Dexamethasone enhances NT-3 expression in rat hippocampus after traumatic brain injury

The cellular events in traumatic brain injury (TBI) are complicated, and the factors mediating neurotrophins to protect and repair the injured brain cells are only beginning to be identified. This study examined the effect of dexamethasone (DEX) on neurotrophin-3 (NT-3) expression following TBI. Levels of NT-3 mRNA and protein in rat hippocampus were measured using in situ hybridization and immunohistochemistry, respectively. After TBI, the NT-3 mRNA expression was down-regulated during the first 24 h. DEX reversed the post-traumatic reduction of NT-3 mRNA expression at 2, 4, 6, and 12 h in the hippocampus, and also decreased the cell death in hippocampal hilum and supraventricular cerebral cortex after 7 days. The NT-3 protein levels generally corresponded to the mRNA levels in the hippocampal region. DEX enhanced the NT-3 expression after TBI, indicating that post-traumatic neuroprotection in the hippocampus is at least partially mediated by NT-3 and thus can be modulated by DEX treatment.     

Dexamethasone enhances NT-3 expression in rat hippocampus after traumatic brain injury

The cellular events in traumatic brain injury (TBI) are complicated, and the factors mediating neurotrophins to protect and repair the injured brain cells are only beginning to be identified. This study examined the effect of dexamethasone (DEX) on neurotrophin-3 (NT-3) expression following TBI. Levels of NT-3 mRNA and protein in rat hippocampus were measured using in situ hybridization and immunohistochemistry, respectively. After TBI, the NT-3 mRNA expression was down-regulated during the first 24 h. DEX reversed the post-traumatic reduction of NT-3 mRNA expression at 2, 4, 6, and 12 h in the hippocampus, and also decreased the cell death in hippocampal hilum and supraventricular cerebral cortex after 7 days. The NT-3 protein levels generally corresponded to the mRNA levels in the hippocampal region. DEX enhanced the NT-3 expression after TBI, indicating that post-traumatic neuroprotection in the hippocampus is at least partially mediated by NT-3 and thus can be modulated by DEX treatment.     

Effect of hyperbaric oxygen on striatal metabolites: a microdialysis study in awake freely moving rats after MCA occlusion

We have shown that hyperbaric oxygen (HBO) reduced cerebral infarction in rat middle cerebral artery occlusion model (MCAO). The present study was undertaken to evaluate the effect of HBO on ischemic striatal metabolites at different times after MCAO and reperfusion. A rat MCAO model was produced via the intraluminal filament method. After 2 h of occlusion the suture was removed and reperfusion was allowed. The rats were sacrificed at 24 h after reperfusion. HBO treatment was administered by putting rats in the HBO chamber at 3 atmospheres absolute (ATA) HBO for 1 h. Glucose, lactate, pyruvate, and glutamate in striatal extracellular fluid were collected and measured by a microdialysis system at 7, 10, and 24 h after reperfusion. Glucose, pyruvate and glutamate concentrations were increased after reperfusion. HBO treatment decreased glucose, pyruvate, and glutamate almost to the control level (preocclusion level). The lactate concentration remained unchanged after ischemic/reperfusion and after HBO treatment. This study suggested that altered brain energy metabolites and excitatory amino acids occurred during cerebral ischemia and and HBO regulated these striatal metabolites, which might contribute to the protective effect of HBO in cerebral ischemia.     

Inhibition of apoptosis by hyperbaric oxygen in a rat focal cerebral ischemic model.

The hypothesis was tested that hyperbaric oxygen therapy (HBO) reduced brain infarction by preventing apoptotic death in ischemic cortex in a rat model of focal cerebral ischemia. Male Sprague-Dawley rats were subjected to middle cerebral artery occlusion/reperfusion (MCAO/R) and subsequently were exposed to HBO (2.5 atmospheres absolute) for 2 h, at 6 h after reperfusion. Rats were killed and brain samples were collected at 24, 48, 72 h, and 7 days after reperfusion. Neurologic deficits, infarction area, and apoptotic changes were evaluated by clinical scores, 2,3,7-triphenyltetrazolium chloride staining, caspase-3 expression, DNA fragmentation assay, and terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling (TUNEL)-hematoxylin and eosin (H&E) costaining. In MCAO/R without HBO treatment animals, DNA fragmentation was observed in injured cortex at 24, 48, and 72 h but not in samples at 7 days after reperfusion. Double labeling of brain slides with NeuN and caspase-3 demonstrated neurons in the injured cortex labeled with caspase-3. TUNEL+H&E costaining revealed morphologic apoptotic changes at 24, 48, and 72 h after reperfusion. Hyperbaric oxygen therapy abolished DNA fragmentation and reduced the number of TUNEL-positive cells. Hyperbaric oxygen therapy reduced infarct area and improved neurologic scores at 7 days after reperfusion. One of the molecular mechanisms of HBO-induced brain protection is to prevent apoptosis, and this effect of HBO might preserve more brain tissues and promote neurologic functional recovery.     

针刺对缺血再灌注大鼠海马脑源性神经营养因子mRNA的影响

目的 探讨针刺对缺血再灌注大鼠海马内脑源性神经营养因子（BONF）基因表达的影响，推测针刺改善缺血再灌注的可能机制。方法 采用4-血管阴断法制备大鼠全脑缺血再灌注模型，电针刺激百会、肾俞、足三里穴后，利用RT-PCR检测BDNF mRNA。结果 正常组大鼠海马BDNF mRNA表达极低，缺血再灌注组大鼠海马BDNF mRNA表达明显增高，治疗15d的针刺1、2组大鼠海马BDNF mRNA表达较缺血再灌注组更高，及早治疗且治疗时间为20d的针刺3组大鼠海马BDNF mRNA表达较降低。结论 缺血再灌注大鼠海马BDNF水平增高有利于损伤的神经元存活、恢复；针刺促进脑内细胞分泌内源性BDNF可能是针刺有效治疗缺血再灌注的机制之一。     

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     

Correlation between delayed neuronal cell death and selective decrease in phosphatidylinositol 4-kinase expression in the CA1 subfield of the hippocampus after transient forebrain ischemia.

Transient forebrain ischemia induces a delayed neuronal death in the CA1 area of the hippocampus. However, the mechanism leading to this phenomenon has yet to be established. The authors used an mRNA differential-display method to isolate genes for which mRNA levels change only in the hippocampus during ischemia/reperfusion. They succeeded in identifying the product of one down-regulated gene as phosphatidylinositol 4-kinase (PI 4-K). Compared with control levels, PI 4-K mRNA expression in the hippocampus, but not the cerebral cortex, was significantly decreased by 30% and about 80% 1 and 7 days after ischemia/reperfusion, respectively. Interestingly, PI 4-K and PI bisphosphate levels were selectively decreased in the CA1 region, but not other regions, whereas TUNEL-positive cells could be detected 3 days after ischemia. Consistent with these results, PI 4-K expression was suppressed by hypoxia in SK-N-MC neuroblastoma cells before loss of cell viability. Overexpression of wild-type PI 4-K, but not the kinase-negative mutant of PI 4-K (K1789A), recovered the loss of viability induced by hypoxia. These findings strongly suggest that a prior decrease in PI 4-K and PI bisphosphate levels caused by brain ischemia/hypoxia is partly involved in delayed neuronal cell death.     

Transient upregulation of the AT2 receptor mRNA level after global ischemia in the rat brain

The present study examined changes in angiotensin receptors (AT1 and AT2) and angiotensinogen mRNA level after global ischemia in the rat brain. The AT2 mRNA level increased by three-fold in both the cortex and hippocampus, which are known to be sensitive to ischemic injury, 3 h after ischemia. The increase thus appeared only during the early reperfusion period. In the striatum, amygdala and cerebellum, the level increased moderately 3 h and/or 24 h after ischemia; there was no change in the hypothalamus. On the other hand, the AT1A and AT1B receptor mRNA levels were not altered in the cortex or hippocampus during the early reperfusion period, even 3 h and 24 h after ischemia. There was no significant alteration in angiotensinogen mRNA level 3 h or 24 h after ischemia. These results suggest that the transient upregulation of AT2 receptor mRNA occurs in the cortex and hippocampus after injury and these changes may be in some way related to the molecular events which lead to delayed neuronal cell death.     

脑源性神经营养因子在大鼠脑缺血再灌注损伤过程中基因表达的变化

目的 :观察脑缺血再灌注损伤后脑皮层、梗塞区和海马神经元脑源性神经营养因子 (BDNF)水平的变化 ,及与脑病理变化的关联性 ;探讨 BDNF在脑缺血再灌注损伤中的可能作用机理。方法 :线栓法复制大鼠大脑中动脉脑缺血再灌注模型 ,原位核酸分子杂交检测脑不同区域 BDNFm RNA,图象分析间接定量其水平。结果 :1.脑缺血及缺血再灌注均能诱导双侧脑皮层、海马和梗塞区及其对侧相应区神经元 BDNFm RNA水平增高。2 .梗塞区因缺血损伤过重 ,神经元 BDNFm RNA水平增高的幅度小。 3.再灌注后神经元 BDNFm RNA的水平继续升高 ;其变化规律在不同脑区大致相似。 4.神经元 BDNFm RNA基础水平与神经元抗损伤力呈正相关。结论 :脑缺血及缺血再灌注损伤均导致双侧大脑 BD-NFm RNA表达的变化 ,BDNFm RNA水平的提高能增强神经元的抗损伤能力。     

细胞外信号调节激酶在NGF/VEGF介导的神经保护作用中的动态变化及其调控机制

目的探讨细胞外信号调节激酶1(ERK1)在局灶性脑缺血/再灌注不同时间、不同脑区的动态时空变化,以及其在NGF/VEGF介导的神经保护作用中的调控表达机制。方法采用兔大脑中动脉阻断(MCAO)局灶性脑缺血再灌注模型,所有动物随机分为假手术组(n=6)、缺血/再灌注组(n=60)、因子干预组(n=40)。应用免疫组化检测ERK1在脑缺血/再灌注损伤不同脑区的动态表达,同时,应用免疫组化、流式细胞术和电镜检测caspase-3表达、凋亡和超微结构的变化。结果免疫组化分析显示,再灌注损伤1hERK1首先在海马CA3和齿状回(DG)表达增加,6h后其它脑区也相继增加,随再灌注时间延长而加剧,1~3d达高峰。再灌注1hcaspase-3活性表达在各脑区迅速增加,3d达高峰。应用神经保护剂(NGF/VEGF)后各脑区ERK1表达呈明显抑制,caspase-3表达同时被抑制。结论ERK信号通路可能通过调节死亡受体途径介导神经保护作用,抑制ERK信号途径可能是减轻脑缺血损伤过程中神经细胞死亡的有效方法。     

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.     

Eag1, Eag2, and SK3 potassium channel expression in the rat hippocampus after global transient brain ischemia

Transient global brain ischemia causes delayed neuronal death in the hippocampus that has been associated with impairments in hippocampus-dependent brain function, such as mood, learning, and memory. We investigated the expression of voltage-dependent Kcnh1 and Kcnh5, ether à go-go-related Eag1 and Eag2 (K(V) 10.1 and K(V) 10.2), and small-conductance calcium-activated SK3 (K(Ca) 2.3, Kcnn3) K(+) channels in the hippocampus in rats after transient global brain ischemia. We tested whether the expression of these channels is associated with behavioral changes by evaluating the animals in the elevated plus maze and step-down inhibitory avoidance task. Seven or tweny-eight days after transient global brain ischemia, one group of rats had the hippocampus bilaterally dissected, and mRNA levels were determined. Seven days after transient global brain ischemia, the rats exhibited a decrease in anxiety-like behavior and memory impairments. An increase in anxiety levels was detected 28 days after ischemia. Eag2 mRNA downregulation was observed in the hippocampus 7 days after transient global brain ischemia, whereas Eag1 and SK3 mRNA expression remained unaltered. This is the first experimental evidence that transient global brain ischemia temporarily alters Eag2. The number of intact-appearing pyramidal neurons was substantially decreased in CA1 and statistically measurable in CA2, CA3, and CA4 hippocampal subfields compared with sham control animals 7 or 28 days after ischemia. mRNA expression in the rat hippocampus. The present results provide further information for the characterization of the physiological role of Eag2 channels in the central nervous system.     

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.     

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.     

Effects of the hyperbaric oxygen treatment on the Na+,K+ -ATPase and superoxide dismutase activities in the optic nerves of global cerebral ischemia-exposed rats

The effects of hyperbaric oxygen (HBO) treatment on the Na+,K+ -ATPase and superoxide dismutase (SOD) activities were examined in the optic nerves of the rats exposed to global cerebral ischemia. Animals were exposed to global cerebral ischemia of 20-min duration and were either sacrificed or exposed to the first HBO treatment immediately, 0.5, 1, 2, 6, 24, 48, 72 or 168 h after ischemic procedure (for Na+,K+ -ATPase activities measurement) or 2, 24, 48 or 168 h after ischemia (for SOD activities measurement). HBO procedure was repeated for 7 consecutive days. It was found that global cerebral ischemia induced a statistically significant decrease in the Na+,K+ -ATPase activity of the optic nerves, starting from 0.5 to 168 h of reperfusion. Maximal enzymatic inhibition was registered 24 h after the ischemic damage. The decline in the Na+,K+ -ATPase activity was prevented in the animals exposed to HBO treatment within the first 6 h of reperfusion. The results of the presented experiments demonstrated also a statistically significant increase in the SOD activity after 24, 48 and 168 h of reperfusion in the optic nerves of non-HBO-treated ischemic animals as well as in the ischemic animals treated with HBO. Our results indicate that global cerebral ischemia induced a significant alterations in the Na+,K+ -ATPase and SOD activities in the optic nerves during different periods of reperfusion. HBO treatment, started within the first 6 h of reperfusion, prevented ischemia-induced changes in the Na+,K+ -ATPase activity, while the level of the SOD activity in the ischemic animals was not changed after HBO administration.     

Somatostatin type 2 receptor expression in the rat hippocampus following cerebral ischemia.

We examined how transient cerebral ischemia affects the mRNA expression, and the immunoreactive distribution, of the somatostatin type 2 (sst2) receptor in the adult rat hippocampus. Following reperfusion, sst2 mRNA levels increased significantly in the CA1 region by 3 h, and were also increased in the CA3 and CA4/hilus subfields at 6 and 12 h. At 24 h, however, sst2 receptor mRNA levels returned to baseline throughout the hippocampus. At the protein level, we found the regional immunoreactivity of the sst2a receptor was maintained, or slightly elevated, throughout the hippocampus at 6 h, but not different from control at 24 h. These results suggest that sst2 receptors maintain their normal distribution and prevalence in the post-ischemic hippocampus before the deterioration of the vulnerable CA1 neurons. Thus, they represent attractive targets for neuroprotective interventions.     

高压氧预处理减少局灶性脑缺血后泛素化蛋白聚集

目的探讨泛素-蛋白酶体系统在重复高压氧（HBO）预处理导致的脑缺血耐受中所起的作用。方法采用大脑中动脉阻闭（MCAO）制备大鼠局灶性脑缺血模型,将33只SD大鼠随机分为假手术组（Sham）组、MCAO组和HBO组,分别观察各组大鼠脑梗死灶的大小和神经功能学评分,并用免疫组织化学染色检测再灌注2h、24h时相点大鼠脑组织CA1区异常泛素化蛋白聚集的变化。结果 HBO组脑梗死容积小于MCAO组,差异有统计学意义（P〈0.05）,其神经功能学评分也明显优于MCAO组（P〈0.05）。再灌注各时相点,HBO组泛素化蛋白聚集明显少于MCAO组（P〈0.05）。结论高压氧预处理所诱导的脑缺血耐受的机制可能与泛素化蛋白聚集降低有关。