Hyperbaric oxygen treatment decreases post-ischemic neurotrophin-3 mRNA down-regulation in the rat hippocampus.

The therapeutic effect of hyperbaric oxygen (HBO) on ischemic injury was investigated using in situ hybridization to detect the mRNA expression of neurotrophin-3 (NT-3), which is thought to play a crucial role in protecting against neuronal death induced by brain ischemia. The rats under investigation were subjected to 10 min transient forebrain ischemia, and subsequently exposed to HBO (100% oxygen, 2.5 atm absolute) for 2 h. Levels of NT-3 mRNA in the CA1, CA2 and CA3 regions, and the dentate gyrus of the hippocampus were measured after various reperfusion periods. Neuronal death in the hippocampal CA1 region was also measured by Nissl staining, seven days post ischemia. The results demonstrated that HBO treatment significantly reduced the ischemia-induced down-regulation of the NT-3 mRNA level at 4 h post ischemia, and significantly increased cell survival 7 days after reperfusion. The findings suggest that an HBO treatment maintaining the NT-3 mRNA level in the hippocampus can be beneficial to the ischemic brain within a certain time frame.     

Spatial and temporal distribution of N-type Ca(2+) channels in gerbil global cerebral ischemia

In the present study, we have investigated the spatial and temporal distribution of voltage-gated calcium channels in the gerbil model of global cerebral ischemia using immunohistochemistry. Distinct localizations of P-type (alpha(1A)), N-type (alpha(1B)), and L-type (alpha(1C) and alpha(1D)) Ca(2+) channels were observed in the hippocampus at days 1-5 after ischemic injury. However, increased expression of N-type Ca(2+) channels was detectable in brain regions vulnerable to ischemia only at days 2 and 3 after ischemic injury. The pyramidal cell bodies of CA1-3 areas and the granule cell bodies of the dentate gyrus were intensely stained at days 2 and 3 following ischemic injury. Transient changes in N-type Ca(2+) channel expression were also observed in the affected cerebral cortex and striatum at days 2 and 3 after ischemic injury. Although the present study has not addressed the multiple mechanisms contributing to the intracellular free Ca(2+) concentration ([Ca(2+)](i)) increase in the ischemic brain, the first demonstration of the transient increase in N-type Ca(2+) channels may prove useful for future investigations.     

Expression of glial cell line-derived neurotrophic factor induced by transient forebrain ischemia in rats.

This study examined the expression of glial cell line-derived neurotrophic factor (GDNF) mRNA and the cellular localization of GDNF production in rats subjected to transient forebrain ischemia induced by four-vessel occlusion. Transient forebrain ischemia induced GDNF mRNA expression in the hippocampus from 3 h to 3 days after the ischemic episode, with peak expression at 6 h. The GDNF mRNA increase in the cerebral cortex was similar to that in the hippocampus, whereas no increase in GDNF mRNA was observed in the striatum and brainstem. Western blot analysis showed that GDNF in the hippocampal CA1 region was increased slightly from 3 to 24 h after the ischemia, and then subsequently declined to below the baseline level. In the hippocampus, GDNF was evenly produced in pyramidal neurons of both sham-operated rats and normal rats, as determined by immunohistochemistry. Interestingly, we found that ischemia-induced reactive astrocytes, as well as surviving neurons, produced GDNF in 3-7 days after the ischemia. On the other hand, in other regions, such as the cerebral cortex, striatum, and brainstem, there was no change in GDNF-positive cells secondary to ischemia. These findings suggest that expression of GDNF mRNA is regulated in part via ischemia-induced neuronal degeneration. They also suggest that ischemia-induced reactive astrocytes may produce GDNF to protect against neuronal death. Therefore, GDNF may play an important role in ischemia-induced neuronal death in the brain.     

Dexamethasone enhances upregulation of nerve growth factor mRNA expression in ischemic rat brain.

Nerve growth factor (NGF) is well-established as a trophic factor that plays a crucial role in neuroregeneration and plasticity after brain insults. Dexamethasone (DEX), a powerful glucocorticoid steroid, has long been used in the clinical management of neurological disorders. We examined the relationship between NGF and DEX after an ischemic insult to the brain. In situ hybridization was used to measure NGF mRNA expression in the rat hippocampus after 20 min of transient forebrain ischemia. Immunostaining for NGF protein was performed using the avidin-biotin peroxidase method. Immunohistochemistry for glial fibrillary acidic protein (GFAP) was also used to study the astrocyte reaction in the hippocampal CA1 area. Ischemic brain from rats not treated with DEX had a 2 and 3 fold increase in NGF mRNA compared to sham-operated rats at 4 and 6 h after ischemia, respectively. The NGF mRNA expression returned to basal levels 12 h to 7 days post-ischemia. Treatment with DEX potentiated the ischemia-induced increase of NGF mRNA to 4 times that of sham-operated rats at 6 h following reperfusion and NGF protein expression was similarly elevated. Additionally, the number of GFAP positive astrocytes in the CA1 region in the ischemic rats was markedly increased. These data suggest that DEX may play a role in modulating NGF mRNA expression in the hippocampal neuronal response to brain ischemia.     

钾通道Kv4.2、Kv1.4在硫化氢后处理对大鼠短暂全脑缺血神经保护中的作用研究

目的研究硫氢化钠(sodium hydrosulfide,Na HS)后处理对短暂全脑缺血大鼠海马中钾通道Kv4.2和Kv1.4 mRNA表达变化的影响及其脑保护作用,从而探讨Na HS对大鼠短暂全脑缺血神经保护作用的机制。方法用4VO方法建立大鼠短暂性全脑缺血(transient global cerebral ischemia,t GCI)模型,大鼠被随机分配到3组,分别为:假手术组(sham)、t GCI组、Na HS后处理组。Na HS后处理组为t GCI之后1 d,给予大鼠腹腔注射Na HS 24μmmol/kg或者180μmmol/kg。通过尼氏染色与Neu N免疫染色确定海马神经元的死亡,通过RT-PCR方法检测海马组织Kv4.2和Kv1.4mRNA水平的表达变化。结果 (1)与t GCI组比较,在t GCI之后1 d给予24μmol/kg Na HS后处理使海马CA1区存活细胞数目显著增加,而高剂量的Na HS(180μmol/kg)后处理对t GCI大鼠海马CA1区则无明显的保护作用。(2)在Re 26 h和Re 48 h,海马组织中Kv4.2、Kv1.4的mRNA表达水平均明显低于假手术组(P<0.05)。在Re 26 h+Na HS组,kv4.2(1.24±0.08)和kv1.4(1.11±0.07)的mRNA表达水平均分别高于Re 26 h组的kv4.2(0.75±0.04)和kv1.4(0.79±0.06),差异均有显著性(P<0.05)。结论外源性Na HS可能通过上调大鼠t GCI后海马区Kv4.2和Kv1.4 mRNA的表达,从而导致膜电位超极化,降低神经元兴奋性和氧耗,继而保护神经元免受脑缺血损伤。     

脑缺血再灌流大鼠脑胶质源性神经营养因子基因表达的变化

探讨脑缺血再灌流不同时程及不同程度缺血对海马及皮层胶质源性神经营养因子（ｇｌｉａｌｃｅｌｌｌｉｎｅ ｄｅｒｉｖｅｄ ｎｅｕｒｏｔｒｏｐｈｉｃ ｆａｃｔｏｒ, ＧＤＮＦ）基因表达的影响,以及Ｎ甲基Ｄ天冬氨酸（Ｎｍ ｅｔｈｙｌＤｓａｐａｒｔａｔｅ, ＮＭＤＡ）受体拮抗剂,钙离子通道阻断剂是否能调节缺血病态下ＧＤＮＦｍ ＲＮＡ的表达。参照Ｓｍ ｉｔｈ 等方法建立大鼠前脑缺血再灌流动物模型。用ＤＩＧＯｌｉｇｏｎｕｃｌｅｏｔｉｄｅ ３′ｅｎｄ ｌａｂｅｌｉｎｇ Ｋｉｔ,标记５１ ｍ ｅｒ的ＧＤＮＦ寡核苷酸探针在含有海马结构的冰冻组织切片上进行原位杂交检测ＧＤＮＦｍ ＲＮＡ的表达。１０ ｍ ｉｎ 缺血再灌流２ ｈ,齿状回ＧＤＮＦｍ ＲＮＡ表达上调。再灌流６ ｈ,ＣＡ１,ＣＡ３ 和皮层ＰＡＲ区ＧＤＮＦｍ ＲＮＡ表达亦见增多,２４ ｈ 达高峰。Ｋｅｔａｍ ｉｎｅ 可使ＧＤＮＦ的基因表达在海马结构及皮层ＰＡＲ区明显低于相应的缺血再灌流组,统计学差异显著（Ｐ＜ ００５）。脑缺血再灌流时ＧＤＮＦ基因表达增加,对缺血神经元可能起保护作用。Ｋｅｔａｍ ｉｎｅ可阻断缺血后ＧＤＮＦｍ ＲＮＡ 的表达增加,提示ＮＭＤＡ谷氨酸受体很可能参与介导了缺     

Differential localization of rat Eag1 and Eag2 K+ channels in hippocampal neurons

Two isoforms of rat ether-a-go-go (Eag) K channels, rEag1 and rEag2, are widely expressed in many regions of the brain. The neurophysiological roles of these channels, however, are unclear. We addressed this issue by studying their subcellular localizations in hippocampal neurons. Immunofluorescence studies using markers for different compartments of neurons demonstrated a differential expression pattern of rEag1 and rEag2 K channels in the somatodendritic region. Furthermore, rEag1 K channels were in close proximity to synaptophysin and densin-180, but not GAD65. Our data suggest that both rEag1 and rEag2 K channels may play a pivotal role in the regulation of the excitability of dendrites and somas, and that rEag1 K channels may modulate the postsynaptic signaling of glutamatergic synapses.     

Alteration of serum/glucocorticoid regulated kinase-1 (sgk-1) gene expression in rat hippocampus after transient global ischemia

Expression of the serum/glucocorticoid regulated kinase-1 (sgk-1) gene has been reported to be induced by various stress stimuli such as hyper- or hypo-osmotic stress, UV irradiation, and heat shock stress; however, its association with global ischemia in the brain has not been studied. Using high-density oligonucleotide array analysis, we found that the sgk-1 gene was one of the genes showing alteration of expression in the rat hippocampus during 1-4 h of reperfusion after 10 min of transient global cerebral ischemia. Using TaqMan RT-PCR analysis, we confirmed an increased level of sgk-1 gene expression with statistical significance in the rat hippocampus at 2 h of reperfusion after 10 min of transient global cerebral ischemia. Using in situ hybridization (ISH) analysis, the increased level of sgk-1 gene expression was found to localize in pyramidal cells of CA2 and CA3 regions of the hippocampus after 2 h of reperfusion. These results provide an insight into the alterations of sgk-1 gene expression in the rat hippocampus after transient global cerebral ischemia.     

Resveratrol prevents CA1 neurons against ischemic injury by parallel modulation of both GSK‐3β and CREB through PI3‐K/Akt pathways

Accumulating evidence indicates that resveratrol potently protects against cerebral ischemia damage due to its oxygen free radicals scavenging and antioxidant properties. However, cellular mechanisms that may underlie the neuroprotective effects of resveratrol in brain ischemia are not fully understood yet. This study aimed to investigate the potential association between the neuroprotective effect of resveratrol and the apoptosis/survival signaling pathways, in particular the glycogen synthase kinase 3 (GSK‐3β) and cAMP response element‐binding protein (CREB) through phosphatidylinositol 3‐kinase (PI3‐K)‐dependent pathway. An experimental model of global cerebral ischemia was induced in rats by the four‐vessel occlusion method for 10 min and followed by different periods of reperfusion. Nissl staining indicated extensive neuronal death at 7 days after ischemia/reperfusion. Administration of resveratrol by i.p. injections (30 mg/kg) for 7 days before ischemia significantly attenuated neuronal death. Both GSK‐3β and CREB appear to play a critical role in resveratrol neuroprotection through the PI3‐K/Akt pathway, as resveratrol pretreatment increased the phosphorylation of Akt, GSK‐3β and CREB in 1 h in the CA1 hippocampus after ischemia/reperfusion. Furthermore, administration of LY294002, an inhibitor of PI3‐K, compromised the neuroprotective effect of resveratrol and decreased the level of p‐Akt, p‐GSK‐3β and p‐CREB after ischemic injury. Taken together, the results suggest that resveratrol protects against delayed neuronal death in the hippocampal CA1 by maintaining the pro‐survival states of Akt, GSK‐3β and CREB pathways. These data suggest that the neuroprotective effect of resveratrol may be mediated through activation of the PI3‐K/Akt signaling pathway, subsequently downregulating expression of GSK‐3β and CREB, thereby leading to prevention of neuronal death after brain ischemia in rats.     

Erratum to: Differential alterations in the distribution of voltage-gated calcium channels in aged rat cerebellum: [Brain Research 903 (2001) 247–252]

In the present study, we have investigated the spatial and temporal distribution of voltage-gated calcium channels in the gerbil model of global cerebral ischemia using immunohistochemistry. Distinct localizations of P-type (α_1A), N-type (α_1B), and L-type (α_1C and α_1D) Ca²⁺ channels were observed in the hippocampus at days 1–5 after ischemic injury. However, increased expression of N-type Ca²⁺ channels was detectable in brain regions vulnerable to ischemia only at days 2 and 3 after ischemic injury. The pyramidal cell bodies of CA1-3 areas and the granule cell bodies of the dentate gyrus were intensely stained at days 2 and 3 following ischemic injury. Transient changes in N-type Ca²⁺ channel expression were also observed in the affected cerebral cortex and striatum at days 2 and 3 after ischemic injury. Although the present study has not addressed the multiple mechanisms contributing to the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) increase in the ischemic brain, the first demonstration of the transient increase in N-type Ca²⁺ channels may prove useful for future investigations.     

Differential expression of HSC73 and HSP72 mRNA and proteins between young and adult gerbils after transient cerebral ischemia: relation to neuronal vulnerability.

This study presents a quantitative comparison of the time courses and regional distribution of both constitutive HSC73 and inducible HSP72 mRNA expression and their respective encoded proteins between young (3-week-old) and adult (3-month-old) gerbil hippocampus after transient global ischemia. The constitutive expression of HSC73 mRNA and protein in the hippocampus of the young sham-operated gerbils was significantly higher than in the adults. The HSC73 mRNA expression after ischemia in the CA1 layer of young gerbils was greater than in adult gerbils. HSC73 immunoreactivity was not significantly changed after ischemia-reperfusion in adult hippocampus, whereas it decreased in young gerbils. Ischemia-reperfusion led to induction of HSP72 mRNA expression throughout the hippocampus of both young and adult gerbils. HSP72 mRNA induction was more intense and sustained in the CA1 subfield of young gerbils; this was associated with a marked induction of HSP72 proteins and neuronal survival. The transient expression of HSP72 mRNA in the CA1 layer of adult gerbils was not associated with a subsequent synthesis of HSP72 protein but was linked to neuronal loss. Expression of HSP72 mRNA was shifted to an earlier period of reflow in CA3 and dentate gyrus (DG) subfields of young animals. These findings suggest that the induction of both HSP72 mRNA and proteins in the CA1 pyramidal neurons of young gerbils, as well as the higher constitutive expression of HSC73, may partially contribute to higher neuronal resistance of young animals to transient cerebral ischemia.     

Delayed neuronal death and damage of GDNF family receptors in CA1 following focal cerebral ischemia

Delayed neuronal death (DND) of pyramidal neurons in the CA1 and CA3 regions of the hippocampus has been extensively studied following global brain ischemia, whereas only little is known about DND in this highly vulnerable brain region after focal brain ischemia. In the present study, the distribution and time course of hippocampal neuronal apoptosis were studied following transient middle cerebral artery occlusion (MCAO) in rats 1, 3, 7, 14, and 30 days after the insult. In 60% of the animals, more than 90% of CA1 pyramidal neurons showed strong nick-end labeling (TUNEL) staining at day 3 with fragmentation and marginalization of the nuclei in approximately 40% of these cells. The number of TUNEL-positive cells decreased within the next days, but 30 days after MCAO, some apoptotic neurons were still present. Analysis of the expression of the glial cell line-derived neurotrophic factor (GDNF) and its receptors GFRalpha1, GFRalpha2, and GFRalpha3 using triple immunofluorescence and confocal laser scanning microscopy revealed that in all animals showing marked hippocampal DND, the neuronal staining for GFRalpha1, GFRalpha3, and GDNF decreased prior to the onset of TUNEL staining in CA1. After 7 days, some apoptotic neurons still expressed GFRalpha3, whereas only few showed GFRalpha1 immunoreactivity, indicating that GFRalpha1 may be beneficial for the survival of hippocampal neurons. The data suggest that reduced expression of GDNF and impairment of GFRalpha1/3 may contribute to hippocampal DND after focal brain ischemia.     

Differential distribution of three Ca(2+)-activated K(+) channel subunits, SK1, SK2, and SK3, in the adult rat central nervous system

Ca(2+)-activated, voltage-independent K(+) channels are present in most neurons and mediate the afterhyperpolarizations (AHPs) following action potentials. They present distinct physiological and pharmacological properties and play an important role in controlling neuronal firing frequency and spike frequency adaptation. We used in situ hybridization to characterize the distribution patterns of the three cloned SK channel subunits (SK1-3), the prime candidates likely to underlie Ca(2+)-dependent AHPs in the central nervous system. We found high levels of expression in regions presenting prominent AHP currents, such as, for example, neocortex and CA1-3 layers of the hippocampus (SK1 and SK2), reticularis thalami (SK1 and SK2), supraoptic nucleus (SK3), and inferior olivary nucleus (SK2 and SK3). Our results reveal the functional role of SK channels with defined subunit compositions in some neurons and open the way to the identification of the molecular determinants of AHP currents in many brain regions.     

Mapping second messenger systems in the rat hippocampus after transient forebrain ischemia: in vitro [3H]forskolin and [3H]inositol 1,4,5-trisphosphate binding

Autoradiographic imaging demonstrated predominant and reciprocal localization of forskolin and inositol 1,4,5-trisphosphate (IP3) binding sites in synaptic areas in the hippocampus. We produced selective damage to the CA1 pyramidal cells in the rat hippocampus by means of transient forebrain ischemia and analyzed the alteration of the intracellular signal transduction using quantitative autoradiography of these second messenger systems. The dendritic fields (stratum oriens, radiatum and lacunosummoleculare) in the CA1 showed 20% decrease in [3H]IP3 binding activity 3 h after ischemia, when no morphological abnormalities were obvious. Thereafter, grain density in these layers decreased and half of the binding sites were lost 2 days after ischemia. By contrast, the stratum pyramidale of the CA1 showed no significant change until 2 days after recirculation. Seven days after ischemia, when CA1 pyramidal cells were depleted, all layers in the CA1 subfield lost 85% of [3H]IP3 binding sites. In the CA3 subfield, only a small and transient alteration in the [3H]IP3 binding was noticed during recirculation. Postischemic reduction of [3H]forskolin binding sites was obvious in the CA1 only 1 h after ischemia followed by loss of 50% of binding activity 7 days after recirculation. These results suggest that forskolin and IP3 binding sites are predominantly distributed on the pyramidal cells in the CA1 subfield and that marked alteration of intracellular signal transduction precedes the delayed CA1 pyramidal cell death.     

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.