Neural stem cells in the subventricular zone of adult rodents produce new striatal neurons that may replace those that have died after stroke; however, the neurogenic response has been considered acute and transient, yielding only small numbers of neurons. In contrast, we show herein that striatal neuroblasts are generated without decline at least for 4 months after stroke in adult rats. Neuroblasts formed early or late after stroke either differentiate into mature neurons, which survive for several months, or die through caspasemediated apoptosis. The directed migration of the new neurons toward the ischemic damage is regulated by stromal cell-derived factor-1␣ and its receptor CXCR4. These results show that endogenous neural stem cells continuously supply the injured adult brain with new neurons, which suggests novel self-repair strategies to improve recovery after stroke. STEM CELLS 2006;24:739 -747

Neurons are continuously generated from stem cells in discrete regions in the adult mammalian brain. We found that ependymal cells lining the lateral ventricles were quiescent and did not contribute to adult neurogenesis under normal conditions in mice but instead gave rise to neuroblasts and astrocytes in response to stroke. Ependymal cell quiescence was actively maintained by canonical Notch signaling. Inhibition of this pathway in uninjured animals allowed ependymal cells to enter the cell cycle and produce olfactory bulb neurons, whereas forced Notch signaling was sufficient to block the ependymal cell response to stroke. Ependymal cells were depleted by stroke and failed to self-renew sufficiently to maintain their own population. Thus, although ependymal cells act as primary cells in the neural lineage to produce neurons and glial cells after stroke, they do not fulfill defining criteria for stem cells under these conditions and instead serve as a reservoir that is recruited by injury.

Neural stem cells (NSCs) in the adult rat subventricular zone (SVZ) generate new striatal neurons during several months after ischemic stroke. Whether the microglial response associated with ischemic injury extends into SVZ and influences neuroblast production is unknown. Here, we demonstrate increased numbers of activated microglia in ipsilateral SVZ concomitant with neuroblast migration into the striatum at 2, 6, and 16 weeks, with maximum at 6 weeks, following 2 h middle cerebral artery occlusion in rats. In the peri-infarct striatum, numbers of activated microglia peaked already at 2 weeks and declined thereafter. Microglia in SVZ were resident or originated from bone marrow, with maximum proliferation during the first 2 weeks postinsult. In SVZ, microglia exhibited ramified or intermediate morphology, signifying a downregulated inflammatory profile, whereas amoeboid or round phagocytic microglia were frequent in the peri-infarct striatum. Numbers of microglia expressing markers of antigen-presenting cells (MHC-II, CD86) increased in SVZ but very few lymphocytes were detected. Using quantitative PCR, strong short- and long-term increase (at 1 and 6 weeks postinfarct) of insulin-like growth factor-1 (IGF-1) gene expression was detected in SVZ tissue. Elevated numbers of IGF-1-expressing microglia were found in SVZ at 2, 6, and 16 weeks after stroke. At 16 weeks, 5% of microglia but no other cells in SVZ expressed the IGF-1 protein, which mitigates apoptosis and promotes proliferation and differentiation of NSCs. The long-term accumulation of microglia with proneurogenic phenotype in the SVZ implies a supportive role of these cells for the continuous neurogenesis after stroke.

Background and Purpose-Stroke induced by middle cerebral artery occlusion (MCAO) triggers increased neurogenesis in the damaged striatum and nondamaged hippocampus of young adult rodents. We explored whether stroke influences neurogenesis similarly in the aged brain. Methods-Young adult (3 months) and old (15 months) rats were subjected to 1 hour of MCAO, and new cells were labeled by intraperitoneal injection of 5-bromo-2Ј-deoxyuridine 5Ј-monophosphate (BrdU), a marker for dividing cells, for 2 weeks thereafter. Animals were euthanized at 7 weeks after the insult, and neurogenesis was assessed immunocytochemically with antibodies against BrdU and neuronal markers with epifluorescence or confocal microscopy. Results-Young and old rats exhibited the same increased numbers of new striatal neurons after stroke, despite basal cell proliferation in the subventricular zone being reduced in the aged brain. In contrast, both the number of stroke-generated granule cells and basal neurogenesis in the dentate subgranular zone were lower in old compared with young animals. Also, the ability of newly formed cells to differentiate into neurons was impaired in the aged dentate gyrus. Conclusions-Basal neurogenesis is impaired in the subgranular and subventricular zones of aged animals, but both regions react to stroke with increased formation of new neurons. The magnitude of striatal neurogenesis after stroke is similar in young and old animals, indicating that this potential mechanism for self-repair also operates in the aged brain. Key Words: cerebral arteries Ⅲ hippocampus Ⅲ ischemia Ⅲ neuronal plasticity Ⅲ stem cells Ⅲ stroke, ischemic T he production of neurons in the mammalian brain continues throughout life. Neurons generated in the subventricular zone (SVZ) migrate to the olfactory bulb, and they differentiate into interneurons. Neurogenesis in the dentate subgranular zone (SGZ) gives rise to neurons in the granule cell layer (GCL). Basal hippocampal and olfactory bulb neurogenesis declines in aged animals, 1-4 but different treatments also increase the production of new neurons in old brains. 1,5,6 Stroke induced by middle cerebral artery occlusion (MCAO) in young adult rodents triggers increased neurogenesis in the SGZ/GCL and SVZ. [7][8][9][10][11] This insult causes neuronal loss in the striatum and cerebral cortex, whereas hippocampal formation is spared. The SVZ neuroblasts migrate into the damaged striatal area and adopt the phenotype of projection neurons. 8 -10 Stroke leads to deficits in hippocampus-associated spatial memory, 12 and the increase in SGZ neurogenesis might aim to counteract cognitive impairments. Striatal neurogenesis, therefore, may contribute to recovery of stroke-impaired motor function.Ischemic stroke occurs more often in aged humans. It is therefore of major clinical interest to explore whether the aged brain retains the capacity for stroke-induced neurogenesis. Here we show that both basal and stroke-induced hippocampal neurogenesis is attenuated in old animals. In contrast, despite redu...

Type 2 diabetes is a strong risk factor for stroke. Linagliptin is a dipeptidyl peptidase-4 (DPP-4) inhibitor in clinical use against type 2 diabetes. The aim of this study was to determine the potential antistroke efficacy of linagliptin in type 2 diabetic mice. To understand whether efficacy was mediated by glycemia regulation, a comparison with the sulfonylurea glimepiride was done. To determine whether linagliptin-mediated efficacy was dependent on a diabetic background, experiments in nondiabetic mice were performed. Type 2 diabetes was induced by feeding the mice a high-fat diet for 32 weeks. Mice were treated with linagliptin/glimepiride for 7 weeks. Stroke was induced at 4 weeks into the treatment by transient middle cerebral artery occlusion. Blood DPP-4 activity, glucagon-like peptide-1 (GLP-1) levels, glucose, body weight, and food intake were assessed throughout the experiments. Ischemic brain damage was measured by determining stroke volume and by stereologic quantifications of surviving neurons in the striatum/cortex. We show pronounced antistroke efficacy of linagliptin in type 2 diabetic and normal mice, whereas glimepiride proved efficacious against stroke in normal mice only. These results indicate a linagliptin-mediated neuroprotection that is glucose-independent and likely involves GLP-1. The findings may provide an impetus for the development of DPP-4 inhibitors for the prevention and treatment of stroke in diabetic patients.

Stroke is a neurodegenerative disorder and the leading cause of disability in adult humans. Treatments to support efficient recovery in stroke patients are lacking. Several studies have demonstrated the ability of grafted neural stem cells (NSCs) to partly improve impaired neurological functions in stroke-subjected animals. Recently, we reported that NSCs from human fetal striatum and cortex exhibit region-specific differentiation in vitro, but survive, migrate and form neurons to a similar extent after intrastriatal transplantation in newborn rats. Here, we have transplanted the same cells into the stroke-damaged striatum of adult rats. The two types of NSCs exhibited a similar robust survival (30%) at 1 month after transplantation, and migrated throughout the damaged striatum. Striatal NSCs migrated farther and occupied a larger volume of striatum. In the transplantation core, cells were undifferentiated and expressed nestin and, to a lesser extent, also GFAP, betaIII-tubulin, DCX and calretinin, markers of immature neural lineage. Immunocytochemistry using markers of proliferation (p-H3 and Ki67) revealed a very low content of proliferating cells (<1%) in the grafts. Human cells outside the transplantation core differentiated, exhibited mature neuronal morphology and expressed mature neuronal markers such as HuD, calbindin and parvalbumin. Interestingly, striatal NSCs generated a greater number of parvalbumin+ and calbindin+ neurons. Virtually none of the grafted cells differentiated into astrocytes or oligodendrocytes. Based on these data, human fetal striatum- and cortex-derived NSCs could be considered potentially safe and viable for transplantation, with strong neurogenic potential, for further exploration in animal models of stroke.

Neural stem cells (NSCs) derived from human fetal striatum and transplanted as neurospheres survive in stroke-damaged striatum, migrate from the implantation site, and differentiate into mature neurons. Here, we investigated how various steps of neurogenesis are affected by intrastriatal transplantation of human NSCs at different time points after stroke and with different numbers of cells in each implant. Rats were subjected to middle cerebral artery occlusion and then received intrastriatal transplants of NSCs. Transplantation shortly after stroke (48 hours) resulted in better cell survival than did transplantation 6 weeks after stroke, but the delayed transplantation did not influence the magnitude of migration, neuronal differentiation, and cell proliferation in the grafts. Transplanting greater numbers of grafted NSCs did not result in a greater number of surviving cells or increased neuronal differentiation. A substantial number of activated microglia was observed at 48 hours after the insult in the injured striatum, but reached maximum levels 1 to 6 weeks after stroke. Our findings show that the best survival of grafted human NSCs in stroke-damaged brain requires optimum numbers of cells to be transplanted in the early poststroke phase, before the inflammatory response is established. These findings, therefore, have direct clinical implications.

Diabetes is a strong risk factor for premature and severe stroke. The GLP-1R (glucagon-like peptide-1 receptor) agonist Ex-4 (exendin-4) is a drug for the treatment of T2D (Type 2 diabetes) that may also have neuroprotective effects. The aim of the present study was to determine the efficacy of Ex-4 against stroke in diabetes by using a diabetic animal model, a drug administration paradigm and a dose that mimics a diabetic patient on Ex-4 therapy. Furthermore, we investigated inflammation and neurogenesis as potential cellular mechanisms underlying the Ex-4 efficacy. A total of seven 9-month-old Type 2 diabetic Goto–Kakizaki rats were treated peripherally for 4 weeks with Ex-4 at 0.1, 1 or 5 μg/kg of body weight before inducing stroke by transient middle cerebral artery occlusion and for 2–4 weeks thereafter. The severity of ischaemic damage was measured by evaluation of stroke volume and by stereological counting of neurons in the striatum and cortex. We also quantitatively evaluated stroke-induced inflammation, stem cell proliferation and neurogenesis. We show a profound anti-stroke efficacy of the clinical dose of Ex-4 in diabetic rats, an arrested microglia infiltration and an increase of stroke-induced neural stem cell proliferation and neuroblast formation, while stroke-induced neurogenesis was not affected by Ex-4. The results show a pronounced anti-stroke, neuroprotective and anti-inflammatory effect of peripheral and chronic Ex-4 treatment in middle-aged diabetic animals in a preclinical setting that has the potential to mimic the clinical treatment. Our results should provide strong impetus to further investigate GLP-1R agonists for their neuroprotective action in diabetes, and for their possible use as anti-stroke medication in non-diabetic conditions.