Neuropeptide Y promotes neurogenesis in murine subventricular zone

Stem cells of the subventricular zone (SVZ) represent a reliable source of neurons for cell replacement. Neuropeptide Y (NPY) promotes neurogenesis in the hippocampal subgranular layer and the olfactory epithelium and may be useful for the stimulation of SVZ dynamic in brain repair purposes. We describe that NPY promotes SVZ neurogenesis. NPY (1 microM) treatments increased proliferation at 48 hours and neuronal differentiation at 7 days in SVZ cell cultures. NPY proneurogenic properties are mediated via the Y1 receptor. Accordingly, Y1 receptor is a major active NPY receptor in the mouse SVZ, as shown by functional autoradiography. Moreover, short exposure to NPY increased immunoreactivity for the phosphorylated form of extracellular signal-regulated kinase 1/2 in the nucleus, compatible with a trigger for proliferation, whereas 6 hours of treatment amplified the phosphorylated form of c-Jun-NH(2)-terminal kinase signal in growing axons, consistent with axonogenesis. NPY, as a promoter of SVZ neurogenesis, is a crucial factor for future development of cell-based brain therapy. Disclosure of potential conflicts of interest is found at the end of this article.     

BM88/Cend1 expression levels are critical for proliferation and differentiation of subventricular zone-derived neural precursor cells

Neural stem cells remain in two areas of the adult mammalian brain, the subventricular zone (SVZ) and the dentate gyrus of the hippocampus. Ongoing neurogenesis via the SVZ-rostral migratory stream pathway maintains neuronal replacement in the olfactory bulb (OB) throughout life. The mechanisms determining how neurogenesis is restricted to only a few regions in the adult, in contrast to its more widespread location during embryogenesis, largely depend on controlling the balance between precursor cell proliferation and differentiation. BM88/Cend1 is a neuronal lineage-specific regulator implicated in cell cycle exit and differentiation of precursor cells in the embryonic neural tube. Here we investigated its role in postnatal neurogenesis. Study of in vivo BM88/Cend1 distribution revealed that it is expressed in low levels in neuronal precursors of the adult SVZ and in high levels in postmitotic OB interneurons. To assess the functional significance of BM88/Cend1 in neuronal lineage progression postnatally, we challenged its expression levels by gain- and loss-of-function approaches using lentiviral gene transfer in SVZ-derived neurospheres. We found that BM88/Cend1 overexpression decreases proliferation and favors neuronal differentiation, whereas its downregulation using new-generation RNA interference vectors yields an opposite phenotype. Our results demonstrate that BM88/Cend1 participates in cell cycle control and neuronal differentiation mechanisms during neonatal SVZ neurogenesis and becomes crucial for the transition from neuroblasts to mature neurons when reaching high levels.     

EphB2 induces proliferation and promotes a neuronal fate in adult subventricular neural precursor cells

The subventricular germinal zone (SVZ) retains an active population of stem cells and neural precursor cells throughout adulthood. EphrinB signaling mediates angiogenesis and vasculogenesis in the developing and adult brain. Recent studies indicate that molecules involved in angiogenesis often influence neurogenesis as well. However, little work has been done considering a role for EphB2/EphrinB in adult neural precursor cells. We therefore examined whether the EphB2 receptor tyrosine kinase could directly effect proliferation of SVZ neural precursors and/or direct the cell fate of SVZ cells in vitro. Here, we found that clustered EphB2 increased bromodeoxyuridine (BrdU) incorporation and proliferation of SVZ neurosphere cultures. Immunostaining and RT-PCR analysis for beta-tubulin III (Tuj1) and GFAP indicated 4-day treatment with EphB2 promoted a neuronal phenotype, suggesting that the EphB2 receptor might also direct SVZ cell fate. EphB2 transiently down-regulated SVZ cell mRNA of Notch1 and Zic1, genes that regulate neurogenesis and neuronal differentiation. Notch1 has been implicated in apoptosis of neural precursors, however, a cell viability assay revealed no statistical difference between EphB2-treated and control cultures. When SVZ neurospheres were cultured upon Matrigel, EphB2 attenuated radial migration of SVZ cells in vitro. These results demonstrate that EphB2/EphrinB signaling directly induces SVZ proliferation, decreases migration, and promotes a neuronal fate of SVZ neural precursors independent of cell survival.     

Histamine stimulates neurogenesis in the rodent subventricular zone

Neural stem/progenitor cells present in the subventricular zone (SVZ) are a potential source of repairing cells after injury. Therefore, the identification of novel players that modulate neural stem cells differentiation can have a huge impact in stem cell-based therapies. Herein, we describe a unique role of histamine in inducing functional neuronal differentiation from cultured mouse SVZ stem/progenitor cells. This proneurogenic effect depends on histamine 1 receptor activation and involves epigenetic modifications and increased expression of Mash1, Dlx2, and Ngn1 genes. Biocompatible poly (lactic-co-glycolic acid) microparticles, engineered to release histamine in a controlled and prolonged manner, also triggered robust neuronal differentiation in vitro. Preconditioning with histamine-loaded microparticles facilitated neuronal differentiation of SVZ-GFP cells grafted in hippocampal slices and in in vivo rodent brain. We propose that neuronal commitment triggered by histamine per se or released from biomaterial-derived vehicles may represent a new tool for brain repair strategies. STEM CELLS 2012; 30:773-784.     

Biphasic effects of exogenous VEGF on VEGF expression of adult neural progenitors

Vascular endothelial growth factor (VEGF) regulates neurogenesis. The present study investigated the direct effect of VEGF on the enhancement of proliferation and differentiation of the adult mouse subventricular zone (SVZ) neural progenitors in vitro. A high dose (500 ng/ml) of VEGF significantly downregulated endogenous VEGF receptors 1 and 2, which was associated with significantly reduced neural progenitor cell proliferation and enhancement of neuronal differentiation. A low dose (50 ng/ml) of VEGF significantly upregulated endogenous VEGF receptors 1 and 2 but did not increase proliferation and differentiation. These data suggest that exogenous VEGF has a biphasic effect on the expression of endogenous VEGF receptors, and the high dose of VEGF enhances adult neural progenitor cell differentiation into neurons.     

Acute intermittent nicotine treatment induces fibroblast growth factor-2 in the subventricular zone of the adult rat brain and enhances neuronal precursor cell proliferation

Over the past years, evidence has accumulated that stem cells are present in the adult brain, and generate neurons and/or glia from two active germinal zones: the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the dentate gyrus of the hippocampus. This study shows that acute intermittent nicotine treatment significantly enhances neuronal precursor cell proliferation in the SVZ of adult rat brain, but not in the SGZ of the hippocampus, and pre-treatment with mecamylamine, a nonselective nAChR antagonist, blocks the enhanced precursor proliferation by nicotine. This effect is supported by up-regulation of fibroblast growth factor-2 (FGF-2) mRNA in the SVZ and the expression of its receptor FGFR-1 in cells of SVZ showing precursor cells profile. It is also demonstrated that the nicotine effect on neuronal precursor proliferation is mediated by FGF-2 via fibroblast growth factor receptor 1 (FGFR-1) activation by showing that i.c.v. pre-treatment with anti-FGF-2 antibodies or with FGFR-1 inhibitor 3-[(3-(2-carboxyethyl)-4-methylpyrrol-2-yl)methylene]-2-indolinone (SU5402) blocks nicotine-induced precursor cell proliferation. This nicotine enhancement of neuronal precursor cell proliferation was not accompanied by an increase in the number of apoptotic cells. Taken together the present findings revealed the existence in the SVZ of the adult rat brain of a trophic mechanism mediated by FGF-2 and its receptor and regulated by nAchR activation. This possibility of in vivo regulation of neurogenesis in the adult brain by exogenous factors may aid to develop treatments stimulating neurogenesis with potential therapeutic implications.     

nNOS deficiency-induced cell proliferation depletes the neurogenic reserve

The consequences of nitric oxide synthase (NOS) gene knockout on proliferation, survival and differentiation of neuronal precursors in the subgranular (SGZ) and subventricular (SVZ) zones were analyzed. Comparative studies were performed in neonatal, adult and old (18-month) wild-type (WT), nNOS, eNOS, and iNOS knockout (KO) mice. Effects on brain cell proliferation were studied by sacrificing animals at 24h after injecting BrdU, while effects on survival and differentiation of dividing brain cells were studied by sacrificing other animals at three weeks after injections and double immunostaining with cell phenotype-specific antibodies. In the neonatal SGZ, cell proliferation was higher than at any other age, with a significantly decreased level in eNOS-KO mice. In the neonatal SVZ, cell proliferation in each of the three NOS-KO strains was significantly lower than in WT. In the adult, in both the SGZ and SVZ, all strains showed lower levels of cell proliferation than in neonates. Thereby, the significant highest cell proliferation was found in the SGZ and SVZ of nNOS-KO mice. In the SGZ and SVZ of old mice, in each strain, BrdU-positive cell counts were further reduced from adult levels, whereby cell proliferation of nNOS-KO mice attained the most massive reduction (in the SGZ almost to zero). In adult animals sacrificed 21 days after BrdU injections, values of BrdU-/NeuN-positive cells in all knockout animals were the same as WT, indicating that the initial cell proliferation changes were not sustained or translated into neuronal differentiation. The effect of nNOS-KO, inducing cell proliferation only temporarily, consists with the concept that neuronal stem cells have a finite proliferation capacity.     

Effects of interferon-gamma and tumor necrosis factor-alpha on clonogenic growth of cell lines and primary cultures from human gliomas and brain metastases

In this study we tested the effect of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and a combination of these immunotherapeutics on clonogenic growth of cell lines and primary cultures of malignant human brain tumors. Out of 29 primary cultures studied, 5 were inhibited 50% or more by IFN-gamma (10 ng/ml), 6 were inhibited 50% or more by TNF-alpha (10 ng/ml) and 14 were inhibited 50% or more by a combination of IFN-gamma (10 ng/ml) and TNF-alpha (10 ng/ml). The doses of the immunotherapeutics used in this in vitro study are achievable in serum after intravenous administration of IFN-gamma and TNF-alpha without causing unacceptable side effects. We believe that some glioma patients and some patients with brain metastases will benefit from receiving treatment with IFN-gamma, TNF-alpha or a combination of these immunotherapeutics. The patients should be selected for treatment with these immunotherapeutics according to in vitro sensitivity results.     

Postnatal neurogenesis generates heterotopias, olfactory micronodules and cortical infiltration following single-cell Tsc1 deletion

Neurological symptoms in tuberous sclerosis complex (TSC) and associated brain lesions are thought to arise from abnormal embryonic neurogenesis due to inherited mutations in Tsc1 or Tsc2. Neurogenesis persists postnatally in the human subventricular zone (SVZ) where slow-growing tumors containing Tsc-mutant cells are generated in TSC patients. However, whether Tsc-mutant neurons from the postnatal SVZ contribute to brain lesions and abnormal circuit remodeling in forebrain structures remain unexplored. Here, we report the formation of olfactory lesions following conditional genetic Tsc1 deletion in the postnatal SVZ using transgenic mice or targeted single-cell electroporation. These lesions include migratory heterotopias and olfactory micronodules containing neurons with a hypertrophic dendritic tree. Most significantly, our data identify migrating glial and neuronal precursors that are re-routed and infiltrate forebrain structures (e.g. cortex) and become glia and neurons. These data show that Tsc1-mutant cells from the neonatal and juvenile SVZ generate brain lesions and structural abnormalities, which would not be visible using conventional non-invasive imaging. These findings also raise the hypothesis that micronodules and the persistent infiltration of cells to forebrain structures may contribute to network malfunction leading to progressive neuropsychiatric symptoms in TSC.     

Neurogenesis in the striatum of the quinolinic acid lesion model of Huntington's disease

The presence of ongoing neurogenesis in the adult mammalian brain raises the exciting possibility that endogenous progenitor cells may be able to generate new neurons to replace cells lost through brain injury or neurodegenerative disease. We have recently demonstrated increased cell proliferation and the generation of new neurons in the Huntington's disease human brain. In order to better understand the potential role of endogenous neuronal replacement in neurodegenerative disorders and extend our initial observations in the human Huntington's disease brain, we examined the effect of striatal cell loss on neurogenesis in the subventricular zone (SVZ) of the adult rodent forebrain using the quinolinic acid (QA) lesion rat model of Huntington's disease. Cell proliferation and neurogenesis were assessed with bromodeoxyuridine (BrdU) labeling and immunocytochemistry for cell type-specific markers. BrdU labeling demonstrated increased cell proliferation in the SVZ ipsilateral to the QA-lesioned striatum, resulting in expansion of the SVZ in the lesioned hemisphere. Quantification revealed that QA lesion-induced striatal cell loss produced a significant increase in the area of BrdU-immunoreactivity in the SVZ ipsilateral to the lesioned hemisphere between 1 and 14 days post-lesion compared with sham-lesioned animals, with the greatest increase observed at 7 days post-lesion. These changes were associated with an increase in cells in the anterior SVZ ipsilateral to the lesioned striatum expressing the antigenic marker for SVZ neuroblasts, doublecortin (Dcx). Importantly, we observed Dcx-positive cells extending from the SVZ into the QA-lesioned striatum where a subpopulation of newly generated cells expressed markers for immature and mature neurons. This study demonstrates that loss of GABAergic medium spiny projection neurons following QA striatal lesioning of the adult rat brain increases SVZ neurogenesis, leading to the putative migration of neuroblasts to damaged areas of the striatum and the formation of new neurons.     

Increased re-entry into cell cycle mitigates age-related neurogenic decline in the murine subventricular zone

Although new neurons are produced in the subventricular zone (SVZ) of the adult mammalian brain, fewer functional neurons are produced with increasing age. The age-related decline in neurogenesis has been attributed to a decreased pool of neural progenitor cells (NPCs), an increased rate of cell death, and an inability to undergo neuronal differentiation and develop functional synapses. The time between mitotic events has also been hypothesized to increase with age, but this has not been directly investigated. Studying primary-cultured NPCs from the young adult and aged mouse forebrain, we observe that fewer aged cells are dividing at a given time; however, the mitotic cells in aged cultures divide more frequently than mitotic cells in young cultures during a 48-hour period of live-cell time-lapse imaging. Double-thymidine-analog labeling also demonstrates that fewer aged cells are dividing at a given time, but those that do divide are significantly more likely to re-enter the cell cycle within a day, both in vitro and in vivo. Meanwhile, we observed that cellular survival is impaired in aged cultures. Using our live-cell imaging data, we developed a mathematical model describing cell cycle kinetics to predict the growth curves of cells over time in vitro and the labeling index over time in vivo. Together, these data surprisingly suggest that progenitor cells remaining in the aged SVZ are highly proliferative.     

Mesenchymal Stem Cells Stimulate Endogenous Neurogenesis in the Subventricular Zone of Adult Mice

Mammalian neurogenesis has been demonstrated in the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the dentate gyrus in the hippocampus. However, the low rate and the restricted long term survival of newborn cells limit the restorative ability of this process. Adult bone marrow derived mesenchymal stem cells (MSCs) have been extensively studied due to their wide therapeutic potential. The aim of this study was to determine if MSC transplantation to the normally restrictive SVZ of mice housed in an enriched environment stimulates endogenous neurogenesis. In the presented study 30 C57BL/6 female mice were divided into 3 groups: standard environment injected with phosphate buffered saline (PBS) and enriched environment injected with either PBS or MSCs. Bromodeoxyuridine was injected for 6 days, and 3 weeks later the mice were sacrificed and the brain tissue analyzed immunohistochemically. PBS-treated mice housed in enriched cages showed augmented neurogenesis in the SGZ but not the SVZ. MSC transplantation was associated with increased proliferation and neuronal differentiation of neural progenitors within the SVZ and an increase in the proportion of the newborn neurons out of the total proliferating cells. Histological analysis confirmed the survival of a significant amount of the transplanted cells at least 3 weeks after transplantation, and the presence of brain-derived neurotrophic factor expression. To our knowledge, this is the first study to show that MSCs might interfere with the tight regulation of the SVZ, independent of the induced brain lesion.     

Endogenous hepatocyte growth factor is a niche signal for subventricular zone neural stem cell amplification and self-renewal

Neural stem cells persist in the adult mammalian brain, within the subventricular zone (SVZ). The endogenous mechanisms underpinning SVZ neural stem cell proliferation, self-renewal, and differentiation are not fully elucidated. In the present report, we describe a growth-stimulatory activity of liver explant-conditioned media on SVZ cell cultures and identify hepatocyte growth factor (HGF) as a major player in this effect. HGF exhibited a mitogenic activity on SVZ cell cultures in a mitogen-activated protein kinase (MAPK) (ERK1/2)-dependent manner as U0126, a specific MAPK inhibitor, blocked it. Combining a functional neurosphere forming assay with immunostaining for c-Met, along with markers of SVZ cells subtypes, demonstrated that HGF promotes the expansion of neural stem-like cells that form neurospheres and self-renew. Immunostaining, HGF enzyme-linked immunosorbent assay and Madin-Darby canine kidney cell scattering assay indicated that SVZ cell cultures produce and release HGF. SVZ cell-conditioned media induced proliferation on SVZ cell cultures, which was blocked by HGF-neutralizing antibodies, hence implying that endogenously produced HGF accounts for a major part in SVZ mitogenic activity. Brain sections immunostaining revealed that HGF is produced by nestin-expressing cells and c-Met is expressed within the SVZ by immature cells. HGF intracerebroventricular injection promoted SVZ cell proliferation and increased the ability of these cells exposed in vivo to HGF to form neurospheres in vitro, whereas intracerebroventricular injection of HGF-neutralizing antibodies decreased SVZ cell proliferation. The present study unravels a major role, both in vitro and in vivo, for endogenous HGF in SVZ neural stem cell growth and self-renewal.     

Pigment epithelium-derived factor is a niche signal for neural stem cell renewal

Adult stem cells are characterized by self-renewal and multilineage differentiation, and these properties seem to be regulated by signals from adjacent differentiated cell types and by extracellular matrix molecules, which collectively define the stem cell "niche." Self-renewal is essential for the lifelong persistence of stem cells, but its regulation is poorly understood. In the mammalian brain, neurogenesis persists in two germinal areas, the subventricular zone (SVZ) and the hippocampus, where continuous postnatal neuronal production seems to be supported by neural stem cells (NSCs). Here we show that pigment epithelium-derived factor (PEDF) is secreted by components of the murine SVZ and promotes self-renewal of adult NSCs in vitro. In addition, intraventricular PEDF infusion activated slowly dividing stem cells, whereas a blockade of endogenous PEDF decreased their cycling. These data demonstrate that PEDF is a niche-derived regulator of adult NSCs and provide evidence for a role for PEDF protein in NSC maintenance.     

新生大鼠脑缺血后脑室下区基因表达谱的生物信息学分析

目的建立3日龄大鼠脑缺血模型,采用基因芯片分析新生大鼠未成熟脑缺血损伤后脑室下区(SVZ)基因表达谱的变化。方法同窝3日龄大鼠随机分为实验组和对照组,采用双侧颈总动脉结扎法制备缺血性脑损伤模型,于不同时点取SVZ组织,采用Affymetrix Rat230 2.0基因表达谱芯片观察SVZ基因表达变化,芯片数据分别用3种不同方法分析,并用实时PCR方法验证芯片结果。结果①通过差异基因筛选,发现3日龄大鼠脑缺血损伤后SVZ有17个基因发生表达变化,其中上调基因10个,下调基因7个,这些基因参与多种功能的调节。②基于基因功能的表达趋势分析显示,在所有参与增殖、凋亡功能的基因中,转化生长因子-β(TGF-β)在3日龄大鼠脑缺血损伤后SVZ微环境基因表达变化中起枢纽作用。实时PCR验证结果显示,TGF-β1及Smad2于缺血后1、4和7 d表达均上升,7 d达高峰。③在参与Wnt、TGF、BMP和血管内皮生长因子(VEGF)通路所有基因组成的基因功能相似性网络中,有13个基因在网络中起核心调控作用,构成信号通路串话节点。结论新生大鼠在脑缺血损伤后SVZ微环境中,参与神经新生的BMP、TGF、VEGF和Wnt通路间的串话...     

Ischemia induces cell proliferation and neurogenesis in the gerbil hippocampus in response to neuronal death

We studied hippocampal cellular proliferation and neurogenesis processes in a model of transient global cerebral ischemia in gerbils by labelling dividing cells with 5'-Bromo-2'-deoxyuridine (BrdU). Surrounding the region of selective neuronal death (CA1 pyramidal layer of the hippocampus), an important increase in reactive astrocytes and BrdU-labelled cells was detected 5 days after ischemia. A similar result was found in the dentate gyrus (DG) 12 days after ischemia. The differentiation of the BrdU+ cells was investigated 28 days after BrdU administration by analyzing the morphology, anatomic localization and cell phenotype by triple fluorescent labelling (BrdU, adult neural marker NeuN and DNA marker TOPRO-3) using confocal laser-scanning microscopy. This analysis showed increased neurogenesis in the DG in case of ischemia and triple positive labelling in some newborn cells in CA1. Seven brain hemispheres from gerbils subjected to ischemia did not develop CA1 neuronal death; hippocampus from these hemispheres did not show any of the above mentioned findings. Our results indicate that ischemia triggers proliferation in CA1 and neurogenesis in the DG in response to CA1 pyramidal neuronal death, independently of the reduced cerebral blood flow or the cell migration from subventricular zone (SVZ).     

Cilostazol attenuates ischemic brain injury and enhances neurogenesis in the subventricular zone of adult mice after transient focal cerebral ischemia

Evidence suggests that neurogenesis occurs in the adult mammalian brain, and that various stimuli, for example, ischemia/hypoxia, enhance the generation of neural progenitor cells in the subventricular zone (SVZ) and their migration into the olfactory bulb. In a mouse stroke model, focal ischemia results in activation of neural progenitor cells followed by their migration into the ischemic lesion. The present study assessed the in vivo effects of cilostazol, a type 3 phosphodiesterase inhibitor known to activate the cAMP-responsive element binding protein (CREB) signaling, on neurogenesis in the ipsilateral SVZ and peri-infarct area in a mouse model of transient middle cerebral artery occlusion. Mice were divided into sham operated (n=12), vehicle- (n=18) and cilostazol-treated (n=18) groups. Sections stained for 5-bromodeoxyuridine (BrdU) and several neuronal and a glial markers were analyzed at post-ischemia days 1, 3 and 7. Cilostazol reduced brain ischemic volume (P<0.05) and induced earlier recovery of neurologic deficit (P<0.05). Cilostazol significantly increased the density of BrdU-positive newly-formed cells in the SVZ compared with the vehicle group without ischemia. Increased density of doublecortin (DCX)-positive and BrdU/DCX-double positive neural progenitor cells was noted in the ipsilateral SVZ and peri-infarct area at 3 and 7 days after focal ischemia compared with the vehicle group (P<0.05). Cilostazol increased DCX-positive phosphorylated CREB (pCREB)-expressing neural progenitor cells, and increased brain derived neurotrophic factor (BDNF)-expressing astrocytes in the ipsilateral SVZ and peri-infarct area. The results indicated that cilostazol enhanced neural progenitor cell generation in both ipsilateral SVZ and peri-infarct area through CREB-mediated signaling pathway after focal ischemia.     

Ischemic preconditioning enhances neurogenesis in the subventricular zone

Ischemic preconditioning (IPC) before subsequent prolonged ischemia is considered an emerging endogenous means of ischemic brain protection. We tested whether IPC induces endogenous neurogenesis in the subventricular zone (SVZ) and angiogenesis in the peri-ischemic area. Middle cerebral artery occlusion was administered to rats by filament insertion for 10 min (IPC) and/or 2 h (prolonged focal ischemia [PFI]). IPC alone increased 5'-bromo-2'-deoxyuridine (BrdU) (+) cells 2.5-fold in the SVZ compared with controls at 7 days. The numbers of BrdU/doublecortin (Dcx) or BrdU/neuronal nuclei (NeuN) double-labeled cells also increased, but extents of BrdU/glial fibrillary acidic protein (GFAP) double-labeling in the SVZ were not different. The IPC+PFI group showed about a 40% reduction in infarct volume. PFI increased BrdU (+) cells in the SVZ, and this was greatly enhanced by IPC treatment. The number of BrdU/Dcx double-labeled cells was strongly increased in ischemic brains administered IPC. Differentiation into mature neurons was also enhanced at 14 and 28 days. In addition, IPC significantly promoted angiogenesis in the ischemic penumbra as indicated by von Willebrand factor (vWF) staining. Our results indicate that IPC enhances neurogenesis in the SVZ even without subsequent PFI, and also enhances neurogenesis and angiogenesis after subsequent PFI. We conclude that IPC confers neuroprotection, and also promotes endogenous neurogenesis and angiogenesis.