谷氨酸转运体在全脑缺血性癫痫中作用的研究

目的比较三种谷氨酸转运体在全脑缺血性癫痫中的动态变化特征,为癫痫治疗提供有意义靶点.方法SD大鼠以胸部压迫8分钟造成全脑缺血性癫痫模型,分对照组、假手术组、全脑缺血无癫痫组和全脑缺血癫痫组.后两组又根据脑缺血后时间分为6h,24h,48h,72h,5d,7d组.应用免疫组化法研究谷氨酸转运体EAAT-1,EAAT-2,EAAT-3在海马CA1及皮质区表达;研究病理形态变化,同时测定大鼠脑电图改变.结果大鼠癫痫发生率为64%,全脑缺血癫痫大鼠神经损害较无癫痫组严重.与全脑缺血无癫痫大鼠比较,癫痫大鼠海马CA1及皮质区EAAT-2显著持续降低及EAAT-3表达明显升高.结论大鼠癫痫发生与脑缺血严重程度密切相关.海马CA1及皮层区EAAT-2、EAAT-3表达变化是抗癫痫治疗的作用靶点.     

Glucocortcoids mediate the stress-induced extracellular accumulation of glutamate

The hippocampal damage caused by stress has been attributed to an increased glutamatergic tone brought about by secretion of glococorticoids. Although exposure to stress has been shown to increase the outflow of glutamate, direct involvement of glucocorticoid in this phenomenon has not been examined. The present study demonstrates that adrenalectomy attenuates the stress-induced outflow of glutamate in the hippocampus and prefrontal cortex and that glucocorticoid replacement abolishes this attenuation.     

Neuroprotection and glutamate attenuation by acetylsalicylic acid in temporary but not in permanent cerebral ischemia

To assess the effects of acetylsalicylic acid (ASA) on glutamate and interleukin-6 (IL-6) release in the striatum of rats suffering from cerebral ischemia, we used the microdialysis technique with probes implanted 2 h prior to stroke onset. A total of 36 rats were randomly assigned to either temporary (90 min, n=18) or permanent (n=18) middle cerebral artery occlusion (MCAO). Animals received either a bolus of 40 mg/kg ASA or saline as control 30 min after stroke onset. Permanent MCAO led to large infarct volumes with no differences between treatment with ASA (239.8+/-4.1 mm3) and saline (230.1+/-3.9 mm3, p=0.15). In contrast, ASA therapy in temporary ischemia (87.2+/-6.2 mm3) reduced infarct size significantly compared to placebo (155.6+/-4.8 mm3, p<0.0001). Only in temporary ischemia, ASA application reduced glutamate significantly at the time points 90, 120, and 150 min after MCAO. Pooled post-ischemic microdialysate concentrations of IL-6 in temporary MCAO were significantly higher after ASA treatment (215+/-81 pg/mL, p=0.0297) than in saline-treated rats (80+/-13 pg/mL). In the permanent MCAO group, no difference in IL-6 between the ASA (125+/-21 pg/mL) and saline group (68+/-34 pg/mL) was noted. No differences were seen for c-fos positive neurons in the penumbra and hippocampus between all groups. These results suggest that the neuroprotective effect of ASA is reflected by glutamate attenuation and IL-6 induction even if given after stroke onset, but only if reperfusion is achieved.     

Lubeluzole attenuates K(+)-evoked extracellular accumulation of taurine in the striatum of healthy rats and rats with hepatic failure

Lubeluzole is a newly designed neuroprotectant which has proved effective in the treatment of experimental stroke in rats, mainly by inhibition of the glutamate-activated NO pathway, but also by counteracting osmotic stress by a mechanism associated with the release of the osmotically active amino acid taurine (Tau). Here we show that lubeluzole administered i.p. decreases by 25% the high (50 mM) K+-evoked accumulation of Tau in striatal microdialysates of healthy rats and by 34% in rats with thioacetamide-induced hepatic failure, where the increased extracellular accumulation of Tau signifies ongoing hepatic encephalopathy. Lubeluzole does not affect the nonstimulated accumulation of Tau in either group of rats. The results indicate that lubeluzole may be effective in ameliorating ionic or osmotic stress in a range of pathological conditions involving the rise of extracellular K+, and also in decreasing the vulnerability to stress in rats with hepatic failure.     

Characterization of transient focal ischemia-induced increases in extracellular glutamate and aspartate in spontaneously hypertensive rats

Using middle cerebral artery occlusion (MCAO) and in vivo microdialysis, we have evaluated the changes in extracellular concentrations of the excitatory amino acids (EAA) glutamate and aspartate during varying periods of MCAO (0, 30, 60 min) in the striatum of spontaneously hypertensive rats (SHR). A positive correlation between occlusion time-dependent elevations in EAAs and the resulting ischemic injury was observed. This is the first demonstration of the temporal profile of EAA efflux during transient focal ischemia in SHRs. Possible sources and mechanisms of ischemia-induced EAA efflux were examined during 60 min of MCAO. Removal of Ca(2+) from the microdialysis infusion media significantly attenuated ischemia-induced increases in both glutamate (from ischemic peak of 4892 +/- 1298 to 1144 +/- 666% of preischemic values) and aspartate (from 2703 +/- 682 to 2090 +/- 599% of preischemic values). Similarly, infusion of the voltage dependent Na(+) channel blocker tetrodotoxin (TTX; 10 microM) significantly attenuated MCAO-induced increases in glutamate (to 1313 +/- 648%) and aspartate (to 359 +/- 114%). Infusion of the GLT-1 selective nontransportable inhibitor, dihydrokainate (DHK; 1 mM) also significantly attenuated the ischemia-induced increases in both EAAs (1285 +/- 508 and 1366 +/- 741% of the preischemic levels, respectively). These results indicate that during transient focal ischemia the increase in extracellular EAAs originates from both the neuronal pool, via conventional exocytotic release, and glial sources via the reversal of the GLT-1 transporter.     

Effects of glutamate receptor agonist on extracellular glutamate dynamics during moderate cerebral ischemia

We performed real-time monitoring of the extracellular glutamate dynamics in the rat striatum in vivo using the microdialysis electrode technique, during an experimental penumbral condition of moderate global cerebral ischemia and activated glutamate receptors. The local cerebral blood flow (CBF) was measured with a laser-Doppler probe. One minute after bilateral common carotid artery occlusion (BCAO), CBF was reduced to approximately 60% of the pre-ischemic value and it remained at this level during the period of occlusion. After BCAO, a transient depolarization and a transient increase in extracellular glutamate concentration ([Glu]e) were seen. In other rats, 500 microM N-methyl-D-aspartate (NMDA) was locally micro-transfused for 30 min prior to BCAO. Upon induction of BCAO, an anoxic depolarization-like depolarization and a gradual increase in [Glu]e that continued over the duration of BCAO were seen. After BCAO was terminated, the direct current (DC) rapidly recovered to the basal level, while [Glu]e gradually decreased to the basal level. In rats that were locally micro-transfused with 500 microM Kainate prior to BCAO, DC and [Glu]e did not differ significantly from control. Pretreatment with MK-801 prior to NMDA treatment completely inhibited the NMDA-induced changes in DC and [Glu]e. Pretreatment with NBQX prior to NMDA treatment did not inhibit the NMDA-induced changes in DC and [Glu]e. Consequently, we found that activation of NMDA receptors by elevated [Glu]e exerts an important effect on [Glu]e dynamics in the spreading stroke region very early in the acute stage of cerebral ischemia in vivo.     

Metabotropic glutamate receptor subtypes are differentially expressed after transient cerebral ischemia without, during and after tolerance induction in the gerbil hippocampus

Postischemic delayed neuronal death (DND) of hippocampal CA1 neurons can be prevented by a preconditioning sublethal ischemic stimulus. To check for possible participation of metabotropic glutamate receptors (mGluRs) in neuronal death or survival, we analyzed postischemic protein expression of subtypes 1b and 5 of group I mGluRs, which are thought to exert neurotoxic effects after pathological activation due to ischemia, and subtypes 2 and 3 of group II mGluRs, which in contrast are thought to be neuroprotective in this state, respectively. Therefore, three groups of gerbils with reperfusion intervals between 8 h and 4 days (n=5 each) were investigated: one group was subjected to 5 min ischemia, resulting in DND of CA1 neurons, a second group to a tolerance inducing 2.5 min period of ischemia and a third group to 5 min ischemia after prior tolerance induction. The major finding was a transient postischemic reduction of mGluR1b and 5 expression in the ischemic tolerant CA1 subfield at 8 h. This downregulation of neurotoxic mGluRs may indicate a contribution to the survival of highly vulnerable CA1 neurons in the ischemic tolerant state.     

Different dynamic patterns of extracellular glutamate release in rat hippocampus after permanent or 30‐min transient cerebral ischemia and histological correlation

The extent and severity of neuronal damage is different in ischemia with reperfusion compared to ischemia and no reperfusion. To investigate the role of glutamate in cerebral ischemia–reperfusion injury, in vivo microdialysis was performed to examine the dynamic profile of glutamate in the hippocampus in a transient (30 min) or permanent middle cerebral artery occlusion (MCAo) in Wistar rats. The extracellular concentration of glutamate in the cornu ammonis (CA)1 sector of the ipsilateral hippocampus showed a significant but transient elevation of glutamate for both groups immediately following ischemic insult. The initial high peak in glutamate levels in the transient MCAo group was followed by two secondary elevations in glutamate at 50 min and 90 min after initialization of reperfusion. The histopathological outcome was also different in the two groups. The observation that glutamate releases occurred in the early reperfusion phase provided an evidence of additional excitotoxicity of glutamate and thereby a therapeutic base for extended use of glutamate antagonist in the ischemia–reperfusion injury.     

星状神经节阻滞对全脑缺血再灌注家兔循环和呼吸的影响

目的　观察星状神经节阻滞 (SGB)对全脑缺血再灌注损伤家兔循环和呼吸的影响。方法　健康日本大耳白兔 15只 ,随机分成 3组 (A、B、C) ,每组 5只。三组动物均使用六血管 (双侧颈内外、椎动脉 )阻断法复制全脑缺血再灌注损伤模型 ,A、B两组分别接受左右侧SGB ,C组作为对照 ,不作SGB。连续观察全脑缺血 2 0min及再灌注 30min期间SGB对家兔心率 (HR)、平均动脉压 (MAP)以及呼吸频率 (RR)的影响。结果　在缺血及再灌注期间的部分时点A组HR、MAP变化较C组有显著意义 (P <0 .0 5 ) ;缺血后 1minB组MAP较C组显著降低 (P <0 .0 5 ) ;与B组比较 ,缺血后与再灌注期间的部分时段A组HR较快 ,MAP较低 (P <0 .0 5 ) ;各组内与缺血前比较 ,缺血后及再灌注开始的部分时段HR明显减慢 (P <0 .0 5 ) ,B、C两组尤为显著 ;MAP显著升高 (P <0 .0 5 ) ,A、C两组更为显著 ,部分时点P <0 .0 1,且C组在缺血一开始即出现显著性差异 (P <0 .0 5 )。组间未见RR的显著性变化。结论　SGB影响全脑缺血再灌注期间的心率和血压 ,倾向于减小其波动幅度 ,左侧对血压的影响以及右侧对心率的影响较对侧明显 ;SGB不改变缺血再灌注损伤对呼吸的影响     

Ultrastructural studies of cerebral vascular spasm after cardiac arrest-related global cerebral ischemia in rats

The present investigation was undertaken to study the ultrastructural morphology of brain blood vessels during vasospasm following total cerebral ischemia. Global cerebral ischemia was produced in rats by compression of the cardiac vessel bundle (i.e., cardiac arrest) using a metal hook that was introduced into the mediastinum. Ischemia lasted for 10 min with blood recirculation for 6, 12 and 24 h. Rat brains were perfusion-fixed and regions from the cerebral cortex and associated leptomeningeal vessels were evaluated by scanning and transmission electron microscopy. We noted three general vasoconstrictive responses in vessels of various sizes including veins and arteries. These alterations related to the smooth muscle cell arrangement associated with each constricted vessel including a circumferential, and longitudinal arrangement, or a combination of both types. Other features in the three types of vasoconstricted vessels included thickening of the vessel basement membranes with increased endothelial microfilaments and vesicular profiles. Our studies present evidence that ischemia of 10-min duration with blood reflow for 6, 12 and 24 h produces profound and variable vasospastic changes in some but not all vessels. These vascular alterations are thought to be caused in part by vasoactive substances released both by endothelial and blood cells and by perivascular cellular elements in response to the ischemic episode.     

小檗碱对小鼠全脑缺血后神经元凋亡的影响

目的观察小檗碱能否对小鼠全脑缺血后细胞凋亡产生抑制作用.方法利用改良的Pulsinelli-Brierley4血管闭塞法制成小鼠全脑缺血再灌注动物模型.TUNEL法原位检测特征性DNA片段.琼脂糖凝胶电泳技术检测DNA梯度带.结果小檗碱组与再灌流各时间段缺血组相比,海马CA1区TUNEL阳性细胞数明显减少(P<0.01).缺血组再灌注24h、48h可见典型的DNA梯度带,分别在180、360、540bp等处.小檗碱组未见特征性DNA梯度带.结论小檗碱可以减少小鼠全脑缺血后海马CA1区细胞凋亡的发生.     

全脑缺血大鼠血液流变学变化特征的实验研究

目的：观察大鼠全脑缺血后血液流变学的动态变化。方法：采用4-VO大鼠急性全脑缺血模型,于4-VO后2d、4d、7d、14d和28d对大鼠血液流变学指标、脑组织含水量进行检测并在光镜下观察细胞形态学的变化。结果：大鼠全脑缺血再灌注后血液流变学指标、脑组织含水量2d时即增高,至7d时达到高峰,14d时逐渐恢复,28d时恢复至假手术组水平。再灌注后2d、4d、7d与假手术组比较有显著差异(P<0．05)。结论：大鼠全脑缺血再灌注后血液流变学指标、脑组织含水量随着再灌注时间的延长而增高,7d时达到高峰。     

Neuroprotective effect of tiagabine in transient forebrain global ischemia: an in vivo microdialysis,behavioral, and histological study

The neuroprotective effect of tiagabine was investigated in global ischemia in gerbils. Two groups of the animals received 15 mg/kg of tiagabine 30 min before ischemia. In the first group, the temperature was controlled at 37 degrees C from time of injection to 1 h after ischemia. In the second group, the temperature was left uncontrolled to see the hypothermic effect of tiagabine. Microdialysis was performed in CA1 region of hippocampus in half of the animals in each group to assess the levels of glutamate and gamma-amino-butyric acid (GABA). Animal behavior was also tested in 28-day groups in a radial-arm maze. Histology was done 7 and 28 days after ischemia in CA1 region of hippocampus to assess early and delayed effect of drug. A significant suppression of glutamate was noted in both groups (P<0.01). Behavioral results showed that in the temperature-uncontrolled treatment group, animals significantly reduced their working memory errors as compared to the temperature-controlled treatment group. Histology revealed a significant neuroprotection (P<0.001) in the temperature-uncontrolled treatment group. In the temperature-controlled treatment group, however, neuroprotection was insignificant (P>0.05). A third group of animals received the same dose of tiagabine 3 h after ischemia. Temperature was not controlled in this group. The animals were sacrificed after 7 days so no behavior testing was carried out. Histology showed no neuroprotection in this group (P>0.05). These results show that tiagabine offers a significant neuroprotection in global ischemia in gerbils when given 30 min before ischemia but not when given 3 h after ischemia.     

利用Affymetrix芯片筛选全脑缺血大鼠海马差异表达基因

目的 筛选出全脑缺血大鼠海马差异表达基因.方法 建立大鼠体外循环模型,并将大鼠按随机数字表法分成两组:对照组(n=3),建立体外循环5min后取大鼠海马组织;实验组(n=3),停循环全脑缺血5 min后取大鼠海马组织.采用Affymetrix大鼠全基因组芯片检测两组大鼠海马基因表达的变化,获取差异表达基因.结果 筛选出差异表达基因共55个(上调30个,下调25个),其中28个基因表达差异有统计学意义(P＜0.05).结论 筛选出的全脑缺血大鼠海马差异表达基因对阐明全脑缺血性损伤的分子生物学机制有重要意义.     

小檗碱对小鼠全脑缺血后神经元凋亡相关基因的影响

目的 探讨小檗碱对小鼠全脑缺血后神经元凋亡相关基因的影响,以了解小檗碱保护脑缺血的机制,为其开发利用提供理论依据。方法 利用改良的Pulsinelli-Brierley4血管闭塞法制成小鼠全脑缺血再灌注动物模型。小檗碱用量为1mg/kg,于术前30min,术后每日1次,腹腔注射。免疫组织化学技术检测凋亡相关基因Bcl-2,Bax蛋白的表达。结果 正常组海马区未见Bcl-2或Bax蛋白表达;缺血组再灌注6h海马CA3区可见Bcl-2阳性细胞,24h达到高峰,48h开始下降;小檗碱治疗组再灌注24h、48h及168hBcl-2阳性细胞明显减少（P＜0．01）。缺血组再灌注6h海马CA1区可见Bax阳性细胞;48h达高峰;168h明显下降;小檗碱组再灌注24h,48h及168hBax阳性细胞数明显减少（P＜0．01）。结论 小檗碱可以增加小鼠全脑缺血后海马CA3区bcl-2基因的表达,降低CA1区Bax基因的表达,从而减少凋亡的发生,可能为其保护脑缺血的机制之一。     

Enriched environment delays the onset of hippocampal damage after global cerebral ischemia in rats

An enriched environment has been shown to improve cognitive, behavioral and histopathological outcome after focal cerebral ischemia and head trauma. The purpose of this study was to determine the effect of an enriched environment on histopathology following global cerebral ischemia. Wistar rats (21 weeks of age) were placed in different environments [standard cages (SC) or enriched environment (EE) cages] for 2 months before and either 6 days or 2 months after ischemia. Rats underwent 10 min of global ischemia by bilateral carotid artery occlusions plus hypotension. Five groups (n=4-5 in each group) were studied: (1) rats kept in SC before and 2 months after ischemia; (2) rats kept in SC before ischemia but transferred to an EE for 2 months after ischemia; (3) rats kept in EE before and after ischemia for 2 months; (4) rats kept in SC before and 6 days after ischemia; (5) rats kept in EE before and 6 days after ischemia. At 7 days or 2 months after ischemia, brains were perfusion-fixed, and ischemic injury was assessed by counting numbers of normal neurons in the hippocampal CA1 sector. Physiological variables showed no inter-group differences. Rats housed in EE for 2 months before and for 6 days (but not 2 months) after global ischemia showed significantly better preservation of pyramidal neurons in the hippocampal CA1 area when compared to control animals (middle CA1, 20.5+/-5.4 vs. 2.8+/-0.6; lateral CA1, 31.5+/-7.2 vs. 2.6+/-0.6, respectively). The present data suggest that housing in EE for 2 months before and 6 days after ischemia can delay the onset of damage to hippocampal pyramidal neurons, which eventually occurs despite 2-month EE.     

脑缺血时皮层区细胞外液中氨基酸的动态变化——微透析观察

应用微透析技术,活体动态观察沙土鼠脑缺血时皮层区细胞外液(ECF)中氨基酸(AA)的变化。结果发现:(1)应用微透析技术在脑皮层中可检测到多种AA,其中包括神经介质类AA和非神经介质类AA;(2)脑缺血时,神经介质类AA显著升高,而非神经介质类AA仅轻度升高。结果提示,脑缺血时神经介质类AA和非神经介质类AA升高的机制可能不同,神经介质类AA在脑缺血损伤中起着重要作用。     

Non-NMDA glutamate receptor binding in canine brain after global cerebral ischemia and reperfusion

We employed a canine model to test the effects of global cerebral ischemia and reperfusion on binding to α-amino-3-hydroxy-5-methyl-4-isoxazole proprionate (AMPA), kainate (KA), and metabotropic glutamate receptors. Ischemia was induced by 10 min of cardiac arrest, followed by restoration of spontaneous circulation for periods of 0, 0.5, 2, 4, and 24 h. Frozen sections were prepared from parietal and temporal cortex, hippocampus, and striatum, and in vitro autoradiography was performed with one of three radioligands: [³H]AMPA, [³H]KA, or [³H]glutamate (using conditions allowing specific labeling of the metabotropic binding site). In striatum, metabotropic binding was unchanged, whereas AMPA and KA binding decreased by 20–30% at 30 min postischemia, remaining depressed through 24h. In cortex, AMPA and metabotropic binding were decreased at several timepoints after ischemia and recirculation, particularly in parietal cortex, whereas KA binding was unaffected in this tissue. Binding to hippocampal regions was largely unchanged, except for a decrease in KA binding at 2 and 4 h postischemia. These findings contrast with results from parallel studies showing increased striatal binding to NMDA receptors following ischemia. Decreased binding to non-NMDA glutamate receptors in striatum and parietal cortex may serve to protect against damage mediated through these receptors.     

Prostacyclin attenuates in the rabbit hippocampus early consequences of transient complete cerebral ischemia

The effects of prostacyclin (PGI2) on ischemic changes of extracellular calcium concentration (CaE+2) and the blood-brain barrier (BBB) permeability were studied by microdialysis of the rabbit hippocampus. This was combined with morphological and neurophysiological observations. Complete cerebral ischemia lasting 15 min was produced by ligation of the brachiocephalic trunk, the left subclavian and both internal thoracic arteries. PGI2 was infused continuously i.v. in the last 3 min of ischemia and for 40 min after it, at a rate of 2 micrograms/kg/min. Control rabbits were submitted to untreated 15-min complete cerebral ischemia. The animals treated with PGI2 were found to have recovered bioelectric activity of the cortex and hippocampus in half the time that it took the untreated group. Application of PGI2 reduced by 60% the depth of ischemia-evoked drop of CaE+2 without acceleration of recovery during recirculation. The postischemic increase of BBB permeability to fluorescein was diminished. The number of morphologically changed neurons in the hippocampus of PGI2-treated animals was significantly lower than in the untreated group.     

Inhibition of glutamate release in rat hippocampus by kynurenic acid does not protect CA1 cells from forebrain ischemia

We assessed the effect of a broad spectrum glutamatergic receptor antagonist, kynurenic acid (500 mg/kg) on ischemia-induced hippocampal glutamate release and neuronal damage. Kynurenic acid significantly decreased glutamate release during ischemia but had no effect on the hippocampal lesion. Some protection was observed in the cortex and in the striatum. These data suggested that the extracellular accumulation of glutamate during forebrain ischemia does not play a major role in the hippocampus.