Transient Cerebral Ischemia Induces Active Astrocytosis Without Distinct Neuronal Death in the Gerbil Main Olfactory Bulb: A Long-Term Analysis

In the present study, we examined ischemia-induced neuronal and glial changes in the gerbil MOB at various time points during 60 days after 5 min of transient cerebral ischemia. The number of neuronal neuclei-immunoreactive neurons was not changed after ischemia/reperfusion (I/R). Myelin basic protein immunoreaction was well preserved after I/R. Five days after I/R, reactive form of GFAP-immunoreactive astrocytes began to increase in the external plexiform layer and granule cell layer: These reactive astrocytes peaked 10 days after I/R, thereafter, they decreased with time after I/R. Iba-1-immunoreactive microglia were ubiquitously distributed in all layers of the MOB. After I/R, significant changes in their morphology and immunoreactivity were not detected. The results of western blot analyses for GFAP, Iba-1 and MBP were similar to the immunohistochemical data. In addition, 8-hydroxy-2′-deoxyguanosine (a marker for DNA damage) immunoreactivity and SOD1, an antioxidant, protein levels were not changed in the ischemic MOB. These results indicate that neurons in the MOB are resistant to ischemic insult, showing that astrocytes are activated late in the ischemic MOB.     

Comparison of Changes in GAD65 and GAD67 Immunoreactivity and Levels in the Gerbil Main Olfactory Bulb Induced by Transient Ischemia

In the present study, we investigated changes in glutamate decarboxylase 65 (GAD65) and GAD67 immunoreactivity and protein levels in the main olfactory bulb (MOB) after 5 min of transient forebrain ischemia in gerbils. GAD65 immunoreactivity in the sham-operated group was shown in neurons and neuropil except for the somata of granule cells. GAD65 immunoreactivity was increased in neurons in the external plexiform layer 60 days after ischemia, and in mitral cells 30 and 60 days after ischemia. GAD67 immunoreactivity in the sham-operated group was shown in periglomerular cells, neuron in the external plexiform layer and granule cells with neuropil. GAD67 immunoreactivity in periglomerular cells was increased 10, 45 and 60 days after ischemia. GAD67 immunoreactivity in neurons in the external plexiform layer was increased 10 and 15 days after ischemia. Mitral cells showed strong GAD67 immunoreactivity 10 days after ischemia. However, GAD67 immunoreactivity in the granule cells was not changed with time after ischemia. In Western blot analysis for GAD65 and GAD67 protein levels in the ischemic gerbil MOB, GAD65 level was not changed after ischemia; GAD67 level was increased 10 days after ischemia. These results suggest that transient ischemia causes changes in GAD65 and GAD67 immunoreactivity in the gerbil MOB, and this change may induce a malfunction in olfaction after an ischemic insult. Ki-Yeon Yoo and In Koo Hwang equally contributed to this article.     

Hypoxic Preconditioning Attenuates Neuronal Cell Death by Preventing MEK/ERK Signaling Pathway Activation after Transient Global Cerebral Ischemia in Adult Rats

Our previous data indicated that hypoxic preconditioning (HPC) ameliorates transient global cerebral ischemia (tGCI)-induced neuronal death in hippocampal CA1 subregion of adult rats. However, the possible molecular mechanisms for neuroprotection of this kind are largely unknown. This study was performed to investigate the role of the mitogen-activated protein kinase/extra-cellular signal-regulated kinase kinase (MEK)/extra-cellular signal-regulated kinase (ERK) pathway in HPC-induced neuroprotection. tGCI was induced by applying the four-vessel occlusion method. Pretreatment with 30 min of hypoxia applied 1 day before 10 min tGCI significantly decreased the level of MEK1/2 and ERK1/2 phosphorylation in ischemic hippocampal CA1 subregion. Also, HPC decreased the expression of phosphorylated ERK1/2 in degenerating neurons and astrocytes. However, the administration of U0126, a MEK kinase inhibitor, partly blocked MEK1/2 and ERK1/2 phosphorylation induced by tGCI. Meanwhile, neuronal survival was improved, and glial cell activation was significantly reduced. Collectively, these data indicated that the MEK/ERK signaling pathway might be involved in HPC-induced neuroprotection following tGCI. Also, HPC resulted in a reduction of glial activation.     

Mitochondria and Neuronal Death/Survival Signaling Pathways in Cerebral Ischemia

Apoptotic cell death pathways have been implicated in acute brain injuries, including cerebral ischemia, brain trauma, and spinal cord injury, and in chronic neurodegenerative diseases. Experimental ischemia and reperfusion models, such as transient focal/global ischemia in rodents, have been thoroughly studied and suggest the involvement of mitochondria and the cell survival/death signaling pathways in cell death/survival cascades. Recent studies have implicated mitochondria-dependent apoptosis involving pro- and anti-apoptotic protein binding, the release of cytochrome c and second mitochondria-derived activator of caspase, the activation of downstream caspases-9 and –3, and DNA fragmentation. Reactive oxygen species are known to be significantly generated in the mitochondrial electron transport chain in the dysfunctional mitochondria during reperfusion after ischemia, and are also implicated in the survival signaling pathway that involves phosphatidylinositol-3-kinase (PI3-K), Akt, and downstream signaling molecules, like Bad, 14-3-3, and the proline-rich Akt substrate (PRAS), and their bindings. Further studies of these survival pathways may provide novel therapeutic strategies for clinical stroke.Special issue dedicated to Lawrence F. Eng.     

TAM Receptors Support Neural Stem Cell Survival,Proliferation and Neuronal Differentiation

Tyro3, Axl and Mertk (TAM) receptor tyrosine kinases play multiple functional roles by either providing intrinsic trophic support for cell growth or regulating the expression of target genes that are important in the homeostatic regulation of immune responses. TAM receptors have been shown to regulate adult hippocampal neurogenesis by negatively regulation of glial cell activation in central nervous system (CNS). In the present study, we further demonstrated that all three TAM receptors were expressed by cultured primary neural stem cells (NSCs) and played a direct growth trophic role in NSCs proliferation, neuronal differentiation and survival. The cultured primary NSCs lacking TAM receptors exhibited slower growth, reduced proliferation and increased apoptosis as shown by decreased BrdU incorporation and increased TUNEL labeling, than those from the WT NSCs. In addition, the neuronal differentiation and maturation of the mutant NSCs were impeded, as characterized by less neuronal differentiation (β-tubulin III⁺) and neurite outgrowth than their WT counterparts. To elucidate the underlying mechanism that the TAM receptors play on the differentiating NSCs, we examined the expression profile of neurotrophins and their receptors by real-time qPCR on the total RNAs from hippocampus and primary NSCs; and found that the TKO NSC showed a significant reduction in the expression of both nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), but accompanied by compensational increases in the expression of the TrkA, TrkB, TrkC and p75 receptors. These results suggest that TAM receptors support NSCs survival, proliferation and differentiation by regulating expression of neurotrophins, especially the NGF.     

Mitochondrial Respiratory Function and Cell Death in Focal Cerebral Ischemia

Pyruvate-supported oxygen uptake was determined as a measure of the functional capacity of mitochondria obtained from rat brain during unilateral middle cerebral artery occlusion and reperfusion. During ischemia, substantial reductions developed in both ADP-stimulated and uncoupled respiration in tissue from the focus of the affected area in the striatum and cortex. A similar pattern of change but with lesser reductions was seen in the adjacent perifocal tissue. Succinate-supported respiration was more affected than that with pyruvate in perifocal tissue at 2 h of ischemia, suggesting additional alterations to mitochondrial components in this tissue. Mitochondrial respiratory activity recovered fully in samples from the cortex, but not the striatum, within the first hour of reperfusion following 2 h of ischemia and remained similar to control values at 3 h of reperfusion. In contrast, impairment of the functional capacity of mitochondria from all three regions was seen in the first 3 h of reperfusion following 3 h of ischemia. Extensive infarction generally affecting the cortical focal tissue with more variable involvement of the perifocal tissue developed following 2 h of focal ischemia. Thus, mitochondrial impairment during the first 3 h of reperfusion was apparently not essential for tissue infarction to develop. Nonetheless, the observed mitochondrial changes could contribute to the damage produced by permanent focal ischemia as well as the larger infarcts produced when reperfusion was initiated following 3 h of ischemia.     

Comparison of Newly Generated Doublecortin-immunoreactive Neuronal Progenitors in the Main Olfactory Bulb among Variously Aged Gerbils

In the present study, we investigated age-related differences in neuronal progenitors in the gerbil main olfactory bulb (MOB) using doublecortin (DCX), a marker for neuronal progenitors which differentiate into neurons in the brain. No difference in the number of neuronal nuclei (NeuN)-immunoreactive neurons was found in the MOB at variously aged gerbils. At postnatal month (PM) 1, DCX immunoreaction was detected in all layers of the MOB except for the olfactory nerve layer. At this time point, DCX-immunoreactive cells (neuronal progenitors) were very abundant; however, they did not have fully developed-processes. From PM 3, the number of DCX-immunoreactive neuronal progenitors was decreased with age. At PM 6, DCX-immunoreactive cells showed very well-developed processes. In western blot analysis, DCX protein level in the MOB was highest at PM 1. Thereafter, levels of DCX protein were decreased with age. In the subventricular zone of the lateral ventricle, the number of Ki-67-immunoractive cells (proliferating cells) was also significantly decreased with age. In addition, increases of α-synuclein-immunoreactive structures were observed in the MOB with age. These results suggest that decrease in DCX-immunoreactive neuronal progenitors and its protein levels in the MOB with age may be associated with reduction of cell proliferation in the SVZ and with an increase in α-synuclein in the MOB.     

Cathepsin B and Phospo-JNK in Relation to Ongoing Apoptosis after Transient Focal Cerebral Ischemia in the Rat

Cathepsin B, one of major lysosomal cathepsins, and JNK, a downstream component of Rho kinase (ROCK), are two families of proteases, which play an important role in ischemic cell apoptosis. However, the interrelationship between Cathepsin B and JNK in apotosis has not been examined. In the present study, rats were decapitated at 0, 2, 6, 24, 48 h of reperfusion after 2 h of middle cerebral artery occlusion (MCAO); TUNEL-positive cells appeared in the ipsilateral preoptic region during reperfusion after 2-h MCAO, and gradually increased to a peak of 24 h after reperfusion; Phospho-JNK (p-JNK) immunoreactivity, occurring after Cathepsin B expression, was gradually increased and peaked altogether with Cathepsin B at 6-h reperfusion; Fasudil (5 mg/kg, intraperitoneally), an inhibitor of ROCK, decreased the level of p-JNK and apoptotic neurons, and had no effect on cathepsin B; Immunofluorescent double labeling showed that the colocalization of cathepsin B with p-JNK appeared in the preoptic region at 2, 6, 24, 48 h of reperfusion. These findings indicate that a signal transduction pathway by ischemia–reperfusion is most likely to exist: lysosomal cathepsin B-Rho/Rho kinase pathway-JNK signaling pathway-mitochondrial-dependent intrinsic pathway.     

Time-course Changes in Immunoreactivities of Glucokinase and Glucokinase Regulatory Protein in the Gerbil Hippocampus Following Transient Cerebral Ischemia

Glucose is a main energy source for normal brain functions. Glucokinase (GK) plays an important role in glucose metabolism as a glucose sensor, and GK activity is modulated by glucokinase regulatory protein (GKRP). In this study, we examined the changes of GK and GKRP immunoreactivities in the gerbil hippocampus after 5 min of transient global cerebral ischemia. In the sham-operated-group, GK and GKRP immunoreactivities were easily detected in the pyramidal neurons of the stratum pyramidale of the hippocampus. GK and GKRP immunoreactivities in the pyramidal neurons were distinctively decreased in the hippocampal CA1 region (CA), not CA2/3, 3 days after ischemia–reperfusion (I–R). Five days after I–R, GK and GKRP immunoreactivities were hardly detected in the CA1, not CA2/3, pyramidal neurons; however, at this point in time, GK and GKRP immunoreactivities were newly expressed in astrocytes, not microglia, in the ischemic CA1. In brief, GK and GKRP immunoreactivities are changed in pyramidal neurons and newly expressed in astrocytes in the ischemic CA1 after transient cerebral ischemia. These indicate that changes of GK and GKRP expression may be related to the ischemia-induced neuronal damage/death.     

Comparison of Neurogenesis in the Dentate Gyrus Between the Adult and Aged Gerbil Following Transient Global Cerebral Ischemia

In the present study, we compared differences in cell proliferation, neuroblast differentiation and neuronal maturation in the hippocampal dentate gyrus (DG) between the adult and aged gerbil induced by 5 min of transient global cerebral ischemia using Ki-67 and BrdU (markers for cell proliferation), doublecortin (DCX, a marker for neuroblast differentiation) and neuronal nuclei (NeuN, a marker for mature neuron). The number of Ki-67-immunoreactive (⁺) cells in the DG of both the groups peaked 7 days after ischemia/reperfusion (I/R). However, the number in the aged DG was 40.6 ± 1.8% of that in the adult DG. Thereafter, the number decreased with time. After ischemic damage, DCX immunoreactivity and its protein level in the adult and aged DG peaked at 10 and 15 days post-ischemia, respectively. However, DCX immunoreactivity and its protein levels in the aged DG were much lower than those in the adult. DCX immunoreactivity and its protein level in the aged DG were 11.1 ± 0.6% and 34.4 ± 2.1% of the adult DG, respectively. In addition, the number of Ki-67⁺ cells and DCX immunoreactivity in both groups were similar to those in the sham at 60 days postischemia. At 30 days post-ischemia, the number of BrdU⁺ cells and BrdU⁺/NeuN⁺ cells in the adult-group were much higher (281.2 ± 23.4% and 126.4 ± 7.4%, respectively) than the aged-group (35.6 ± 6.8% and 79.5 ± 6.1%, respectively). These results suggest that the ability of neurogenesis in the ischemic aged DG is much lower than that in the ischemic adult DG.     

Targeted Deletion of the ERK5 MAP Kinase Impairs Neuronal Differentiation,Migration, and Survival during Adult Neurogenesis in the Olfactory Bulb

Recent studies have led to the exciting idea that adult-born neurons in the olfactory bulb (OB) may be critical for complex forms of olfactory behavior in mice. However, signaling mechanisms regulating adult OB neurogenesis are not well defined. We recently reported that extracellular signal-regulated kinase (ERK) 5, a MAP kinase, is specifically expressed in neurogenic regions within the adult brain. This pattern of expression suggests a role for ERK5 in the regulation of adult OB neurogenesis. Indeed, we previously reported that conditional deletion of erk5 in adult neurogenic regions impairs several forms of olfactory behavior in mice. Thus, it is important to understand how ERK5 regulates adult neurogenesis in the OB. Here we present evidence that shRNA suppression of ERK5 in adult neural stem/progenitor cells isolated from the subventricular zone (SVZ) reduces neurogenesis in culture. By contrast, ectopic activation of endogenous ERK5 signaling via expression of constitutive active MEK5, an upstream activating kinase for ERK5, stimulates neurogenesis. Furthermore, inducible and conditional deletion of erk5 specifically in the neurogenic regions of the adult mouse brain interferes with cell cycle exit of neuroblasts, impairs chain migration along the rostral migratory stream and radial migration into the OB. It also inhibits neuronal differentiation and survival. These data suggest that ERK5 regulates multiple aspects of adult OB neurogenesis and provide new insights concerning signaling mechanisms governing adult neurogenesis in the SVZ-OB axis.     

Protective Effect of Oren-gedoku-to Against Induction of Neuronal Death by Transient Cerebral Ischemia in the C57BL/6 Mouse

We examined the neuroprotective effects of oren-gedoku-to (TJ15), a herbal medicine, after transient forebrain ischemia. Transient forebrain ischemia was induced by occlusion of both common carotid arteries for 15 min in C57BL/6 mice treated with TJ15. In the control ischemic group without TJ15 treatment, histologic examination of brain tissue collected seven days after reperfusion showed death of pyramidal cells in CA2-3 area of the hippocampus, unilaterally or bilaterally. In mice treated with oral TJ15 (845 mg/kg/day) for five weeks, the frequency of ischemic neuronal death was significantly lower. Immunohistochemistry for Cu/Zn-superoxide dismutase (Cu/Zn-SOD) showed strongly reactive astrocytes in the hippocampus of ischemic mice treated with TJ15. Damage to nerve cells by free radicals plays an important role in the induction of neuronal death by ischemia-reperfusion injury. Our results suggest that TJ15 protects against ischemic neuronal death by increasing the expression of Cu/Zn-SOD and suggest that oren-gedoku-to reduces the exposure of hippocampal neurons to oxidative stress.     

Neuronal Damage Using Fluoro-Jade B Histofluorescence and Gliosis in the Gerbil Septum Submitted to Various Durations of Cerebral Ischemia

The extent of neuronal damage/death in some brain regions is highly correlated to duration time of transient ischemia. In the present study, we carried out neuronal degeneration/death and glial changes in the septum 4 days after 5, 10, 15, and 20 min of transient cerebral ischemia using gerbils. To examine neuronal damage, Fluoro-Jade B (F-J B, a marker for neuronal degeneration) histofluorescence staining was used. F-J B positive (⁺) cells were detected in the septo-hippocampal nucleus (SHN) of the septum only in the 20 min ischemia-group; the mean number of F-J B⁺ neurons was 14.9 ± 2.5/400 μm² in a section. Gliosis of astrocytes and microglia was examined using anti-glial fibrillary acidic protein (GFAP) and anti-ionized calcium-binding adapter molecule 1 (Iba-1), respectively. In all the ischemia-groups, GFAP- and Iba-1-immunoreactive astrocytes and microglia, respectively, were increased in number, and apparently tended to be increased in their immunoreactivity. Especially, in the 20 min ischemia-group, the number and immunoreactivity of Iba-immunoreactive microglia was highest and strongest in the ischemic SHN 4 days after ischemia–reperfusion. In brief, our findings showed that neuronal damage/death in the SHN occurred and gliosis was apparently increased in the 20 min ischemia-group at 4 days after ischemia–reperfusion.     

Rapid Down-Regulation of GABAA Receptors in the Gerbil Hippocampus Following Transient Cerebral Ischemia

Abstract: During transient cerebral ischemia, there is a temporary and robust accumulation of extracellular GABA in the hippocampus. We examined whether the acute exposure of GABA_A/benzodiazepine receptors to high concentrations of GABA early after ischemia results in receptor down-regulation as observed in vitro. Gerbils were killed 30 and 60 min following a 5-min bilateral carotid occlusion, and their brains were prepared for receptor autoradiography. The hydrophilic GABA_A receptor antagonist [³H]SR-95531 and the hydrophobic benzodiazepine agonist [³H]flunitrazepam were used to distinguish between cell surface and internalized receptors. Ischemia significantly decreased [³H]SR-95531 binding in hippocampal areas CA1 and CA3 and in the dentate gyrus 30 min after ischemia. Scatchard analysis in area CA1 revealed that ischemia decreased the B _max as low as 44%. The affinity of the remaining sites was increased substantially (72% decrease in K _D). As expected, there were no changes in the binding of [³H]flunitrazepam to hippocampus in the early postischemic period because the benzodiazepine could bind to both internalized receptors and those on the cell surface. We hypothesize that prolonged exposure (∼30–45 min) of GABA_A receptors to high concentrations of synaptic GABA in vivo causes receptor down-regulation, perhaps via receptor internalization.     

美满霉素对沙鼠急性脑缺血后脑皮质小白蛋白表达的影响

目的：观察美满霉素对蒙古沙鼠短暂性脑缺血再灌后前额皮质中小白蛋白Parvalbumin（PV）表达的影响,为进一步研究其生理功能变化及治疗提供参考。方法：42只健康雄性蒙古沙鼠随机分为：正常组对照组（6只）、缺血再灌组（18只）,美满霉素治疗组（18只）。夹闭蒙古沙鼠双测颈总动脉10min诱导前脑缺血后,动物分别存活1天、3天或7天。用免疫组化方法检测前额皮质中PV的表达,用图像分析仪测定灰度值。结果：与正常组相比,各缺血再灌组与美满霉素治疗组中前额皮质中PV表达均先减少后回升。缺血再灌1天组,PV表达缓慢减少（P〉0.05）;3天组PV表达减少到低谷（P〈0.05）;7天组PV表达有明显的恢复,但仍低于正常组（P〈0.05）;然而美满霉素治疗各组中的PV表达均强于对应的缺血组（P〈0.05）。结论：美满霉素能抑制蒙古沙鼠脑缺血再灌注后脑皮质中小白蛋白的表达,发挥脑保护作用。     

Chronic Effects of Pyridoxine in the Gerbil Hippocampal CA1 Region after Transient Forebrain Ischemia

In a previous study, we reported that the administration of pyridoxine (vitamin B₆) to mice for 3 weeks significantly increased cell proliferation and neuroblast differentiation in the dentate gyrus without any neuronal damage. In the present study, we investigated the restorative potentials of pyridoxine on ischemic damage in the hippocampal CA1 region of Mongolian gerbils. Gerbils were subjected to 5 min of transient ischemia, and surgical operation success was assessed by ophthalmoscope during occlusion of common carotid arteries and spontaneous motor activity at 1 day after ischemia/reperfusion. Pyridoxine (350 mg/kg) or its vehicle (physiological saline) was intraperineally administered to ischemic gerbils twice a day starting 4 days after ischemia/reperfusion for 30 or 60 days. The repeated administration of pyridoxine for 30 and 60 days significantly increased doublecortin-immunoreactive neuroblasts in the dentate gyrus and increased NeuN-immunoreactive mature neurons and βIII-tubulin-immunoreactive dendrites in the hippocampal CA1 region. Furthermore, brain-derived neurotrophic factor (BDNF) protein levels were significantly increased in pyridoxine-treated groups compared to those in the vehicle-treated groups. These results suggest that chronic administration of pyridoxine enhances neuroblast differentiation in the dentate gyrus and induces new mature neurons in the hippocampal CA1 region by up-regulating BDNF expression in hippocampal homogenates.     

Cryptic Expression of the 70-kDa Heat Shock Protein,hsp72, in Gerbil Hippocampus after Transient Ischemia

The 70 kDa heat shock protein, hsp72, is known to be induced following transient global ischemia in brain, as detected by immunocytochemistry and in situ hybridization techniques. However, while hsp72 mRNA is expressed rapidly following postischemic recirculation, immunocytochemistry fails to detect hsp72 protein for many hours after such insults, even in cell populations that readily express Fos and other proteins encoded by ischemia-induced mRNAs. In the present study, hsp72 expression in gerbil hippocampus was compared by immunocytochemistry and immunoblot methods at several intervals following 10 min ischemia. As established in previous studies, hsp72 immunoreactivity remained undetectable in postischemic neurons at 6 h following such insults. In contrast, immunoblots of dissected gerbil hippocampus demonstrated nearly maximal accumulation of hsp72 at this time point. These results indicate that the protein is present, but cryptic to detection in perfusion-fixed sections, during early recirculation. The constitutively expressed heat shock cognate protein, hsc70, did not show significant changes in level or distribution by either method, except for a decrease in CA1 staining at 48 h. These results confirm that hsp72 rapidly accumulates to high levels in postischemic hippocampus, and suggest that further studies of its subcellular localization during this interval may offer insight into its functional role as a component of the stress response in neurons after such insults.     

Synthesis of a Stress Protein Following Transient Ischemia in the Gerbil

In vitro translation products of gerbil brain preparations, obtained from animals killed during recirculation following transient ischemia, showed increased synthesis of a 70-kilodalton stress protein, identified by two-dimensional gel electrophoresis. Stimulation of stress protein synthesis was evident as early as 2 h after recirculation, at which time overall translation activity remained low. Expression of the 70-kilodalton protein reached a maximum at 8 h recirculation, when incorporation into other translation products had returned to essentially control levels. Increased incorporation into the stress protein was still detectable after 24 h recirculation. Although the functional consequences of increased expression of this stress protein remain unknown, these results suggest that the gerbil ischemia model may provide a useful experimental system in which to study the involvement of this phenomenon in processes related to postischemic cell damage and recovery.