NMDA and AMPA/kainate glutamate receptors modulate dentate neurogenesis and CA3 synapsin-I in normal and ischemic hippocampus.

The effect of N-methyl-D-aspartate (NMDA) and 2-(aminomethyl)phenylacetic acid/kainate (AMPA/kainate) glutamate receptors on dentate cell proliferation and hippocampal synapsin-I induction was examined after global ischemia. Cell proliferation was assessed using BrdU labeling, and synaptic responses were assessed using synapsin-I expression. Systemic glutamate receptor antagonists (MK-801 and NBQX) increased BrdU-labeled cells in the dentate subgranular zone (SGZ) of control adult gerbils (30% to 90%, P < 0.05). After global ischemia (at 15 days after 10 minutes of ischemia), most CA1 pyramidal neurons died, whereas the numbers of BrdU-labeled cells in the SGZ increased dramatically (>1000%, P < 0.0001). Systemic injections of MK801 or NBQX, as well as intrahippocampal injections of either drug, when given at the time of ischemia completely blocked the birth of cells in the SGZ and the death of CA1 pyramidal neurons at 15 days after ischemia. Glutamate receptor antagonists had little effect on cell birth and death when administered 7 days after ischemia. The induction of synapsin-I protein in stratum moleculare of CA3 at 7 and 15 days after global ischemia was blocked by pretreatment with systemic or intrahippocampal MK-801 or NBQX. It is proposed that decreased dentate glutamate receptor activation--produced by glutamate receptor antagonists in normal animals and by chronic ischemic hippocampal injury--may trigger dentate neurogenesis and synaptogenesis. The synapsin-I induction in mossy fiber terminals most likely represents re-modeling of dentate granule cell neuron presynaptic elements in CA3 in response to the ischemia. The dentate neurogenesis and synaptogenesis that occur after ischemia may contribute to memory recovery after hippocampal injury caused by global ischemia.     

Three steps of neural stem cells development in gerbil dentate gyrus after transient ischemia.

The stage of neurogenesis can be divided into three steps: proliferation, migration, and differentiation. To elucidate detailed relations between these three steps after ischemia, the authors evaluated the three steps in the adult gerbil dentate gyrus (DG) after 5 minutes of transient global ischemia using bromodeoxyuridine (BrdU), highly polysialylated neural cell adhesion molecule (PSA-NCAM), and neuronal nuclear antigen (NeuN) and glial fibrillary acidic protein (GFAP) as markers for proliferation, migration, and differentiation, respectively. Bromodeoxyuridine-labeled cells increased approximately sevenfold, and PSA-NCAM-positive cells increased approximately threefold in the subgranular zone (SGZ) with a peak 10 days after ischemia. Bromodeoxyuridine-labeled cells with PSA-NCAM expression were first detected both in the SGZ and the granule cell layer (GCL) 20 days after ischemia and gradually decreased after that, whereas BrdU-labeled cells with NeuN gradually increased in the GCL until 60 days after ischemia. A few BrdU-labeled cells with GFAP expression were detected in DG after ischemia; no PSA-NCAM-positive cells with GFAP expression were detected, but the radial processes of glial cells were partly in contact with PSA-NCAM-positive cell bodies and dendrites. These results suggest that neural stem cell proliferation begins at the SGZ, and that the cells then migrate into the GCL and differentiate mainly into neuronal cells. The majority of these three steps finished in 2 months after transient global ischemia.     

大鼠脑缺血再灌注诱导自体神经干细胞原位增殖的研究

目的研究缺血性脑损伤对内源性神经干细胞增殖、迁移的影响。方法参照Pulsinelli-Brierley法制作短暂性全脑缺血动物模型,全脑缺血10min后再灌注,采用SABC免疫组化染色显示5'-溴脱氧尿嘧啶(BrdU)阳性细胞和神经巢蛋白(Nestin)阳性细胞,光镜下观察并统计分析脑缺血损伤后内源性神经干细胞增殖、迁移的变化过程。结果脑缺血再灌流24h后,海马、齿状回和室管膜下区的BrdU阳性细胞和Nestin阳性细胞增多,7～10d达到高峰,术后20d仍有表达;在室管膜下区,BrdU阳性细胞和Nestin阳性细胞有向皮质、海马迁移的现象。结论①成年大鼠全脑缺血后7～10d,内源性神经干细胞的增殖达到高峰。②增殖的内源性神经干细胞存在由增殖区向靶区迁移的现象。     

Autoradiographic and histological studies of postnatal neurogenesis. I. A longitudinal investigation of the kinetics,migration and transformation of cells incoorporating tritiated thymidine in neonate rats,with special reference to postnatal neurogenesis in some brain regions

Neonate rats were injected systemically with thymidine-H³ and killed after different periods of survival. Cell proliferation, migration and transformation in the brain were studied autoradiographically. It was established that cells multiplying in the ependymal and subependymal walls of the olfactory ventricle migrate outward into the olfactory bulb, where they become differentiated into granule cells. These postnatally formed granule cells contribute to the formation of the granular and several other layers of the olfactory bulb. Cells multiplying at a high rate in the wall of the lateral ventricle migrate to the hippocampus and contribute to the formation of the granule cells in the granular layer of the dentate gyrus. Cells multiplying at a high rate in the external and internal granular layers of the cerebellum become differentiated into granule cells, and, to a lesser extent, other types of nerve cells of the cerebellar cortex. Evidence was also obtained of the postnatal origin of many of the granule cells of the cochlear nucleus. Postnatal neurogenesis is restricted to these short-axoned granule cells or microneurons; the long-axoned nerve cells or macroneurons of the brain are formed prenatally.     

Neurogenesis in the adult mammalian brain

The production of neurons in the mammalian brain is typically restricted to a discrete developmental period ending, for the most part, prior to parturition. However, in certain regions of the brain, including the dentate gyrus, new neurons continue to be produced well into adulthood. In the adult brain, new cells arise from progenitors located within the hilus and subgranular zone of the dentate gyrus, and then migrate into the dentate granule cell layer. Morphological, biochemical, and ultrastructural evidence indicate that many of these new cells become granule neurons, the principal projection neuron of the dentate gyrus. Anatomic studies have demonstrated that adult-generated granule neurons contribute axonal projections to area CA3 of Ammon's horn, while electron microscopy studies have revealed that these cells possess dendritic processes that extend into the dentate molecular layer to form synapses. Collectively, these data indicate that adult-generated granule neurons become functionally incorporated into the pre-existing neural circuitry of the dentate gyrus. The production and survival of adult-generated granule neurons are significantly influenced by experiential and neuroendocrine factors, suggesting that adult neurogenesis represents a substrate by which the environment may affect the structure and function of the adult brain. Although the precise function of adult-generated granule neurons is unknown, the formation of entirely novel neural circuits, and the regulation of this process by neuroendocrine and experiential factors, is likely to represent an important mode of neural plasticity. Moreover, the persistence of neural progenitors within the adult brain provides hope that an understanding of the process of adult neurogenesis may ultimately be of therapeutic relevance.     

Modification of [3H]inositoltrisphosphate binding in kainic acid-lesioned and postischemic rat hippocampus

A quantitative autoradiographic study was made on the binding of the phosphatidylinositol system ligand [3H]inositol(1,4,5)-trisphosphate (IP3) to forebrain sections from rats decapitated various times after 10 min of forebrain ischemia. To investigate the effect of a deafferentation of the hippocampal CA1, kainic acid-induced CA3-lesioned rats with or without 10 min of cerebral ischemia, were also included. The highest binding was found in the hippocampal CA1. Ten min of cerebral ischemia did not change the binding significantly. Between 5 min and 1 h of recirculation there was a 25-35% binding decline in all regions. In the CA1, where the pyramidal cells became necrotic 6 days after ischemia, there was a further decline to 16% of control. In the cortex, where there is no necrosis in this model, binding did not return to control values until day 14. Four days after a selective CA3 lesion with kainic acid, there was a significant 25% decline in the cortex, dentate gyrus and CA1, whereas in the necrotic CA3 binding declined to 54% of control. Ten min of ischemia did not alter this binding significantly. This decrease in calcium mobilizing intracellular receptors after ischemia and seizures could be due to increased membrane degradation, or to a more specific down-regulation following increased intracellular concentration of calcium and IP3.     

Induction of highly polysialylated neural cell adhesion molecule (PSA-NCAM) in postischemic gerbil hippocampus mainly dissociated with neural stem cell proliferation

We investigated a possible expression of highly polysialylated neural cell adhesion molecule (PSA-NCAM) in gerbil hippocampus after 5 min of transient global ischemia in association to the proliferation of neural stem cell labeled with bromodeoxyuridine (BrdU). The number of PSA-NCAM positive cells increased in the granule cell layer (GCL) of dentate gyrus (DG) by 1.9 to 2.7-fold at 10 and 20 days after the reperfusion. The number of BrdU-labeled cells increased mainly in the subgranular zone of DG by 7.2 to 8.0-fold at 5 and 10 days after the reperfusion. Immunofluorescence for PSA-NCAM and BrdU showed that the majority of DG cells were not double labeled, while one or two cells per section were double labeled in the deepest portion of the GCL only at 10 days after the reperfusion. These results suggest different predominant spatial distribution and chronological change of PSA-NCAM positive and BrdU-labeled cells in DG after transient ischemia.     

Increased proliferation of neural progenitor cells but reduced survival of newborn cells in the contralateral hippocampus after focal cerebral ischemia in rats.

Recent studies demonstrated that neurogenesis in the adult hippocampus increased after transient global ischemia; however, the molecular mechanism underlying increased neurogenesis after ischemia remains unclear. The finding that proliferation of progenitor cells occurred at least a week after ischemic insult suggests that the stimulus was not an ischemic insult to progenitor cells. To clarify whether focal ischemia increases the rate of neurogenesis in the remote area, the authors examined the contralateral hemisphere in rats subjected to permanent occlusion of the middle cerebral artery. In the subgranular zone of the hippocampal dentate gyrus, the numbers of bromodeoxyuridine (BrdU)-positive cells increased approximately sixfold 7 days after ischemia. In double immunofluorescence staining, more than 80% of newborn cells expressed Musashi1, a marker of neural stem/progenitor cells, but only approximately 10% of BrdU-positive cells expressed glial fibrillary acidic protein (GFAP), a marker of astrocytes. The number of BrdU-positive cells markedly decreased 28 days after BrdU administration after ischemia, but it was still elevated compared with that of sham-operated rats. In double immunofluorescence staining, 80% of newborn cells expressed NeuN, a marker of differentiated neurons, and 10% of BrdU-positive cells expressed GFAP. However, in the other areas of the contralateral hemisphere including the rostral subventricular zone, the number of BrdU-positive cells remained unchanged. These results showed that focal ischemia stimulated the proliferation of neuronal progenitor cells, but did not support survival of newborn cells in the contralateral hippocampus.     

Implication of "Down syndrome cell adhesion molecule" in the hippocampal neurogenesis of ischemic monkeys

Molecular signals regulating adult neurogenesis in primates are largely unknown. Here the authors used differential display to analyze gene expression changes that occur in dentate gyrus of adult monkeys after transient global cerebral ischemia. Among 14 genes upregulated, the authors focused on Down syndrome cell adhesion molecule (DSCAM) known to play crucial role during neuronal development, and characterized its expression pattern at the protein level. In contrast with approximately threefold upregulation of Dscam gene on days 5 and 7, immunoblotting and immunofluorescence analyses using specific antibodies showed a gradual decrease of DSCAM after ischemia until day 9 followed by recovery on day 15. In the control, immunofluorescence reactivity of DSCAM was detected in dentate gyrus granule cells and CA4 neurons but decreased after ischemia, being compatible with the immunoblotting data. However, in the subgranular zone, cerebral ischemia led to a marked increase of DSCAM-positive cells on days 9 and 15. DSCAM upregulation was seen in two cell types: one is immature neurons positive for polysialylated neural cell adhesion molecule or betaIII-tubulin, while another is astrocytes positive for S100beta. Young astrocytes were in intimate contact with newly generated neurons in the subgranular zone. These data suggest implication of DSCAM in the adult neurogenesis of primate hippocampus upregulated after ischemia.     

Different thresholds of HSP70 and HSC70 heat shock mRNA induction in post-ischemic gerbil brain.

Thresholds of induction of heat shock protein (HSP) 70 and heat shock cognate protein (HSC) 70 mRNAs after transient global ischemia in gerbil brain were investigated by in situ hybridization using cloned cDNA probes selective for each mRNA species. In sham control brain, HSP70 mRNA was little present, while HSC70 mRNA was present in most cell populations. A 0.5-min occlusion of bilateral common carotid arteries did not affect the amount of HSP70 and HSC70 mRNAs. The selective induction of HSC70 mRNA was observed in dentate granule cells at 1 h, and in most cells of hippocampus especially dentate gyrus at 3 h after 1 min of ischemia when induction of HSP70 mRNA was not evident in the identical brain. The selective induction diminished by 2 days. However, after 2 min of ischemia, HSP70 and HSC70 mRNAs were induced together in hippocampal cells from 1 h of the reperfusion, and the co-induction prolonged in CA1 cells until 2 days. Body temperatures monitored at rectum increased after the reperfusion with a peak at 30 min. The degree of increase of the body temperature was significantly higher in the case after 2-min ischemia than in the cases after 0.5- and 1-min ischemia. Although HSP70 and HSC70 mRNAs are generally co-induced in stressful conditions, our results suggest the different thresholds of the induction between HSP70 and HSC70 mRNAs after transient brain ischemia. The selective induction of HSC70 mRNA which is not accompanied by the induction of HSP70 mRNA may relate to the differences of the duration of ischemia and the degree of the increase of body temperature after ischemia.     

Induction of heat shock protein 72-like immunoreactivity in the hippocampal formation following transient global ischemia

Global ischemia was produced in adult rats by combining bilateral carotid artery occlusions with systemic hypotension for 5 or 10 minutes. Induction of the 72 kD heat shock protein (HSP72) in the hippocampus was examined immunocytochemically 18-24 hours later. Several patterns of HSP72-like immunoreactivity (HSP72LI) were observed. Five minutes of ischemia induced HSP72 in isolated columns of CA1a pyramidal neurons, or throughout CA1 pyramidal neurons and dentate hilar neurons. Ten minutes of ischemia induced marked HSP72LI in CA3 pyramidal neurons, moderate HSP72LI in dentate granule cells, and minimal HSP72LI in CA1 pyramidal, dentate hilar neurons, and hippocampal glia. Two hippocampi subjected to 10 minutes of ischemia exhibited marked HSP72LI in capillary endothelial cells but no neuronal or glial HSP72LI. It is proposed that (a) the induction of HSP72 in hippocampal sectors correlates with their vulnerability to global ischemia (CA1 greater than hilus greater than CA3 greater than dentate gyrus); (b) the induction of HSP72 in hippocampal cells correlates with their vulnerability to global ischemia in that mild ischemia induced HSP72 only in neurons, moderate ischemia in neurons and glia, and severe ischemia only in capillary endothelial cells; (c) the failure to induce HSP72 in hippocampal neurons in 2 cases of 10 min ischemia may be related to severe injury causing disruption of protein synthesis in these cells.     

Effects of CXCR7-neutralizing antibody on neurogenesis in the hippocampal dentate gyrus and cognitive function in the chronic phase of cerebral ischemia

Stromal cell-derived factor-1 and its receptor CXCR4 are essential regulators of the neurogenesis that occurs in the adult hippocampal dentate gyrus.However,the effects of CXCR7,a new atypical receptor of stromal cell-derived factor-1,on hippocampal neurogenesis after a stroke remain largely unknown.Our study is the first to investigate the effect of a CXCR7-neutralizing antibody on neurogenesis in the dentate gyrus and the associated recovery of cognitive function of rats in the chronic stage of cerebral ischemia.The rats were randomly divided into sham,sham+anti-CXCR7,ischemia and ischemia+anti-CXCR7 groups.Endothelin-1 was injected in the ipsilateral motor cortex and striatum to induce focal cerebral ischemia.Sham group rats were injected with saline instead of endothelin-1 via intracranial injection.Both sham and ischemic rats were treated with intraventricular infusions of CXCR7-neutralizing antibodies for 6 days 1 week after surgery.Immunofluorescence staining with doublecortin,a marker for neuronal precursors,was performed to assess the neurogenesis in the dentate gyrus.We found that anti-CXCR7 antibody infusion enhanced the proliferation and dendritic development of doublecortin-labeled cells in the dentate gyrus in both ischemic and sham-operated rats.Spatial learning and memory functions were assessed by Morris water maze tests 30-32 days after ischemia.CXCR7-neutralizing antibody treatment significantly reduced the escape latency of the spatial navigation trial and increased the time spent in the target quadrant of spatial probe trial in animals that received ischemic insult,but not in sham operated rats.These results suggest that CXCR7-neutralizing antibody enhances the neurogenesis in the dentate gyrus and improves the cognitive function after cerebral ischemia in rats.All animal experimental protocols and procedures were approved by the Institutional Animal Care and Use Committee of China Medical University(CMU16089 R)on December 8,2016.     

Acetylsalicylic acid reduces ischemia-induced proliferation of dentate cells in gerbils

Transient global ischemia causes neurogenesis in the dentate gyrus of adult rodents. Ischemic insults to rodents also induce cyclooxygenase-2 (COX-2), an isoform of cyclooxygenases (COXs) and a rate-limiting enzyme for prostanoid synthesis. In the present experiments, adult Mongolian gerbils were chronically treated with acetylsalicylic acid (ASA), a non-selective COX inhibitor, and the proliferation of cells in the dentate gyrus was examined under ischemia. It was proved that BrdU-labeled cells in the dentate gyrus were significantly reduced in number following ASA treatment after 10 min global ischemia. The result strongly suggests that COX, probably COX-2, and prostanoids play an important role in the proliferation of neural cells after ischemia in gerbils.     

Proliferation of neural and neuronal progenitors after global brain ischemia in young adult macaque monkeys

To investigate the effect of global cerebral ischemia on brain cell proliferation in young adult macaques, we infused 5-bromo-2'-deoxyuridine (BrdU), a DNA replication indicator, into monkeys subjected to ischemia or sham-operated. Subsequent quantification by BrdU immunohistochemistry revealed a significant postischemic increase in the number of BrdU-labeled cells in the hippocampal dentate gyrus, subventricular zone of the temporal horn of the lateral ventricle, and temporal neocortex. In all animals, 20-40% of the newly generated cells in the dentate gyrus and subventricular zone expressed the neural progenitor cell markers Musashi1 or Nestin. A few BrdU-positive cells in postischemic monkeys were double-stained for markers of neuronal progenitors (class III beta-tubulin, TUC4, doublecortin, or Hu), neurons (NeuN), or glia (S100beta or GFAP). Our results suggest that ischemia activates endogenous neuronal and glial precursors residing in diverse locations of the adult primate central nervous system.     

Short-term and long-term survival of new neurons in the rat dentate gyrus

New neurons continue to be generated in the dentate gyrus throughout adulthood. Previous studies have shown that a significant proportion of new granule cells labeled with the thymidine analogue bromodeoxyuridine (BrdU) are lost from the adult dentate gyrus within 2 weeks. How long this loss continues and the extent to which it represents cell death, as opposed to dilution of label, is unclear. To address these questions, adult rats were injected with BrdU, and BrdU labeling in the dentate gyrus was compared at several survival time points. Double labeling with BrdU and the cell cycle marker Ki-67 showed that BrdU is detectable for up to 4 days in some cells that continue to divide, indicating that any decrease in the number of BrdU-labeled cells after 4 days is likely to reflect cell death rather than BrdU dilution. Death of new cells in the granule cell layer occurred at a steady rate between 6 and 28 days after labeling, resulting in loss of 50% of BrdU-labeled cells over this 22-day period. New granule cells that survived this first month lived for at least 5 additional months. In contrast, 26% of the granule cells labeled with BrdU at the peak of dentate gyrus development on postnatal day (P) 6 died between 1 and 6 months after labeling. These findings suggest that granule cells born during adulthood that become integrated into circuits and survive to maturity are very stable and may permanently replace granule cells born during development.     

Nature and fate of proliferative cells in the hippocampal dentate gyrus during the life span of the rhesus monkey

The nature of proliferative cells in the subgranular zone (SGZ) of the hippocampal region and the fate of their progeny was analyzed by 3H-thymidine (3H-TdR) autoradiography combined with immunocytochemistry at the light and electron microscopic levels in 18 rhesus monkeys ranging in age from late gestation to 17 years. Our analysis indicates that, during the last quarter of gestation and the first 3 postnatal months, the SGZ produces both glial and neuronal cells. These 2 major classes of cells originate from the 2 precursor lines and, following their mitotic division, migrate to the granular layer. During the juvenile period (4-6 months of age), neuronal production tapers off and most postmitotic cells remaining within the SGZ differentiate into glial elements. In postpubertal animals (3 years and older), the 3H-TdR-labeled cells in the dentate gyrus belong to several non-neuronal classes. The largest group was immunoreactive to the glial fibrillary acidic protein (GFAP) at both the light and electron microscopic levels, indicating their astrocytic nature. The remaining 3H-TdR-labeled, GFAP-negative cells had ultra-structural characteristics of either microglia, oligodendroglia, or their progenitory stem cells. Therefore, there is a continuing addition and/or turnover of the glial cells in the dentate gyrus of sexually mature monkeys, but, in contrast to the massive neurogenesis reported in adult rodents, the production of new neurons could not be detected after puberty. The significance of a stable population of neurons in the hippocampal formation of mature primates is discussed in relation to its possible function in memory.     

Granule-like neurons at the hilar/CA3 border after status epilepticus and their synchrony with area CA3 pyramidal cells: functional implications of seizure-induced neurogenesis.

A group of neurons with the characteristics of dentate gyrus granule cells was found at the hilar/CA3 border several weeks after pilocarpine- or kainic acid-induced status epilepticus. Intracellular recordings from pilocarpine-treated rats showed that these "granule-like" neurons were similar to normal granule cells (i. e., those in the granule cell layer) in membrane properties, firing behavior, morphology, and their mossy fiber axon. However, in contrast to normal granule cells, they were synchronized with spontaneous, rhythmic bursts of area CA3 pyramidal cells that survived status epilepticus. Saline-treated controls lacked the population of granule-like cells at the hilar/CA3 border and CA3 bursts. In rats that were injected after status epilepticus with bromodeoxyuridine (BrdU) to label newly born cells, and also labeled for calbindin D(28K) (because it normally stains granule cells), many double-labeled neurons were located at the hilar/CA3 border. Many BrdU-labeled cells at the hilar/CA3 border also were double-labeled with a neuronal marker (NeuN). Taken together with the recent evidence that granule cells that are born after seizures can migrate into the hilus, the results suggest that some newly born granule cells migrate as far as the CA3 cell layer, where they become integrated abnormally into the CA3 network, yet they retain granule cell intrinsic properties. The results provide insight into the physiological properties of newly born granule cells in the adult brain and suggest that relatively rigid developmental programs set the membrane properties of newly born cells, but substantial plasticity is present to influence their place in pre-existing circuitry.     

Apoptosis and necrosis occur in separate neuronal populations in hippocampus and cerebellum after ischemia and are associated with differential alterations in metabotropic glutamate receptor signaling pathways.

It was evaluated whether postischemic neurodegeneration is apoptosis and occurs with alterations in phosphoinositide-linked metabotropic glutamate receptors (mGluRs) and their associated signaling pathways. A dog model of transient global incomplete cerebral ischemia was used. The CA1 pyramidal cells and cerebellar Purkinje cells underwent progressive delayed degeneration. By in situ end-labeling of DNA, death of CA1 and Purkinje cells was greater at 7 days than 1 day after ischemia, whereas death of granule neurons in dentate gyrus and cerebellar cortex was greater at 1 than at 7 days. Ultrastructurally, degenerating CA1 pyramidal neurons and cerebellar Purkinje cells were necrotic; in contrast, degenerating granule neurons were apoptotic. In agarose gels of regional DNA extracts, random DNA fragmentation coexisted with internucleosomal fragmentation. By immunoblotting of regional homogenates, mGluR1alpha, mGluR5, phospholipase Cbeta (PLCbeta), and Galphaq/11 protein levels in hippocampus at 1 and 7 days after ischemia were similar to control levels, but in cerebellar cortex, mGluR1alpha and mGluR5 were decreased but PLCbeta was increased. By immunocytochemistry, mGluR and PLCbeta immunoreactivity dissipated in CA1 and cerebellar Purkinje cell/ molecular layers, whereas immunoreactivities for these proteins were enhanced in granule neurons. It was concluded that neuronal death after global ischemia exists as two distinct, temporally overlapping forms in hippocampus and cerebellum: necrosis of pyramidal neurons and Purkinje cells and apoptosis of granule neurons. Neuronal necrosis is associated with a loss of phosphoinositide-linked mGluR transduction proteins, whereas neuronal apoptosis occurs with increased mGluR signaling.