大鼠局灶性脑缺血再灌注损伤的研究
摘要
脑缺血再灌注后第二信使Ca~(2+)、c-AMP等的变化激活细胞信号转导途径，最终使CREB磷酸化启动c-fos，Bcl-2，BDNF，NGF等基因的转录，参与神经元的再生、损伤后修复。而星形胶质细胞的激活支持和保护大脑免受脑缺血损伤。本实验通过MCAO大鼠模型的制作，观察脑缺血再灌注及药物干预后CREB的磷酸化与c-Fos蛋白以及GFAP的表达，结合神经功能损害评分，HE染色观察缺血后组织形态学变化，立体测量脑梗死体积，探讨脑缺血再灌注及脑保护剂的作用机理，探索尼莫地平对不同程度的局灶性脑缺血的保护作用及其治疗时间窗。实验共分为四个部分：
实验一：目的 为临床脑缺血研究提供合理实用的-动物模型。方法30只S-D雄性大鼠采用颈内动脉尼龙线线栓法MCAO造模，随机均分为5组：A.MCAO 60min组；B．MCAO 90min组；L．MCAO 120min组；D．NCAO 180min组；E．假手术组。再灌注后24h行脑功能障碍评分。再灌注72h 15只处死测量脑组织梗死体积，另15只行HE染色。结果 HE染色示A至D组缺血半暗区神经元损伤及再灌注24h后神经功能障碍依次加重：脑梗死体积A至D组依次增大，而B与C缺血组无统计学差界。结论 大鼠MCAO 90min为较佳脑缺血模型。
第四军医大学硕士学位论文
--........月翩确脚口..甲甲....偏甲，.口.叫禅.
实验二:目的研究c一FoS和GFAP在FCI再灌注中的表达，尼莫地平脑
保护作用的时机。方法采用线栓法MeAo gom in加BeeAL 6omin造模，18
只S一D大鼠随机等分为6组:对照组，缺血再灌注(卜R)组，预防组，缺血
治疗组，I一R治疗组，再灌注后治疗组。尼莫地平颈内动脉给药，再灌注后
24h脑功能障碍评分，再灌注72h处死测量脑组织梗死体积，并行HE染色，
c一Fos，GFAP免疫组化染色。另选18只动物，I一R组9只和卜R治疗组9只
各再分为再灌注后Zh(AI组)，24h(AZ组)，48h(A3组)，行HE染色，e一Fos，
GFAP免疫组化染色。结果HE染色示预防组，卜R治疗组，再灌注后治疗组
较之卜R组缺血半暗区神经元损伤明显减轻。再灌24h后神经功能障碍评分
为:缺血治疗组妻卜R组>再灌注后治疗组>预防组>I一R治疗组>对照组，
治疗组中除缺血治疗组外均较卜R组梗死体积小，但较对照组大，差异有统
计学意义。治疗组皮质缺血_半暗带，海马cAI区GFAP阳性细胞数均较I一R
组少(p<0.05)，但齿状回减少程度较轻:给药后皮质缺血半暗带以及海马
各区C一FoS阳性细胞数明显减少.G队p在再灌注2h大量表达，48h达高峰，
且反应性星型胶质细胞居多，72h持续表达:c一FoSZh反应最为强烈，24h
仍大量表达，48h后明显下降，72h少呈表达。结论预防性及再灌注早期尼
莫地平动脉给药对FCI再灌注治疗效果较好;反应性胶质细胞对脑缺血后神
经元存活起着重要作用;C一fos基因参与了脑缺血损伤的信号转导。
实验三:目的研究尼莫地平对不同程度的FCI的保护作用及其治疗时
间窗，pCREB，C一FoS在大鼠脑缺血再灌注中的表达和脑保护剂对其影响。方
法24只S一D雄性大鼠采用右侧颈内动脉尼龙线线栓法MCAO gomin造模，缺
血组和治疗组均各分四组:A.MCAO加单侧颈总动脉结扎6Omin组;B.MCAO
力[}BCCAI二30min组;C.MCA()加BCCAI二60m in组;D.MCA()加BCCAL 90min组。
尼莫地平颈内动脉给药，再灌注后24h行脑功能障碍评分，再灌注72h处死
测量脑组织梗死体积，行HE染色，pcREI飞，C一1:05免疫组化染色。另选6只
第四军医大学硕士学位论文
~.-.....，.........耳翩叫娜.....叫.口甲..圈口........................口...............
动物，均分为A缺血组和A治疗组，再灌注后2h(A1组)，24h(A2组)，48h(A3
组)处死，行HE染色，pCREB，c一Fos免疫组化染色。结果HE染色示尼莫地
平干预后缺血半暗区神经元损伤明显减轻。再灌注24h后A至D组神经功能
障碍依次加重，治疗组中相应A，B，C组别均较缺血相应组别梗死体积小，
而D缺血组与治疗组差异无统计学意义。治疗组A，B，C组大鼠缺血半暗带
边缘区pCREB表达均较相应缺血组别强;c一FoS表达与给药前比较减弱
(p(0.05)。A缺血组半暗区pCREB再灌注Zh大量表达，24h达高峰，72h
后开始下降:c一Fos Zh最高，24h仍高，72h后明显下降。结论早期尼莫地
平动脉给药对轻度，中度脑缺血再灌注损伤治疗效果较好:CREB磷酸化后在
脑缺血损伤中的神经元存活起着重要作用。
实验四:目的研究尼莫地平、胞磷胆碱单用或二药合用对Fcl再灌注
的保护作用。方法16只S一D雄性大鼠随机均分为缺血对照组(A组)，尼莫
地平组(B组)，胞磷胆碱组(C组)，合并用药组(D组)。线栓法MCAO gomin
加BCCAI6Omin造模，尼莫地平颈内动脉给药，再灌注后24h行脑功能障碍
评分，再灌注72h处死测量脑?
BACKGROUD: After cerebral ischemia/reperfusion,the changes of second second messenger systems including Ca2+, c-AMP in neurons activate signal transductions of cells, make CREB phosphorylated and start up gene transcriptions of c-fos,Bcl-2,BDNF,NGF, which take part in neurons regeneration and repair following injury.The activation of astrocytes help ischemic brain repair and prevent from injury.To discuss mechanism of cerebral ischemia/reperfusion and effect of neuroprotective medications, to study the neuroprotection and therapeutic time windows of nimodipine for different degrees focal cerebral ischemia,and to establish experiment base for application of nimodipine clinically, MCAO rats models were made , immunohistochemical expressions of CREB phosphorylation. c-Fos and GFAP were observed between cerebral ischemia-reperfusion rats and medication intervation ones,combining with neurologic deficit score,HE staining for examining morphologic changes after ischemia and brain infarct volumes stereotically meas
ured.The experiments were totally divide into four parts as following:
EXPERIMENT 1: AIM To provide reasonable and practicable animal
models for clinical cerebral ischemia research.METHODS Middle Cerebral Artery Occlusion (MCAO) were carried out on 30 male S-D rats by suture occlusion via intra artery methods.The animals were divided into 5 groups randomly and equally (n=6):group A with 60-min MCAO, group B with 90-min MCAO,group C with 120-min MCAO, group D with 180-min MCAO, group D with sham operation. The neurological outcomes were evaluated at 24 h after the reperfusion.After 72h of reperfusion the brain infarct volumes were measured and HE staining performed respectively on 15 rats. RESULTS HE staining showed penumbra neuron damages aggravated in turns from A to D.Neurological dysfuctions and brain infarct volumes gradually increased from A to D,but there was no significance in brain infarct volume between group B and C. CONCLUSION 90-min-MCAO rats were perfect cerebral ischemia models.
EXPERIMENT 2: AIM To investigate c-Fos and GFAP expression following focal cerebral ischemia/reperfusion, To study the suitable neuro-protective time of nimodipine for FCI. METHODS MCAO model was carried out for 90 min by suture occlusion with 60-min BCCAL. 18 male S-D rats were allocated to 6 groups randomly and equally (n=3):control group, ischemia/ reperfusion (I-R) group, preventive group, ischemia treatment group, I-R treatment group and post-reperfusion treatment group.Nimodipine was injected by an intracarotid route. The neurological outcomes and the brain infarct volumes were evaluated respectively at 24h and 72h after the reperfusion. HE staining, c-Fos and GFAP immonohistochemical staining were performed simultaneously. Another 18 same models were equally divided into two groups ,I-R and I-R treatment groups ,each group with 3 subdivions. At 2h (groupAl), 24h (groupA2) and 48h (groupA3) after reperfusion, rats (n=3) were killed and above stainings
were performed. RESULTS HE staining showed penumbra neuron damages alleviated in preventive group,I-R treatment one and post-reperfusion treatment one compared with those in I-R group. Neurological dysfuctions took turns as following after 24 h of reperfusion: ischemia treatment group I-R group> post-reperfusion treatment group > prevention group > I-R treatment group > control group.Infarct volume of each treatment group except that of ischemia treatment one notably decreased (p<0.05) compared with that of I-R group, while increased compared with that of control group. There were significantly differences among those volumes. After therapies, GFAP positive cells in cortex penumbra and CA1 region more reduced than those of I-R group (p<0.05) ,but there were gently reductions of those cells in dentate gyrus. C-Fos expressions of same regions had significant decrease after treatment. Following reperfusion, GFAP clearly expressed at 2h,peaked at 48h and persisted at 72h,while c-Fos reactived strongest at 2h, keep increased till 24h and obviously decreased a
实验一：目的 为临床脑缺血研究提供合理实用的-动物模型。方法30只S-D雄性大鼠采用颈内动脉尼龙线线栓法MCAO造模，随机均分为5组：A.MCAO 60min组；B．MCAO 90min组；L．MCAO 120min组；D．NCAO 180min组；E．假手术组。再灌注后24h行脑功能障碍评分。再灌注72h 15只处死测量脑组织梗死体积，另15只行HE染色。结果 HE染色示A至D组缺血半暗区神经元损伤及再灌注24h后神经功能障碍依次加重：脑梗死体积A至D组依次增大，而B与C缺血组无统计学差界。结论 大鼠MCAO 90min为较佳脑缺血模型。
第四军医大学硕士学位论文
--........月翩确脚口..甲甲....偏甲，.口.叫禅.
实验二:目的研究c一FoS和GFAP在FCI再灌注中的表达，尼莫地平脑
保护作用的时机。方法采用线栓法MeAo gom in加BeeAL 6omin造模，18
只S一D大鼠随机等分为6组:对照组，缺血再灌注(卜R)组，预防组，缺血
治疗组，I一R治疗组，再灌注后治疗组。尼莫地平颈内动脉给药，再灌注后
24h脑功能障碍评分，再灌注72h处死测量脑组织梗死体积，并行HE染色，
c一Fos，GFAP免疫组化染色。另选18只动物，I一R组9只和卜R治疗组9只
各再分为再灌注后Zh(AI组)，24h(AZ组)，48h(A3组)，行HE染色，e一Fos，
GFAP免疫组化染色。结果HE染色示预防组，卜R治疗组，再灌注后治疗组
较之卜R组缺血半暗区神经元损伤明显减轻。再灌24h后神经功能障碍评分
为:缺血治疗组妻卜R组>再灌注后治疗组>预防组>I一R治疗组>对照组，
治疗组中除缺血治疗组外均较卜R组梗死体积小，但较对照组大，差异有统
计学意义。治疗组皮质缺血_半暗带，海马cAI区GFAP阳性细胞数均较I一R
组少(p<0.05)，但齿状回减少程度较轻:给药后皮质缺血半暗带以及海马
各区C一FoS阳性细胞数明显减少.G队p在再灌注2h大量表达，48h达高峰，
且反应性星型胶质细胞居多，72h持续表达:c一FoSZh反应最为强烈，24h
仍大量表达，48h后明显下降，72h少呈表达。结论预防性及再灌注早期尼
莫地平动脉给药对FCI再灌注治疗效果较好;反应性胶质细胞对脑缺血后神
经元存活起着重要作用;C一fos基因参与了脑缺血损伤的信号转导。
实验三:目的研究尼莫地平对不同程度的FCI的保护作用及其治疗时
间窗，pCREB，C一FoS在大鼠脑缺血再灌注中的表达和脑保护剂对其影响。方
法24只S一D雄性大鼠采用右侧颈内动脉尼龙线线栓法MCAO gomin造模，缺
血组和治疗组均各分四组:A.MCAO加单侧颈总动脉结扎6Omin组;B.MCAO
力[}BCCAI二30min组;C.MCA()加BCCAI二60m in组;D.MCA()加BCCAL 90min组。
尼莫地平颈内动脉给药，再灌注后24h行脑功能障碍评分，再灌注72h处死
测量脑组织梗死体积，行HE染色，pcREI飞，C一1:05免疫组化染色。另选6只
第四军医大学硕士学位论文
~.-.....，.........耳翩叫娜.....叫.口甲..圈口........................口...............
动物，均分为A缺血组和A治疗组，再灌注后2h(A1组)，24h(A2组)，48h(A3
组)处死，行HE染色，pCREB，c一Fos免疫组化染色。结果HE染色示尼莫地
平干预后缺血半暗区神经元损伤明显减轻。再灌注24h后A至D组神经功能
障碍依次加重，治疗组中相应A，B，C组别均较缺血相应组别梗死体积小，
而D缺血组与治疗组差异无统计学意义。治疗组A，B，C组大鼠缺血半暗带
边缘区pCREB表达均较相应缺血组别强;c一FoS表达与给药前比较减弱
(p(0.05)。A缺血组半暗区pCREB再灌注Zh大量表达，24h达高峰，72h
后开始下降:c一Fos Zh最高，24h仍高，72h后明显下降。结论早期尼莫地
平动脉给药对轻度，中度脑缺血再灌注损伤治疗效果较好:CREB磷酸化后在
脑缺血损伤中的神经元存活起着重要作用。
实验四:目的研究尼莫地平、胞磷胆碱单用或二药合用对Fcl再灌注
的保护作用。方法16只S一D雄性大鼠随机均分为缺血对照组(A组)，尼莫
地平组(B组)，胞磷胆碱组(C组)，合并用药组(D组)。线栓法MCAO gomin
加BCCAI6Omin造模，尼莫地平颈内动脉给药，再灌注后24h行脑功能障碍
评分，再灌注72h处死测量脑?
BACKGROUD: After cerebral ischemia/reperfusion,the changes of second second messenger systems including Ca2+, c-AMP in neurons activate signal transductions of cells, make CREB phosphorylated and start up gene transcriptions of c-fos,Bcl-2,BDNF,NGF, which take part in neurons regeneration and repair following injury.The activation of astrocytes help ischemic brain repair and prevent from injury.To discuss mechanism of cerebral ischemia/reperfusion and effect of neuroprotective medications, to study the neuroprotection and therapeutic time windows of nimodipine for different degrees focal cerebral ischemia,and to establish experiment base for application of nimodipine clinically, MCAO rats models were made , immunohistochemical expressions of CREB phosphorylation. c-Fos and GFAP were observed between cerebral ischemia-reperfusion rats and medication intervation ones,combining with neurologic deficit score,HE staining for examining morphologic changes after ischemia and brain infarct volumes stereotically meas
ured.The experiments were totally divide into four parts as following:
EXPERIMENT 1: AIM To provide reasonable and practicable animal
models for clinical cerebral ischemia research.METHODS Middle Cerebral Artery Occlusion (MCAO) were carried out on 30 male S-D rats by suture occlusion via intra artery methods.The animals were divided into 5 groups randomly and equally (n=6):group A with 60-min MCAO, group B with 90-min MCAO,group C with 120-min MCAO, group D with 180-min MCAO, group D with sham operation. The neurological outcomes were evaluated at 24 h after the reperfusion.After 72h of reperfusion the brain infarct volumes were measured and HE staining performed respectively on 15 rats. RESULTS HE staining showed penumbra neuron damages aggravated in turns from A to D.Neurological dysfuctions and brain infarct volumes gradually increased from A to D,but there was no significance in brain infarct volume between group B and C. CONCLUSION 90-min-MCAO rats were perfect cerebral ischemia models.
EXPERIMENT 2: AIM To investigate c-Fos and GFAP expression following focal cerebral ischemia/reperfusion, To study the suitable neuro-protective time of nimodipine for FCI. METHODS MCAO model was carried out for 90 min by suture occlusion with 60-min BCCAL. 18 male S-D rats were allocated to 6 groups randomly and equally (n=3):control group, ischemia/ reperfusion (I-R) group, preventive group, ischemia treatment group, I-R treatment group and post-reperfusion treatment group.Nimodipine was injected by an intracarotid route. The neurological outcomes and the brain infarct volumes were evaluated respectively at 24h and 72h after the reperfusion. HE staining, c-Fos and GFAP immonohistochemical staining were performed simultaneously. Another 18 same models were equally divided into two groups ,I-R and I-R treatment groups ,each group with 3 subdivions. At 2h (groupAl), 24h (groupA2) and 48h (groupA3) after reperfusion, rats (n=3) were killed and above stainings
were performed. RESULTS HE staining showed penumbra neuron damages alleviated in preventive group,I-R treatment one and post-reperfusion treatment one compared with those in I-R group. Neurological dysfuctions took turns as following after 24 h of reperfusion: ischemia treatment group I-R group> post-reperfusion treatment group > prevention group > I-R treatment group > control group.Infarct volume of each treatment group except that of ischemia treatment one notably decreased (p<0.05) compared with that of I-R group, while increased compared with that of control group. There were significantly differences among those volumes. After therapies, GFAP positive cells in cortex penumbra and CA1 region more reduced than those of I-R group (p<0.05) ,but there were gently reductions of those cells in dentate gyrus. C-Fos expressions of same regions had significant decrease after treatment. Following reperfusion, GFAP clearly expressed at 2h,peaked at 48h and persisted at 72h,while c-Fos reactived strongest at 2h, keep increased till 24h and obviously decreased a
引文
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Neuropharmacology, 1997;36(1): 107-113.
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