局灶性脑缺血再灌注大鼠皮质中谷氨酸相互作用蛋白和谷氨酸受体2的表达

目的 研究脑缺血再灌注模型半暗带皮质中谷氨酸相互作用蛋白(glutamate receptor interacting protein,GRIP)表达的变化及其与谷氨酸受体2(glutamate receptor 2,GluR2)的关系.方法 36只SD雄性大鼠随机分为缺血再灌注1、6、12、24、72 h组和假手术组...     

Homer与缺血性脑损伤中谷氨酸、γ-氨基丁酸相关性研究

目的探讨Homer与神经元缺血性损伤中谷氨酸（Glu）、γ-氨基丁酸（GABA）相互关系。方法培养大鼠脑皮层原代神经元,先分为对照组、空载体组和Homeda siRNA转染组,检测Homefla变化。再将上述三组各分成正常组,缺氧缺血再灌注损伤后30min、1h、3h、12h、24h、48h共7小组,并进行Glu、GABA和Homeda的检测。结果①转染组神经元HomeHa表达量明显减少;②损伤中,乳酸脱氢酶（LDH）活性增强;与其它两组比较,转染组培养液LDH活性最低;③损伤中,Glu和GABA与LDH变化正相关;Glu和Homerla的变化成反比;而GABA和Homerla的变化成正比。结论缺血性神经元损伤后,Homerla蛋白不仅调控Glu变化,还通过调控GABA变化保护神经元,在兴奋性和抑制性神经递质平衡中发挥重要作用。     

缺血缺氧后海马神经元突触后膜谷氨酸受体-2含量变化的研究

目的探讨缺血缺氧后大鼠海马神经元突触后膜谷氨酸受体-2 (GluR2)含量变化情况.方法体外培养胚胎大鼠海马神经元,模拟临床缺血过程致神经元缺氧损伤,运用双重免疫荧光技术标记和共聚焦检测技术观察缺血缺氧后不同时间点海马神经元突触后膜GluR2含量变化情况.结果体外培养海马神经元进行模拟缺血处理后,膜表面的GluR2总含量、含有GluR2突触的相对含量以及含有突触部位GluR2的相对含量均明显降低,而且上述变化随着模拟缺血时间的延长而增加,各组间均存在显著性差异(P＜0.05).结论缺血缺氧损伤可致突触后膜表面G1uR2含量降低并随着缺血时间的延长而增加,形成缺乏GluR2的新的AMPA受体通道,介导Ca2 的快速内流,引起神经元的延迟性死亡.     

大鼠全脑缺血再灌流后4个脑区的谷氨酸、门冬氨酸、甘氨酸及γ—氨基丁酸含量的测定

应用高效液相色谱仪,紫外分光光度检测器监测,测定大鼠全脑缺血再灌流后6h～7d的海马、纹状体、丘脑和新皮层组织匀浆中谷氨酸(Glu)、门冬氨酸(Asp)、甘氨酸(Gly)和γ-氨基丁酸(GABA)含量变化。结果显示在海马和丘脑Glu含量于再灌流后6h～3d下降(P<0.05和0.01),GABA含量升高(P<0.05和0.01),在纹状体和新皮层除GABA外,分别于6h～5d均有不同呈度的升高。比较有规律的变化是当GABA升高时Glu含量下降。这可能是由于缺血及再灌流早期Glu等兴奋性氨基酸(EAAs)大量释放,反射性GABA的合成与释放增多使Glu向GABA方向转化有关。     

马桑内酯激活的星形胶质细胞条件培养液对大鼠脑内谷氨酸及其受体GluR2表达的影响

目的 揭示马桑内酯(CL)激活的星形胶质细胞(Ast)条件培养液(ACM)对大鼠脑内谷氨酸(Glu)及其受体GluR2表达的影响.方法 取成年健康雄性SD大鼠48只,采用随机数字表法分为对照组(16只)和CL组(32只),对照组侧脑室注射未加任何刺激物的ACM 10μL,CL组侧脑室注射CL激活的ACM 10μL;按注射后取材时间不同又分为2h、4h、8h和12h四个亚组,对照组每亚组4只,CL组每亚组8只.观察两组大鼠的行为表现,用免疫组化、免疫荧光检测脑内Glu和GluR2表达的变化,Western blot检测脑内GluR2含量的变化.结果 CL组大鼠有痫样发作,而对照组无痫样发作;免疫组化和免疫荧光检测结果显示,CL组皮质和海马区Glu表达较对照组显著增强,4h时差异有统计学意义(P<0.05),而CL组皮质和海马区GluR2的表达较对照组弱,4h时差异有统计学意义(P<0.05).Western blot结果显示,CL组4个时间点的GluR2表达均较对照组含量显著降低,差异有统计学意义(P<0.05).结论 CL激活的ACM能显著增强脑内神经元Glu的表达,降低GIuR2的表达,进而诱发癫痫.     

实验性脑缺血急性期大鼠脑甲状腺激素受体初步研究

运用４血管闭塞法制造ＳＤ大鼠全脑缺血动物模型，并经受体放射分析法测定大鼠脑甲状腺激素受体（Ｔ３Ｒ）的最大结合容量和亲和力的变化。结果显示：与假手术组相比较，缺血５ｍｉｎ脑Ｔ３Ｒ即开始上升，缺血１５ｍｉｎＴ３Ｒ含量明显增高（Ｐ＜０．０１），受体亲和力无改变。大鼠脑缺血急性期脑甲状腺激素受体向上调节为机体的重要代偿反应。     

谷氨酸对丘脑束旁核痛兴奋神经元放电的影响

目的：观察脑室注射谷氨酸（Glu)对大鼠丘脑束旁核（PF）痛兴奋神经元（PEN）电变化的影响。方法：以电脉冲刺激右侧坐骨神经作为伤害性痛刺激，用玻璃微电极细胞外记录神经元放电的变化。结果：（1）伤害性刺激使大鼠丘脑PF的PEN诱发放电频率增加；（2）脑室注射Glu(1.5μg/10μl）加强PEN的电活动，使PEN放电频率的净增值增加，潜伏期缩短；（3）这种作用可被Glu的NMDA受体拮抗剂MK－801（0．17μg/0.5μl)所阻断。结论：Glu在中枢痛沉调制中可能起兴奋作用，而NMDA受体参与介导中枢伤害性信息的传递过程。     

黄芪皂甙和神经节苷脂GM1对大鼠MCAO/IR模型细胞外液氨基酸含量的影响

目的　动态观察黄芪皂甙 (ASS)和神经节苷脂GM1对插线法大鼠MCAO IR模型细胞外液氨基酸含量的影响 ,研究两者单用或合用时对脑缺血 再灌流的保护作用。　　方法　采用缺血 1h再灌流 2h模型 ,检测缺血皮层内脑组织细胞外液内氨基酸含量。　　结果　 1 ASS组谷氨酸 (Glu)峰值浓度下降了 1 3 2 % ,但无统计学差异 ;2 GM1组各类氨基酸都明显下降 ;3 ASS GM1组较单用ASS或GM1组Glu下降有统计学差异。　　结论　尽管ASS未能使Glu产生明显下降 ,但能增加GM1的抗兴奋毒作用 ,两者合用比各自单独使用的脑保护作用更显著。     

氮苯二羧基钒酸盐调控谷氨酸作用下海马神经元内钙离子的研究

目的：探讨第10号染色体缺失的磷酸酶和张力蛋白同源等位基因抑制剂氮苯二羧基钒酸盐（bpv）调控谷氨酸 （Glu）作用下海马神经元内Ca^2＋的作用及机制.方法：用孕12～14 d大鼠原代培养海马神经元,经bpv和Glu受体（GluR）阻断剂预处理后,通过激光共聚焦扫描检测高浓度Glu刺激后的细胞内Ca^2＋浓度变化.结果：Glu可诱导神经元细胞Ca^2＋ 内流,bpv可通过对Glu a-氨基羟甲基恶唑丙酸（AMDA）受体的作用而减少Ca^2＋内流.结论：bpv在脑创伤后对神经元的保护作用可能是通过抑制Glu所引起的Ca^2＋内流而实现的.     

一氧化氮合酶、谷氨酸在局灶脑缺血中的变化

目的　观察一氧化氮合酶 (NOS)和谷氨酸 (Glu)在脑缺血时的改变。方法　应用大鼠大脑中动脉闭塞局灶脑缺血模型 ,观察脑缺血 1h后NOS和Glu含量的变化。结果　缺血 1h后NOS活性显著升高(P <0 .0 5 )、Glu含量亦显著升高 (P <0 .0 1) ;用L NMMA处理后 ,NOS活性显著降低 (P <0 .0 1) ,Glu含量亦降低 (P <0 .0 5 )。结论　Glu生成过多可激活NOS ;抑制NOS活性可减少Glu的生成。     

硫酸镁在大鼠局灶脑缺血中的保护作用

目的 研究非竞争性谷氨酸受体拮抗剂－－硫酸镁在大鼠局灶脑缺血中的作用。方法 采用线栓法建立大鼠右侧大脑中动脉区永久脑缺血模型,分别于缺血前半小时,、缺血后第１、３、６、１２小时静滴１０％硫酸镁溶液,滴速１．５ｍｌ／ｈ,通过神经功能评分、梗死体积及含水量的改变、病理学检查,探讨硫酸镁对脑缺血的保护作用及治疗时间窗。结果 缺血６小时内应用硫酸镁能改善运动功能,减轻脑水肿,缩经体积。结论 硫酸镁具有明显     

头孢曲松钠对大鼠脑缺血损伤的保护作用及其机制

目的 探讨在大鼠局灶性脑缺血模型中应用头孢曲松钠对脑缺血损伤的保护作用及其相关机制.方法 制备Wistar大鼠局灶性脑缺血模型,并按随机数字表法分为单纯缺血组(MCAO组)、头孢曲松钠治疗组(MCAO+CTX组)和盐水对照组,其中MCAO+CTX组为缺血90min时给予头孢曲松钠200 mg/kg.缺血后24 h、48 h、7 d时对各组大鼠进行神经行为学评分和脑水肿程度测定,同时比较各组大鼠皮层和海马谷氨酸转运体功能的差异.结果 随着缺血时间延长,各组大鼠神经行为学评分逐渐提高;脑水肿在缺血后24 h、48 h时逐渐加重,至7 d时已逐渐消退.与MCAO组比较,各时间点MCAO+CTX组大鼠神经行为学评分明显提高,脑水肿程度明显减轻,伤侧皮层及海马谷氨酸转运体功能明显增强,差异均有统计学意义(P＜0.05).结论 头孢曲松钠对大鼠局灶性脑缺血损伤具有保护作用,其机制可能与增强谷氨酸转运体功能从而减轻谷氨酸神经毒性作用有关.

Abstract：

Objective To explore the neuroprotective effect of ceftriaxone on cerebral ischemia injury in rats with focal cerebral ischemia and its possible mechanism. Methods Focal cerebral ischemic models were established in Wistar rats and randomly divided into ischemic group (performed middle cerebral artery occlusion [MCAO]), ceftriaxone (CTX) therapy group (given CTX at a dosage of 200 mg/kg 90 min after MCAO) and control group (given physiological saline only). Twenty-four and 48 h, and 7 d after MCAO, neurological behaviors and cerebral edema level were evaluated in these 3 groups;glutamate transporter function in the cortex and hippocampus of rats was compared between each 2 groups. Results With time extended, neurological behaviors scores were obviously elevated in every group;and cerebral edema became worse at 24 and 48 h and decreased 7 d after MCAO. As compared with that in the ischemic group, glutamate transporter function, level of edema and neurological behaviors scores in cortex and hippocampus of rats in the CTX therapy group were statistically increased at different ischemic time points (P<0.05). Conclusion Ceftriaxone has a neuroprotective effect against focal cerebral ischemia in rats, which may relate to increased glutamate transporter function and reduced glutamate neurotoxicity.     

Uptake of glutamate is impaired in the cortical penumbra of the rat following middle cerebral artery occlusion: an in vivo microdialysis extraction study

By using microdialysis extraction of (3)H-D-aspartate and concomitant recordings of extracellular direct current (DC) potentials, the effect of middle cerebral artery occlusion (MCAO) was studied continuously over a period of 100 min in the cerebral cortex of rats. From analysis of the DC potentials, rats subjected to MCAO could be divided into three groups, one in which the dialysis probe was located in the ischemic core, one in which the probe was in the penumbra, and one in which the probe was in nonischemic tissue. In general, extraction of (3)H-D-aspartate was positively correlated with the DC potential; i.e., changes in the extraction were concurrent with changes in the DC potential. Comparing the different animal groups by integration of all extraction values obtained during MCAO over time, (3)H-D-aspartate extraction was reduced by 40% in the penumbra, and by 58% in the ischemic core, compared with the sham-operated controls. No changes was found in the nonischemic group. In the penumbra group, extraction of (3)H-D-aspartate was reduced initially upon institution of MCAO but recovered to control-like levels over a period of 15-40 min, despite ongoing MCAO. In addition, extraction was reduced transiently during periinfarct depolarizations. A mean of all extraction values obtained during MCAO in the penumbra group was reduced by 47% compared with a mean of values obtained before institution of MCAO. Induction of death resulted in a reduction of (3)H-D-aspartate extraction by 86%. The present results provide direct evidence that uptake of Glu is reduced both in the ischemic core and in the penumbra of the cerebral cortex following MCAO in rats, possibly contributing to the initiation and spread of infarction. The results further indicate that uptake of Glu in the penumbra recovers to control-like levels, despite ongoing MCAO, providing evidence that Glu uptake by the Glu transporter proteins is reinstituted and/or up-regulated.     

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.     

Lipid peroxidation in rat brain during focal cerebral ischemia: prevention of malondialdehyde and lipid conjugated diene production by a novel antiepileptic,lamotrigine

The effects of Lamotrigine (LTG) which blocks ischemia induced glutamate (Glu) release, on lipid peroxidation have been evaluated in cortical and cerebellar tissues of rat brain during focal cerebral ischemia. A total of 45 rats were randomly assigned into one of four groups; sham operated animals as controls, animals subjected to middle cerebral artery occlusion (MCAO) and treatment groups with LTG (20 mg/kg i.p.) either 30 min before or just after MCAO. Changes in lipid peroxides were expressed as nanomoles of malondialdehyde (MDA) and conjugated diene (CD) per milligram of protein. MDA values following 60 min of ischemia relative to contralateral cortex and CD levels in 0, 10 and 60 min of ischemia were found to be higher in the ipsilateral cortex than those in the contralateral cortex. On the other hand, contralateral cerebellar MDA levels after 0 and 60 min of ischemia and CD levels after 0, 10 and 60 min of ischemia were higher than those in the ipsilateral cerebellum. Pharmacological inhibition of Glu release significantly decreased the MDA and CD production in both cortex and cerebellum. Pre- or post-ischemic administration of LTG did not significantly change CD levels, but MDA levels in contralateral cortex were found to be significantly decreased than those in ischemic cortex in both pre- and post-treated group.     

Serum amino acid levels after permanent middle cerebral artery occlusion in the rat

BACKGROUND AND PURPOSE: High levels of glutamate in plasma and cerebrospinal fluid (CSF) have been demonstrated in patients with acute ischemic stroke. Whereas this glutamate increase in CSF is only evidenced during the first 6 h in stable ischemic stroke, it is sustained for 24 h in progressing stroke. The aim of this investigation was to study the evolution of serum glutamate levels after stroke in a rat model of permanent cerebral artery occlusion. METHODS: Glutamate, glycine, aspartate, taurine and tryptophan were measured by high-performance liquid chromatography from serum samples taken before and at different times after permanent middle cerebral artery occlusion (MCAO) and from sham-operated rats. RESULTS: After MCAO, a 3-fold increase in glutamate and a 2-fold increase in glycine and aspartate were observed in rat serum. The onset of this amino acid increase began 4-6 h after ischemic induction, reached peak values at 8-24 h and returned to preischemic values by 48-72 h. Serum concentrations of taurine and tryptophan were not modified after MCAO. Sham-operated rats did not exhibit changes of basal amino acid concentrations in serum. CONCLUSIONS: The serum excitatory amino acid profile in this experimental model confirms that the early detection of increased concentrations of glutamate and glycine at systemic circulation observed in patients with acute stroke is a consequence of the cerebral ischemic process.     

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.     

Chemokine CXCL8 modulates GluR1 phosphorylation

The chemokine interleukin 8/CXCL8 induces the phosphorylation of the GluR1 subunit of the AMPA-type glutamate receptor in neurons and transfected HEK cells, on both serine 845 (S845) and 831 (S831) residues. We previously described that CXCL8 receptor CXCR2 and GluR1 co-precipitate and that GluR1/CXCR2 co-expression both in HEK cells and neurons impairs CXCL8-induced cell migration. Here we show that replacement of S845 with Ala (A), but not with Glu (E), strongly reduces GluR1/CXCR2 interaction and abolishes the impairment of CXCL8-induced cell migration. Considered together our findings point to the phosphorylated state of S845GluR1 as a determinant of GluR1-CXCR2 physical coupling.     

Add-on lamotrigine treatment and plasma glutamate levels in epilepsy: relation to treatment response

We investigated the effects of add-on lamotrigine treatment on plasma glutamate (Glu) levels, in 29 epileptic patients. Plasma Glu levels were determined by high-performance liquid chromatography at baseline and at 1 and 3 months post-treatment. In patients with a seizure reduction of ≥66% a decrease of Glu at month 1 was noted, followed by return to baseline levels at month 3. In the remaining patients a gradual increase of Glu was noted throughout the 3 months of the study. The above findings indicate that an excellent clinical response to add-on lamotrigine may be characterized by a statistically significant, yet transient decrease of plasma Glu levels, while increasing Glu levels may accompany a response that is moderate at best. The combination of lamotrigine with valproate was more frequent in patients with excellent clinical response and tended to result in glutamate decrease.