摘要
谷氨酸受体（glutamate receptors, GluRs）是脑内广泛存在的受体，可分为离子型和代谢型两大类。离子型谷氨酸受体包括NMDA受体、AMPA受体、海人藻酸受体三种亚型。关于代谢型谷氨酸受体（metabotropic glutamate receptor, mGluRs），已克隆出8种亚型，分别为mGluR1, mGluR2, mGluR3, mGluR4, mGluR5, mGluR6, mGluR7, mGluR8。根据氨基酸的序列相似性，信号转导机制及激动剂的选择性不同，可将这些mGluRs分为G-Ⅰ（mGluR1/5）、G-Ⅱ（mGluR2/3）和G-Ⅲ（mGluR4/6/7/8）三组。这些受体被其配体谷氨酸和门冬氨酸激活后，通过不同的细胞内信号传导系统，参与体内众多的生理和病理过程。近年来，这些受体，特别是离子型谷氨酸受体在脑缺血耐受（brain ischemic tolerance, BIT）诱导中的作用引起各国学者的关注，已发现NMDA受体在一定程度上参与BIT的诱导。但是，关于mGluRs是否参与BIT诱导尚未见确切报道。国外学者曾在沙土鼠全脑缺血模型中，运用免疫组化方法研究发现，G-Ⅰ组mGluRs在单纯全脑缺血模型和BIT模型中的表达有微弱差别，提示mGluR1/5可能参与BIT的诱导，但尚无其它更确切的实验依据。而关于G-Ⅱ组mGluRs是否参与BIT的诱导，国内外文献尚未见报道。鉴于mGluRs在脑内广泛存在，且类型众多，对神经元可塑性、兴奋性以及细胞膜离子通道（如钙通道等）功能的广泛影响，有理由推测其在BIT的诱导中可能发挥作用。因此，本研究的目的在于确定mGluRs在BIT诱导中的作用，为最终阐明BIT的形成机制提供依据。
1 mGluRs参与BIT诱导并影响GFAP表达
采用大鼠四血管闭塞全脑缺血模型，应用硫堇染色法和免疫组化SP法，观察mGluR1/5阻断剂(s)-4-carboxy-3-hydroxy-phenylglycine（(s)-4C3HPG）和mGluR2/3阻断剂α-methyl-(4-tetrazolyl-phenyl) glycine (MTPG)对BIT诱导和胶原纤维酸性蛋白（glial fibrillary acidic
protein, GFAP）表达的影响，以确定mGluRs在BIT诱导中的作用。
1.1 mGluR1/5阻断剂(s)-4C3HPG参与BIT诱导并影响GFAP表达
将36只永久凝闭椎动脉的 Sprague Dawley (SD)大鼠（体重280g～320g）随机分为以下四组：①sham组（n=6）：游离双侧颈总动脉，但不夹闭；②损伤性缺血组（n=6）：夹闭双侧颈总动脉8 min；③脑缺血预处理（cerebral ischemic preconditioning，CIP）+损伤性缺血组（n=6）：夹闭双侧颈总动脉3 min，恢复再灌流24 h后再行夹闭8 min；④(s)-4C3HPG组（n=18）：右侧脑室注射(s)-4C3HPG后20 min 行CIP，恢复再灌流24 h后再行夹闭双侧颈总动脉8 min；依据应用(s)-4C3HPG的剂量不同，进一步将该组动物分为0.2 mg 组（n=6）、0.04 mg 组（n=6）和0.008 mg组（n=6）。夹闭颈总动脉过程中，观察到大鼠瞳孔散大，脑电波频率变慢，波幅逐渐缩小甚至呈等电位线，证明产生脑缺血。各组大鼠均在术后或末次缺血再灌后7天灌注取材观察。
硫堇染色显示，sham组海马CA1区锥体细胞可见2～3层，组织学分级（0级：无神经元死亡；1级：散在的神经元死亡；2级：成片的神经元死亡；3级：几乎全部神经元死亡。以下同。）为0～1级，神经元密度（1 mm长度内锥体神经元的个数。以下同）为181±10.5。损伤性缺血组，海马CA1区锥体细胞稀疏，层次不清楚，有大片缺失，组织学分级为2～3级，神经元密度为42±6.7，与sham组相比有显著差异（P<0.05）。CIP+损伤性缺血组海马组织学特征与sham组相似，表明3 min CIP可对其后间隔1天的8 min缺血造成的海马CA1区神经元损伤产生明显的保护作用。(s)-4C3HPG组中，0.2 mg组的形态学改变最为明显，表现为海马CA1区锥体细胞稀疏，排列松散，有大量锥体细胞缺失，组织学分级明显升高（P<0.05）,神经元密度明显下降（P<0.05）。0.04 mg组、0.008 mg组的形态学改变依次减轻。各剂量组组织学分级和神经元密度的变化与(s)-4C3HPG的剂量呈现明显的相关性，与sham组相比均有显著差异（P<0.05）。
GFAP免疫组化染色显示星形胶质细胞呈星形或蜘蛛状，有明显的突起。sham组海马CA1区仅见少量GFAP阳性细胞，突起较少、短，染色较淡。损伤性缺血组海马CA1区GFAP阳性细胞数目明显
增多，胞体肥大，突起增粗、变长现象明显，与sham组相比，其阳性细胞数目、总面积、平均光密度显著增加（P<0.05）。CIP+损伤性缺血组海马CA1区阳性细胞数与sham组相比有增多趋势，但无统计学差异（P >0.05）。(s)-4C3HPG组海马CA1区GFAP阳性细胞数目明显增多，染色加深，表现为胞体肥大，突起增多，变粗，延长，尤以大剂量(s)-4C3HPG时明显，GFAP阳性细胞数目、总面积、平均光密度与sham组、CIP+损伤性缺血组相比均明显增加（P<0.05）。
1.2 mGluR2/3阻断剂MTPG参与BIT诱导并影响GFAP表达
将70只永久凝闭椎动脉的SD大鼠（体重280g～320g）随机分为以下七组：①sham组（n=8）：游离双侧颈总动脉，但不夹闭；②CIP组（n=8）：游离并夹闭双侧颈总动脉3 min；③损伤性缺血组（n=8）：夹闭双侧颈总动脉8 min；④CIP+损伤性缺血组（n=8）：夹闭双侧颈总动脉3 min，恢复再灌流24 h后再行夹闭8 min；⑤MTPG+CIP+损伤性缺血组（n=22）：首先右侧脑室注射MTPG，20 min?
Glutamate receptors (GluRs), widespread in the brain, can be classified into ionotropic glutamate receptors (iGluRs) and metabotropic glutamate receptors (mGluRs). The iGluRs can be further classified into N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazol e (AMPA), and kainate receptors. mGluRs family comprises 8 subtypes, mGluR1, mGluR2, mGluR3, mGluR4, mGluR5, mGluR6, mGluR7 and mGluR8. According to the degree of sequence homology, pharmacological properties and signal transduction mechanisms, they can be classified into three groups: group Ⅰ, including mGluR1/5; group Ⅱ, including mGluR2/3; and group Ⅲ, including mGluR4/6/7/8. These receptors participate in many physiological or pathological processes via different intracelluar signal transduction systems after activated by glutamic acid and aspartic acid, the specific ligands of the receptors. Recently, the roles of these receptors, especially the iGluRs in the induction of brain ischemic tolerance (BIT) have aroused the attention of the scholars all over the world. Many experimental results indicated that NMDA receptors participated in the induction of BIT to some extent. However, few researches addressed the problem whether mGluRs participate in the induction of BIT. The mere relevant report showed that there were minor differences in the expressions of group Ⅰ mGluRs between the global cerebral ischemic and BIT models in gerbils by using immunohistochemistry method, which suggested that mGluR1/5 might participate in the induction of BIT. No further relevant experimental data has been reported until now. There is even no report about whether group Ⅱ mGluRs really participate in the induction of BIT. Since mGluRs widely distribute in the brain, and
they have comprehensive influence on the neuronal plasticity, excitability and ionic channels on the cell membrane, it is reasonable to speculate that the mGluRs may play roles in the induction of BIT. Therefore, the aim of our study is to make sure the role of mGluRs in the induction of BIT, thereby providing experimental evidence for clarifying the mechanism of BIT.
1 The effect of mGluRs on the induction of BIT and the expression of glial fibrillary acidic protein
To determine the role of mGluRs in the induction of BIT, we observed the influence of (s)-4-carboxy-3-hydroxy-phenylglycine （(s)-4C3HPG）, antagonist of mGluR1/5, and α-methyl-(4-tetrazolyl-phenyl)glycine （MTPG）, antagonist of mGluR2/3, on the induction of BIT and the expression of glial fibrillary acidic protein (GFAP) by using thionine staining and immunohistochemistry in a rat model of four-vessel occlusion global cerebral ischemia.
1.1 Effects of (s)-4C3HPG (antagonist of mGluR1/5) on the induction of BIT and the expression of GFAP
Thirty-six Spague Dawley (SD) rats (weighting 280g～320g), whose vertebral arteries were permanently occluded, were randomly divided into 4 groups: ①sham group (n=6)：exposing the bilateral common carotid arteries (BCCA) but without blocking the blood flow; ②ischemic insult group（n=6）：blocking the blood flow of the BCCA for 8 min；③CIP+ischemic insult group ( n=6)：blocking the blood flow of the BCCA for 3 min as a cerebral ischemic preconditioning (CIP), then an ischemic insult was given by blocking the blood flow for 8 min 24 h after CIP; ④ (s)-4C3HPG group（n=18）：20 min after the administration of (s)-4C3HPG via the right lateral cerebral ventricle (RLCV) route, procedures for CIP and ischemic insult were performed as in CIP+ischemic insult group;
According to the dosage of (s)-4C3HPG used, the group was further divided into 0.2 mg（n=6）, 0.04 mg（n=6）and 0.008 mg（n=6）groups. During the occlusion of BCCA, the pupil enlarged and EEG showed decreases in frequency and amplitude, and even an isoelectric level. These phenomena indicated the occurrence of global brain ischemia. Examination for morphological changes in CA1 subfield was performed 7 days after the sham operation or last time of ischemia using thionine staining.
It was found by thionine
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