) and its receptor, c-Met, play pivotal roles in the nervous system during development and in disease states. However, the physiological roles of HGF in the adult brain are not well understood. In the present study, to assess its role in learning and memory function, we used transgenic mice that overexpress HGF in a neuron-specific manner (HGF-Tg) to deliver HGF into the brain without injury. HGF-Tg mice displayed increased alternation rates in the Y-maze test compared with age-matched wild-type (WT) controls. In the Morris water maze (MWM) test, HGF-Tg mice took less time to find the platform on the first day, whereas the latency to escape to the hidden platform was decreased over training days compared with WT mice. A transfer test revealed that the incidence of arrival at the exact location of the platform was higher for HGF-Tg mice compared with WT mice. These results demonstrate that overexpression of HGF leads to an enhancement of both short- and long-term memory. Western blot analyses revealed that the levels of N-methyl-D-aspartate (NMDA) receptor subunits NR2A and NR2B, but not NR1, were increased in the hippocampus of HGF-Tg mice compared with WT controls, suggesting that an upregulation of NR2A and NR2B could represent one mechanism by which HGF enhances learning and memory performance. These results demonstrate that modulation of learning and memory performance is an important physiological function of HGF that contributes to normal CNS plasticity, and we propose HGF as a novel regulator of higher brain functions.

Immunodeficiency reduces neural stem/progenitor cell apoptosis and enhances neurogenesis in the cerebral cortex after stroke.

Acute inflammation in the poststroke period exacerbates neuronal damage and stimulates reparative mechanisms, including neurogenesis. However, only a small fraction of neural stem/progenitor cells survives. In this report, by using a highly reproducible model of cortical infarction in SCID mice, we examined the effects of immunodeficiency on reduction of brain injury, survival of neural stem/progenitor cells, and functional recovery. Subsequently, the contribution of T lymphocytes to neurogenesis was evaluated in mice depleted for each subset of T lymphocyte. SCID mice revealed the reduced apoptosis and enhanced proliferation of neural stem/progenitor cells induced by cerebral cortex after stroke compared with the immunocompetent wild-type mice. Removal of T lymphocytes, especially the CD4(+) T-cell population, enhanced generation of neural stem/progenitor cells, followed by accelerated functional recovery. In contrast, removal of CD25(+) T cells, a cell population including regulatory T lymphocytes, impaired functional recovery through, at least in part, suppression of neurogenesis. Our findings demonstrate a key role of T lymphocytes in regulation of poststroke neurogenesis and indicate a potential novel strategy for cell therapy in repair of the central nervous system.

Behavioral despair during a water maze learning task in mice.

In the case of mice, when the difficulty of a water maze learning task is increased, some animals gradually cease to swim, abandon adaptive learning, and become immobile. We trained 99 male C57BL/6N mice in a pool containing a hidden platform. The pool was surrounded by white featureless walls, and almost all external cues were removed. On the eighth day of escape training, 36 inferior-learners exhibited behavioral despair. The predictive validity of the inferior-learners as a depression model was verified by testing their sensitivity to clinically efficacious antidepressants. The inferior-learners treated with a selective serotonin reuptake inhibitor (SSRI), fluvoxamine, or a serotonin noradrenaline reuptake inhibitor (SNRI), milnacipran, resumed swimming and adaptive learning. Because of facial similarities between inferior-learners and depressive patients and their sensitivity to antidepressant drugs, our experimental method is expected to be an effective tool in basic research on depression.

Timp-3 deficiency impairs cognitive function in mice.

Extracellular matrix (ECM) degradation is performed primarily by matrix metalloproteinases (MMPs). MMPs have recently been shown to regulate synaptic activity in the hippocampus and to affect memory and learning. The tissue inhibitor of metalloproteinase (Timp) is an endogenous factor that controls MMP activity by binding to the catalytic site of MMPs. At present, four Timp isotypes have been reported (Timp-1 through Timp-4) with 35-50% amino-acid sequence homology. Timp-3 is a unique member of Timp proteins in that it is bound to the ECM. In this study, we used the passive avoidance test, active avoidance test, and water maze test to examine the cognitive function in Timp-3 knockout (KO) mice. Habituation was evaluated using the open-field test. The water maze test showed that Timp-3 KO mice exhibit deterioration in cognitive function compared with wild-type (WT) mice. The open-field test showed decreased habituation of Timp-3 KO mice. Immunostaining of brain slices revealed the expression of Timp-3 in the hippocampus. In situ zymography of the hippocampus showed increased gelatinolytic activity in Timp-3 KO mice compared with WT mice. These results present the first evidence of Timp-3 involvement in cognitive function and hippocampal MMP activity in mice. Moreover, our findings suggest a novel therapeutic target to be explored for improvement of cognitive function in humans.