生理学报Acta Physiologica Sinica,   June  25, 2003, 55(3): 303－310

研究论文

代谢型谷氨酸受体阻断剂α-methyl-(4-tetrazolyl-phenyl) glycine对大鼠海马脑缺血耐受诱导的影响

冯荣芳, 李文斌*, 刘惠卿, 李清君, 陈晓玲, 周爱民, 赵红岗, 艾洁

河北医科大学基础医学研究所病理生理学研究室,  石家庄 050017

 

摘要:  实验采用大鼠四血管闭塞全脑缺血模型, 用硫堇染色法和胶质纤维酸性蛋白(GFAP)免疫组化法, 观察右侧脑室内注射Ⅱ型代谢型谷氨酸受体(metabotropic glutamate receptor 2/3, mGluR2/3)阻断剂α-methyl-(4-tetrazolyl-phenyl)glycine (MTPG)对海马CA1区神经元缺血耐受(BIT)诱导的影响, 以探讨mGluR2/3在BIT诱导中的作用。54只大鼠椎动脉凝闭后分为5组: (1)假手术组(n=8): 游离双侧颈总动脉, 但不夹闭;  (2)单纯缺血组(n=8): 夹闭双侧颈总动脉8 min;  (3)缺血预处理组(n=8): 夹闭双侧颈总动脉3 min作为脑缺血预处理(CIP), 再灌注24 h后再行夹闭8 min;  (4) MTPG+缺血预处理组(n=22): CIP前20 min右侧脑室注射MTPG, 其余步骤同缺血预处理组;  MTPG的剂量分别为0.4、0.2、0.04和0.008 mg, 以观察其剂量效应关系;  (5) MTPG+单纯缺血组(n=8): 右侧脑室注射MTPG 0.2 mg 24 h后, 夹闭双侧颈总动脉8 min。所有动物均在手术后或末次缺血后7 d处死, 取材观察。结果如下: (1) 与假手术组相比, 单纯8 min缺血组海马CA1区组织学分级升高、锥体神经元密度降低, GFAP阳性表达增加(P<0.05); (2) 缺血预处理组的组织学分级、神经元密度及GFAP表达与假手术组相似, 未见单纯缺血组的上述变化, 表明CIP可防止后续8 min缺血造成的神经元损伤; (3) MTPG+缺血预处理组海马CA1区组织学分级明显增加、锥体神经元密度降低, 并且GFAP表达也明显增加(P<0.05), 这种变化与MTPG的剂量呈明显正相关, 表明CIP对神经元的保护作用可被MTPG阻断; (4) MTPG+单纯缺血组海马CA1区组织学分级和神经元密度以及GFAP的表达与单纯缺血组相似。上述结果提示,  3 min CIP 可诱导BIT的形成, MTPG可阻断CIP诱导BIT的作用, 表明mGluR 2/3参与BIT的诱导。

 

关键词:  脑预缺血处理;  代谢型谷氨酸受体;   α-甲基-(4-四唑基-苯)甘氨酸;  脑缺血耐受;  海马

中图分类号: Q426;  R338

 

Effects of α-methyl-(4-tetrazolyl-phenyl) glycine on the induction of hippocampal ischemic tolerance in the rat

FENG Rong-Fang, LI Wen-Bin*,  LIU Hui-Qing, LI Qing-Jun,  CHEN Xiao-Ling, ZHOU Ai-Min, ZHAO Hong-Gang,  AI Jie

Department of Pathophysiology, Institute of Basic Medicine, Hebei Medical University,  Shijiazhuang  050017

 

Abstract:  To explore the role of metabotropic glutamate receptor 2/3（mGluR 2/3）in the induction of brain ischemic tolerance (BIT), the influences of mGluR2/3 antagonist α-methyl-(4-tetrazolyl-phenyl) glycine (MTPG) on the induction of BIT and expression of glial fibrillary acidic protein (GFAP) in the hippocampus were observed using thionin staining and GFAP immunohistochemical staining in a rat brain ischemic model with four-vessel occlusion (4VO). Fifty-four rats, of which bilateral vertebral arteries were occluded permanently by electrocautery, were divided into 5 groups: (1) sham operated group (n=8): the bilateral carotid common arteries (BCCA) were separated, but the blood flow was not blocked; (2) ischemia group (n=8): the blood flow of BCCA was blocked for 8 min; (3) ischemic preconditioning (IP) group (n=8): the blood flow of BCCA was  occluded for 3 min as a cerebral ischemic preconditioning (CIP),   and then the rats were exposed to an 8-min brain ischemic insult 24 h after the CIP; (4) MTPG+IP group (n=22): MTPG was administered 20 min before the CIP, then the rats were exposed to an 8-min brain ischemia insult 24 h after the CIP . In order to examine  dosage dependency in the effect of MTPG, 4 dosages of MTPG (0.4, 0.2, 0.04 and 0.008 mg) were administered; (5) MTPG+ischemia group (n=8): an ischemic insult for 8 min was given 24 h after the administration of MTPG (0.2 mg). MTPG was injected into the right lateral cerebral ventricle. The results obtained are as follows.  (1) Ischemic insult for 8 min increased the histological grade (HG) and reduced the neuronal density (ND) significantly, and also  increased the expression of GFAP significantly (P<0.05 vs  sham-operated group). (2) In the IP group, the above changes were not observed, indicating that  CIP could protect pyramidal neurons against the ischemic insult. (3) The protective effects of  CIP were blocked by MTPG, as manifested by the significant increase in HG and decrease in ND in the MTPG+IP group (P<0.05 vs  sham-operated group). The changes were dose-dependent . (4) No obvious difference in the HG, ND and expression of GFAP was detected between the groups of MTPG+ischemia and ischemia. The above results indicate that MTPG blocks the induction of BIT induced by CIP, suggesting that mGluR2/3 participates in the induction of BIT.

 

Key words:  cerebral ischemic preconditioning; metabotropic glutamate receptors; α-methyl-(4-tetrazolyl-phenyl)glycine; brain ischemic tolerance; hippocampus

 

1990年Kitagawa等首先报道了脑缺血耐受现象 (brain ischemic tolerance, BIT)[1]。 已发现BIT的诱导涉及神经递质、 受体、 即刻早期基因及蛋白等众多因素。我室最近应用大鼠四血管闭塞(4 vessel occlusion, 4VO)模型研究发现腺苷受体的上调参与BIT的诱导[2]。兴奋性氨基酸受体是脑内广泛存在的受体, 可分为代谢型和离子型两大类。近年来, 这些受体在BIT诱导中的作用引起各国学者的关注。对离子型兴奋性氨基酸受体中的N-甲基-D-天冬氨酸(NMDA)受体作用的研究取得了较为肯定的结果, 发现NMDA受体拮抗剂对脑缺血有保护作用, 但是, 其在BIT模型中却能阻断CIP的保护作用, 表明NMDA受体参与了BIT的诱导[3,4]。关于代谢型谷氨酸受体(metabotropic glutamate receptors, mGluRs), 应用脑缺血损伤模型的研究表明, 抑制mGluR1/5和激活mGluR2/3可使缺血引起的梗塞灶体积及神经元死亡数目明显下降[5,6], 提示抑制mGluR1/5和兴奋mGluR2/3可保护脑细胞。但在CIP及BIT模型中, 免疫组化检测并未显示出mGluR2/3的表达有变化, mGluR1/5也仅在缺血耐受后有一过性短暂降低[7]。因此, mGluRs在BIT诱导中的作用远未被阐明。本实验观察了mGluR2/3阻断剂α-methyl-(4-tetrazolyl-phenyl)glycine (MTPG)对BIT模型中海马CA1区迟发性神经元死亡的变化, 以进一步探讨mGluR2/3在BIT诱导中的作用。此外, 鉴于mGluR2/3在星形胶质细胞上有大量分布[8,9], 应用MTPG对星形胶质细胞的活动是否有影响？这种影响与BIT的诱导是否有关？为明确这些问题, 本实验同时观察了MTPG对BIT过程中星形胶质细胞胶质纤维酸性蛋白(glial fibrillary acidic protein, GFAP)表达的变化。

 

1  材料和方法

1.1 动物模型及分组

实验用健康雄性SD大鼠(280－320 g)54只, 由河北医科大学实验动物中心提供。

1.1.1 椎动脉凝闭及硬膜外固定电极

在10%水合氯醛350 mg/kg腹腔麻醉下, 颈部背侧纵切口长约1.5 cm, 分离椎旁肌暴露第一颈椎横突;  在体视显微镜下寻找翼状孔, 插入电烙针凝闭双侧椎动脉。在颅骨钻孔, 硬膜外包埋、固定银制电极以供监测脑电。动物恢复2 d后用于下一步实验。

1.1.2 侧脑室注射

在10%水合氯醛350 mg/kg腹腔麻醉下, 将动物固定于脑立体定位仪上, 颅骨钻孔, 参照脑立体定位图谱, 将外径0.2 mm的不锈钢细管插入右侧脑室。细管外侧端连接50  μl微量进样器进行注射, 每次注射液体量为10  μl, 1 min内注完, 留针2 min。

1.1.3 全脑缺血

参照文献[10,11]进行。要点是在水合氯醛麻醉下, 游离双侧颈总动脉, 待动物清醒后夹闭双侧颈总动脉阻断血流。

术中及术后用白炽灯照射动物以维持其肛温在37℃左右, 直到恢复活动。4VO前后观察动物脑电及瞳孔变化, 用以判定脑缺血情况。

将大鼠随机分为以下5组。(1) 假手术组(sham operated, n=8): 游离双侧颈总动脉, 但不夹闭; (2) 单纯缺血组(ischemia, n=8): 夹闭双侧颈总动脉8 min; (3) 缺血预处理组( ischemic preconditioning, IP, n=8): 首先夹闭双侧颈总动脉3 min作为预缺血处理(CIP),  再灌流24 h后再行夹闭8 min; (4) MTPG+缺血预处理组(MTPG+IP, n=22): CIP前20 min右侧脑室注射MTPG, 其余步骤同缺血预处理组,  MTPG的剂量分别为0.4、0.2、0.04和0.008 mg,以观察其效应的剂量关系; (5) MTPG+单纯缺血组(MTPG+ischemia, n=8): 右侧脑室注射MTPG 0.2 mg后24 h夹闭双侧颈总动脉8 min。非给药组均右侧脑室注射等量生理盐水作为MTPG脑室注射的对照。

各组大鼠均在术后或末次缺血再灌后, 常温饲养7 d灌注取材, 行脑组织切片, 硫堇染色和GFAP免疫组化染色, 观察海马CA1区组织学改变及GFAP表达情况。

1.2 试剂

GFAP抗体和MTPG均购自Sigma公司。

1.3 硫堇染色

在戊巴比妥钠麻醉下, 开胸经升主动脉依次灌注下列溶液:  4℃生理盐水100 ml; 4℃ 4%多聚甲醛250 ml。取出大脑, 冠状切取视交叉后1至4 mm脑组织, 用4%多聚甲醛固定2－4 h, 常规脱水, 石蜡包埋。连续切片(片厚6 μm)、 展片、 脱蜡、 硫堇染色、 脱水、 封片。

1.4 GFAP免疫组化染色

灌注、取材、固定及切片同硫堇染色。10%的山羊血清37℃孵育30 min, 兔抗GFAP抗体(1∶200) 4℃过夜, 0.01 mol/L的PBS冲洗, 用生物素标记的羊抗兔的IgG 37℃孵育30 min, 0.01 mol/L的PBS冲洗, 用辣根过氧化物酶标记的链酶卵白素37℃孵育30 min, 冲洗、 DAB显色、 苏木精复染、 脱水封片。省略GFAP抗体的一组切片无阳性染色。

1.5 观察指标及方法

参照Kato分级方法[12], 在光学显微镜下对海马CA1区组织学改变进行分级, 标准如下: 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。取双侧平均值作为统计值。高倍镜下计数双侧海马CA1区1 mm长度区段内存活的锥体细胞数, 每侧计数三个区段, 取其平均数为神经元密度(neuronal density, ND)[5]。

在光学显微镜下观察大鼠海马CA1区GFAP阳性细胞的表达。通过彩色图文分析系统, 在40倍物镜下摄取CA1区图像传送至计算机显示屏, 计数视野内阳性细胞数目, 并测量阳性细胞截面总面积、平均光密度以反映免疫组化染色的程度。每个标本测量5个视野取其平均值。总面积、平均光密度数值越大, 表示GFAP表达越强。

1.6 数据处理及统计学检验

组织学分级采用多样本等级资料的秩和检验;  其它数据以mean±SD表示, 并采用ANOVA及Scheffe法进行统计学分析(stata软件4.0)。

 

2  结果

2.1 海马CA1区组织学分级及神经元密度

硫堇染色显示, 假手术组海马CA1区锥体细胞核大而圆, 核仁明显, 可见浅染的突起, 细胞排列较紧密整齐, 锥体细胞可见2－3层, 组织学分级为0－1级, 神经元密度为181±10.5 (图1A)。单纯缺血组海马CA1区锥体细胞稀疏, 有片状缺失, 有的细胞旁有空染区, 可见核固缩, 胞质浓染, 胞体变小的细胞;  细胞层次不清楚, 组织学分级为2－3级, 神经元密度为42±6.7(图1B), 与假手术组相比有显著差异(P<0.05)。缺血预处理组海马组织学特征与假手术组相似, 组织学分级为0－1级, 神经元密度为174±6.0, 与假手术组相比无统计学差别(图1C), 表明3 min CIP可防止后续8 min缺血造成的神经元损伤。MTPG+缺血预处理组各剂量组大鼠海马CA1区均出现明显损伤性变化, 表现为锥体细胞稀疏、松散, 层次不清, 有的细胞旁有空染区, 其中可见核固缩, 胞质浓染, 胞体变小的细胞(图1D);  组织学分级和神经元密度与假手术组相比均有明显差异(表1)(P<0.05)。该组海马CA1区的组织学分级和神经元密度的变化与MTPG的剂量呈现明显的相关性, 即剂量越大, 海马的组织学分级越高, 神经元密度越低(图2)。MTPG+单纯缺血组大鼠海马组织学特征与单纯缺血组相似(图1E), 组织学分级为2－3 级, 神经元密度为43±8.1, 与单纯缺血组相比无显著差异(P>0.05)(表1)。

2.2 海马CA1区GFAP表达的改变

GFAP免疫组化染色显示形态较为完整的星形胶质细胞, 具有大量放射状突起。假手术组海马CA1区仅见少量GFAP阳性细胞, 突起细小, 染色较淡(图3A, 表2)。单纯缺血组海马CA1区GFAP阳性细胞数目明显增多, 胞体肥大, 突起明显增粗, 染色加深, 与假手术组相比有显著性差异(P<0.05)(图3B, 表2)。缺血预处理组海马CA1区GFAP表达似有增多趋势, 但与假手术组相比无统计学差异(P>0.05)(图3C, 表2)。MTPG+缺血预处理组海马CA1区GFAP阳性细胞数目增多, 胞体肥大, 突起增粗, 染色加深, 尤以大剂量组明显(图3D, 0.2 mg组),    计数和测量海马CAI区GFAP阳性细胞数目、总面积、平均光密度, 与假手术组相比明显增加(表2)(P<0.05)。MTPG+ischemia组海马CA1区GFAP阳性细胞表达同单纯缺血组(图3E, 表2)。

图1.右侧脑室注射MTPG后海马CA1区的组织学分级和神经元密度的变化

Fig. 1.Representative photomicrographs of the hippocampal CA1 subfield showing the changes in histological grades (HG) and neuronal density (ND) of the CA1 hippocampus after administration of MTPG via right lateral cerebral ventricle. Compared with sham operated group, the HG in groups of ischemia, MTPG+IP, MTPG+ischemia is much high, and the ND is very low. The HG and ND in the IP group are similar to those in the sham operated group. A1 and A2: Sham operated group; B1 and B2: Ischemia group; C1 and C2: IP group; D1 and D2: MTPG+IP group (0.2 mg); E1 and E2: MTPG+ischemia group. Thionin staining, the left column×10, and the right column are magnified pictures(×100) of the square in the left column. Scale bar=439 μm in left column; and 28.3 μm in right column. HG: histological grade, ND: neuronal density.

图2.MTPG+IP 组右侧脑室注射MTPG的剂量与海马CA1区组织学分级的均值 (表1中频数与分级的乘积除以例数)和神经元密度的量效关系

Fig. 2.Dose-dependent relationship between the MTPG dosages and mean of histological grades(quotient obtained by dividing the product of the grade and its frequency by the number in table 1) and neuronal density of the hippocampal CA1 subfield in the MTPG+IP group after administration of MTPG via right lateral cerebral ventricle (abscissa is expressed with logarithm value). HG:histological grades, ND:neuronal density.

图3.右侧脑室注射MTPG后海马CA1区GFAP表达的改变

Fig. 3.Representative photomicrographs of immunohistochemical staining showing the changes in the expression of GFAP in the hippocampal CA1 subfield after administration of MTPG via right lateral cerebral ventricle. Compared with the sham operated group, the number of the GFAP positive cells and the staining density are increased significantly in the groups of ischemia, MTPG+IP and MTPG+ischemia, some hypertrophy in the soma and processes could be detacted in the groups. A: Sham operated group; B: Ischemia group; C: IP group; D: MTPG+IP group. E: MTPG+ischemia group. F: Ischemia group. The pictures of A－E (×100) were drew from the level showed in F (×10). Scale bar=43.9 μm in A－E and 439  μm in F.

 

表1. 右侧脑室注射MTPG后海马CA1区组织学分级和锥体神经元密度的改变

Table 1. Changes in the histological grades and pyramidal neuronal density in the hippocampal CA1 subfield after administration of MTPG via the right lateral cerebral ventricle. The standard of the grading was as follows: grade 0, no cell death; grade 1, scattered cell death; grade 2, mass cell death; grade 3, almost complete cell death

Group

n

Histological grade

Neuronal density(mean±SD)

0

1

2

3

Sham-operated

8

6

2

0

0

181±10.5

Ischemia

8

0

0

2

6*

42±6.7*

IP

8

5

3

0

0

174±6

MTPG+IP0.4 mg

4

0

0

3

1*

61±6.2*

0.2 mg

6

0

2

3

1*

86±15.4*

0.04 mg

6

0

4

2

0*

100±13.4*

0.008 mg

6

2

3

1

0

150±13*

MTPG+ischemia

8

0

0

2

6*

43±8.1*

*P<0.05 vs sham-operated group.

 

表2. 右侧脑室注射MTPG后海马CA1区GFAP阳性细胞数、阳性细胞总面积和光密度的改变

Table 2. Changes in number, total area and light density of GFAP immunoreactive cells in the hippocampal CA1 subfield after administration of MTPG via right lateral cerebral ventricle (mean±SD)

Group

n

Numbers of positive cells

Total area(μm2)

Light density

Sham-operated

8

9.2±0.5

2012±170.5

0.10±0.023

Ischemia

8

19.3±2.0*

3214±120.9*

0.16±0.025*

IP

8

12.0±0.7

2441±200.0

0.11±0.021

MTPG+IP 0.4 mg

4

16.9±1.1*

3076±210.1*

0.15±0.014*

0.2 mg

6

19.2±0.6*

3200±200.8*

0.16±0.022*

0.04 mg

6

19.5±1.1*

3250±220.7*

0.16±0.031*

0.008 mg

6

14.2±1.4*

2743±190.2*

0.13±0.027*

MTPG+ischemia

8

18.4±1.7*

3281±198.6*

0.16±0.021*

*P<0.05 vs  sham-operated group.

 

3讨论

mGluRs是G蛋白偶联的膜受体, 种类繁多, 根据其对第二信使的作用, 可将其分为三类: 第一类(包括mGluR1和mGluR5)可刺激磷酸肌醇水解,增加胞内三磷酸肌醇的含量, 而使细胞功能活跃;  第二类(包括mGluR2、mGluR3)和第三类则通过抑制腺苷酸环化酶, 减少胞内cAMP, 而使细胞功能抑制[7, 13]。

MTPG是目前合成的mGluR2/3的强效选择性拮抗剂。本实验中, 于CIP前给予不同剂量的MTPG, 观察其对BIT诱导的影响, 以探讨mGluR2/3在BIT中的作用。研究发现, 3 min CIP可对其后间隔1 d的8 min缺血造成的迟发性神经元损伤产生明显的保护作用。给予MTPG后, 这种保护作用减弱或消失, 表现为海马CA1区组织学分级升高, 神经元密度降低。本研究同时发现, 直接给予MTPG, 不进行CIP, 对8 min损伤性缺血引起的神经元损伤程度无影响, 据此可排除MTPG对8 min缺血损伤的直接作用。因此, 给予MTPG后, CIP保护作用的消失或减弱是MTPG直接阻断了CIP诱导的保护作用所致。鉴于MTPG是mGluR2/3强效选择性阻断剂, 因而这些结果表明mGluR2/3参与BIT的诱导。

mGluR2/3参与BIT的诱导的机制尚不清楚。研究发现, 离子型GluRs占据突触后膜的中心, 而mGluRs分布在突触前、后膜的外周。mGluRs通常不参与海马谷氨酸能神经元之间正常的兴奋传递, 只有当刺激引起突触间隙内谷氨酸浓度达到足够高水平时, 才可激活mGluRs[14]。根据海马锥体神经元上有大量mGluR2/3分布和突触活动增强可使谷氨酸释放明显增加的事实[15,16], 推测CIP是否作为应激刺激首先使胞外谷氨酸浓度增加, 增加的谷氨酸进一步激活mGluR2/3, 参与BIT的诱导。CIP也可能通过G-蛋白偶联的其它信使, 增加mGluR2/3的合成和/或降低其分解;  或改变mGluR2/3的活性, 使其与配体的亲和力增高来参与BIT的诱导。进一步观察CIP诱导BIT时, 脑组织中谷氨酸的浓度及mGluR2/3表达及功能的改变, 可对上述推测作出确切回答。

GFAP是反映星形胶质细胞活化状态的重要物质[9,17]。本实验观察到缺血预处理组与假手术组相比, GFAP表达阳性的星形胶质细胞数目呈增多趋势, 染色稍深, 提示星形胶质细胞可能参与BIT。Kato[17]等在沙鼠CIP模型中发现海马CA1、CA3及齿状回等区域可见明显的星形胶质细胞活化表达, 提示CIP后活化的星形胶质细胞参与BIT的诱导。国内学者也先后证实反应性星形胶质细胞的增生、活化在BIT诱导中可能起重要作用[18]。这些研究与我们的实验相符。研究发现星形胶质细胞的突起包围着谷氨酸能突触[8,9], 因此星形胶质细胞的活化状态对BIT的诱导可能有利[17]。单纯缺血组、MTPG+缺血预处理组和MTPG+缺血组星形胶质细胞数目则明显增多、浓染, 胞体明显增生、肥大, 且伴随着明显的锥体细胞缺失。由于活性星形胶质细胞也能释放毒性细胞因子[19], 参与吞噬、清除变性组织, 因此其过度激活也能造成神经元损伤。星形胶质细胞在缺血肿胀状态可抑制谷氨酸摄取, 同时增加其释放[20], 因此这种肥大、浓染的星形胶质细胞可能是对缺血造成的锥体细胞缺失的代偿反应, 对脑锥体细胞存活无保护作用。

我们的实验在整体水平证实, mGluR2/3参与大鼠海马CA1区锥体细胞BIT的诱导, 为阐明与完善BIT的诱导机制提供了实验依据。

 

致谢: 感谢李陈莉教授、 赵春芳和李英敏技师在技术方面给予的指导

 

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