眼针对大鼠局灶性脑缺血再灌注损伤保护作用及凋亡机制研究
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摘要
目的及背景:缺血性脑血管疾病治疗的关键在于挽救缺血半暗区濒临死亡的神经元和促进损伤后神经功能的恢复。脑缺血再灌注损伤是指脑组织经一定时间的缺血后恢复血流供应,使缺血性损伤进一步加重的病理现象。脑缺血损伤的机制十分复杂。脑缺血再灌注损伤缺血半暗区神经元死亡的主要形式是细胞凋亡。眼针疗法治疗中风疗效肯定。积累了丰富的临床经验,但作用机制研究尚不深入,本课题从抑制细胞凋亡及调控细胞凋亡死亡受体通路信号传导探讨眼针对缺血再灌注损伤的保护作用机制,揭示眼针治疗缺血性中风部分作用机制,更好为临床治疗中风病提供科学依据。
方法:分组方法:将健康雄性SD大鼠240只随机分为假手术组66只,模型组和眼针组各87只。每组又分成脑缺血再灌3h, 24h,72h等时间点。
模型制作方法:参照Longa改良的线栓方法制作大鼠局灶性脑缺血再灌注模型。
试验方法:眼针组取穴:上焦区、下焦区、肝区、肾区。刺法:用35x25mm毫针在相应眼穴区距眶内缘2mm处,平刺,由该区始点向该区终点方向,刺入0.3cm,行捻转手法,留针30分钟,留针15分钟时行针一次,持续时间1分钟。针刺时机:再灌注即刻对动物神经功能缺损评分,随机分到模型组与眼针组,眼针组各时间点在缺血再灌注即刻及处死动物前针刺,动物存活期间每12小时针刺一次。假手术组及模型组不做任何处理,常规喂养。
检测方法及指标:参照Longa 5分制评分标准评价神经功能缺损症状评分;采用TTC染色测量梗死体积;光镜及电镜观察缺血半暗带脑组织病理改变及超微结构;应用TUNEL法观察缺血半暗区脑组织神经元凋亡;采用免疫组化法检测缺血半暗区脑组织Fax,Faxl表达;采用RT一PCR法检测缺血半暗区脑组织caspase-3,caspase-8 mRNA蛋白表达;采用Elisa方法检测缺血半暗区脑组织及血清肿瘤坏死因子-α(TNF-α)含量。
数据统计方法:所有实验数据以均值土标准差( x土s)表示。所得数据用SPSS 10.0软件进行分析,组间比较采用单因素方差分析。
结果
1.眼针对神经功能缺损症状评分的影响
假手术组大鼠手术完成后,无神经缺损体征出现。眼针组、模型组再灌注3h出现明显神经功能缺损。3h时间点,眼针组与模型组两组间比较无显著差异; 24h、72h时间点眼针组与模型组两组间比较有极显著差异。
2、眼针对脑梗死体积的影响
再灌注24h,假手术组未发现有梗死病灶,模型组和眼针组梗死病灶主要位于右侧额、颞、顶叶皮质及尾状核、壳核区域。眼针组梗死体积显著小于模型组。
3、眼针对缺血半暗带区神经元病理改变及超微结构的影响
眼针治疗组与模型组比较,脑组织形态有明显改善。肿胀神经元少见,细胞边界清晰,核仁明显。
电镜观察:眼针组半暗区神经元细胞可见较多线粒体、粗面内质网、核糖体;与模型组比较明显改善,与假手术组比较有轻微改变。神经胶质细胞较假手术组比较可见少量高尔基体、粗面内质网、线粒体、核糖体,胶质细胞基本正常。毛细血管内皮光滑,基本正常。
4、眼针对缺血半暗区神经元细胞凋亡的影响
模型组及眼针组凋亡细胞于脑缺血再灌注后3h开始增多,模型组、眼针组各时间点凋亡细胞与假手术组相比均有显著性差异;眼针组与模型组比较,3h时间点,无显著性差异,24h、72h时间点凋亡细胞数明显减少,有显著性差异。
5、眼针对缺血半暗区神经元Fax Faxl表达的影响
免疫组化染色显示:模型组、眼针组阳性目标表达均于脑缺血再灌注后3h开始增加,各时间点模型组、眼针组与假手术组比较阳性细胞表达均有显著性差异;眼针组与模型组比较,3h时间点阳性目标表达无显著性差异;24h、72h时间点阳性目标表达显著减少,有显著性差异。
6、眼针对缺血半暗带区Caspase-3, Caspase-8 mRNA表达的影响
RT-PCR结果显示各时间点假手术组Caspase-3, Caspase-8 mRNA仅见微弱表达;各时间点模型组、眼针组与假手术组比较Caspase-3,Caspase-8 mRNA表达有显著差异;眼针组与模型组比较,3小时无显著性差异;24小时、72小时有非常显著性差异。
7、眼针对缺血半暗区脑组织及血液肿瘤坏死因子(TNF-α)含量的影响
缺血半暗带区皮层脑组织中TNF-α含量,各时间点模型组、眼针组与假手术组比含量有非常显著性差异。3小时时间点眼针组与模型组比较,无显著性差异;24小时时间点有非常显著性差异;72小时有显著性差异。血液中TNF-α含量与脑组织中的含量趋势基本相同。
结论
1.改良线拴法制作脑缺血再灌注大鼠模型组出现神经功能缺损症状及脑梗死病灶,证明模型成功。
2.中风病的病位在脑,与肝肾关系密切;眼针可改善脑缺血再灌注大鼠神经功能缺损.提示眼针可醒脑开窍,调脏腑虚实、通经络阻滞,因此提出:“眼络于脑,通调脏腑”假说。
3.眼针可降低脑缺血再灌注大鼠神经功能缺损症状评分,缩小脑梗死体积,改善缺血半暗带区神经元受损程度。
4.脑缺血再灌注大鼠模型缺血半暗区细胞凋亡严重,眼针可抑制缺血半暗带区细胞凋亡。
5.脑缺血再灌注大鼠模型缺血半暗区Fax,Faxl表达明显增高;眼针可下调缺血半暗带区Fax,Faxl表达。
6.脑缺血再灌注大鼠模型缺血半暗区Caspase-3, Caspase-8 mRNA表达明显增高;眼针可下调缺血半暗带区Caspase-3, Caspase-8 mRNA表达。
7.脑缺血再灌注大鼠模型缺血半暗区脑组织及血液中肿瘤坏死因子(TNF-α)含量明显升高,眼针可使其表达下调。
8.本课题揭示了眼针治疗缺血性脑血管病可能是通过抑制凋亡信号传导,从而抑制细胞凋亡,挽救缺血半暗区,从而对脑缺血再灌注损伤起保护作用。
Objective and background:
The key to the treatment of Ischemic Cerebrovascular Disease is to rescue the dying neurons in ischemic penumbra and promote the recovery of injured nerve function. Cerebral ischemia-reperfusion injury is the pathological phenomenon that after a short time of ischemia, the brain tissue restores the blood supply while to make the ischemic injury more serious. The mechanism of cerebral ischemic injury is much complicated. The death of the neurons in ischemic penumbra of cerebral ischemia-reperfusion injury takes the apoptosis as the main form for dying. Eye-acupuncture therapy is effective to cure stroke. During that, a lot of experiences are accumulated. But the function mechanism is still in study. The subject is to probe the protective mechanism of eye-acupuncture in the treatment of cerebral ischemia-reperfusion injury by restricting neuron apoptosis and regulating the death receptor signal path of neuron apoptosis. At the same time, find the function mechanism of eye-acupuncture treatment to Ischemic Stroke, in order to provide scientific evidence for the clinical experience.
Method:
Group division: Divided the healthy male SD rats into 3 groups, that is, the sham-operated group, model group and eye-acupuncture group. Each group was divided into groups with different test period like 3h, 24h, or 72h etc. for the cerebral ischemia-reperfusion.
Model Making: Make the model rat of focal cerebral ischemia-reperfusion according to the improved method of Longa.
Experimental method: The points searching of eye-acupuncture group: Shangjiao area, Xiajiao area, liver area, kidney area . Method of acupuncturing: Acupuncture the point area levelly with 2mm distance from the internal orbital margin with the 0.25mmx40mm tiny needles, from starting to the ending in the point area. Until 0.3cm into the area, change to take the twisting practices for 30 minutes. During that time, the needle should be practiced for 1 minute every 15 minutes. Acupuncturing timing: Give the assessment to neurological function deficits at the starting moment of reperfusion. Give the assessment to neurological function deficits at the starting moment of reperfusion. The rats are divided into every group at random. The test hours for acupuncture should be chosen at the moment of reperfusion and before death. Give the acupuncture every 12 hours in the period of living. The rats of sham-operated and model group are fed as usual without any treatment.
Method of testing and indicators:Make the scores of neurological function deficits according to Longa 5-score standards; adopt the TTC staining to measure the infarct volume; observe the pathological changes and the ultra-structure of the brain tissue in ischemic penumbra by micro-spectroscope and electron microscope; detect neuronal apoptosis of the brain tissue in ischemic penumbra by the TUNEL method; test the expression of Fax/Faxl of the brain tissue in ischemic penumbra by immunohistochemistry; test the protein expression of caspase-3mRNA and caspase-8mRNA by RT-PCR of the brain tissue in ischemic penumbra by RT-PCR methods; test the content of TNF-αin ischemic penumbra of the brain tissue and in serum by using the Elisa method.
Method of data statistic: All the data, in the expression form of±SD, is analyzed by spss10.0 software. And the group comparison adopts the single factor variance analysis.
Results:
1.The effect of eye-acupuncture on neurological function deficits
No neurological function deficits appear after the operation in sham-operated group. At the point of 3h is chemia-reperfusion in model group and eye-acupuncture group,The symptom of neurological function deficits is the most serious one in the 24h and 72h ischemia-reperfusion in model group, so the difference is remarkable between eye-acupuncture group and model group.
2. The effect of eye-acupuncture on Infarct volumes
No infarct lesion is found in sham-operated group after 24h following ischemia-reperfusion. The infarct lesion in model and eye-acupuncture group lies in the right part of forehead, temporal, parietal cortex, caudate nucleus and Putamen region. The Infarct volume in eye-acupuncture group is obviously less than that in model group.
3.The effect of eye-acupuncture on the pathological changes and the ultra-structure of the neurons in ischemic penumbra
The model group shows the typical infarct changes. The necrotic foci can be seen clearly in the parietal part of the ischemic side, the lateral striatum and the prefrontal. Fewer cells exist in the necrotic foci.
Observation by micro-spectroscope:There are rich organelles in neuron cytoplasm in penumbra of the sham-operated group. The nucleus is big and elliptic, with the clear and smooth structure of two membranes. The chromatin in the nuclear has uniform distribution.
4.The effect of eye-acupuncture on neuron apoptosis in ischemia penumbra
Apoptotic cells of model group and eye-acupuncture group just begin to increase after 3h following ischemia-reperfusion. It shows no statistically significant difference at 3h in eye-acupuncture group, compared with the model group. The number of apoptotic cells is obviously less at 24h and 72h in eye-acupuncture group, compared with the model group. There is significant difference by statistics .
5. The effect of eye-acupuncture on the expression of Fax/Faxl of the neurons in ischemicpenumbra
The model group, eye needle group masculine goal expression After Nao ischemia pours into again 3h to start to increase, various time model group, the eye needle group and the sham-operation group compare the masculine cell expression to have the significance difference; The eye needle group and the model group compare, a 3h time masculine goal expresses the non-significance difference; 24h, the 72h time masculine goal expression remarkable reduction, has the significance difference
6.The effect of eye-acupuncture on the expression of caspase-3mRNA caspase-8mRNA in penumbra
Various time model group, the eye needle group and the sham-operation group compare Caspase-3, Caspase-8 the mRNA expression to have the remarkable difference; The eye needle group and the model group compare, 3 hour non-significance difference; 24 hour, 72 hours have the unusual significance difference.
7. The effect of eye-acupuncture on TNF-αof blood and brain tissue in penumbra
Various time model group, the eye needle group and the sham-operation group compare Caspase-3, Caspase-8 the mRNA expression to have the remarkable difference; The eye needle group and the model group compare, 3 hour non-significance difference; 24 hour, 72 hours have the unusual significance difference.
Conclusion:
1. When making the model rat of cerebral ischemia-reperfusion with the improved line-tying method, it appears the symptom of neurological deficit and the cerebral infarction lesion. It proves that the model making is successful.
2. Stroke disease lies in the brain and has close relationship with the liver and kidney. Eye- acupuncture can improve the scores of neurological function deficit of the cerebral ischemia-reperfusion rats and many molecular biology dictators. It proves that eye-acupuncture can regulate the actual situation of viscera and clear the block of the meridian. Thus, we have the belief that“The eye connects with the brain with the meridian and regulates the viscera.”
3. Eye- acupuncture can reduce the scores of neurological function deficit of the cerebral ischemia-reperfusion rats, decrease the infarct volume, improve the injury of impaired neuron in penumbra and at the same time protect the impaired brain caused by ischemia-reperfusion.
4. Apoptosis cells have serious condition in penumbra of the cerebral ischemia-reperfusion rats in model group. It shows that can exist in the cerebral ischemia-reperfusion injury, while eye- acupuncture can restrict the apoptosis in the penumbra to protect the impaired brain caused by ischemia-reperfusion.
5. The expression of Fax and Faxl in the ischemic penumbra of the cerebral ischemia-reperfusion rats in model group increases obviously, which proves that the high expression of the promoted apoptosis signals exist in the cerebral ischemia-reperfusion injury. Eye- acupuncture may decrease the expression of Fax and Faxl in the ischemic penumbra to restrict the conduct of the apoptosis signals, so that it can protect the impaired brain caused by ischemia-reperfusion.
6. The expression of Caspase-3 mRNA and Caspase-8mRNA in the ischemic penumbra of the cerebral ischemia-reperfusion rats in model group increases remarkably, which proves that the high expression of the promoted apoptosis signals exist in the cerebral ischemia-reperfusion injury. Eye- acupuncture may decrease the expression of Caspase-3 mRNA and Caspase-8mRNA in the ischemic penumbra to restrict the conduct of the apoptosis signals, so that it can protect the impaired brain caused by ischemia-reperfusion.
7. The content of TNF-αof blood and brain tissue in the ischemic penumbra of the cerebral ischemia-reperfusion rats in model group increases remarkably, which proves that the content of TNF-αexisting in the cerebral ischemia-reperfusion injury increase obviously. Eye-acupuncture can decrease the expression. Eye- acupuncture may decrease the content of TNF-αof blood and brain tissue in the ischemic penumbra of the cerebral ischemia-reperfusion to restrict the apoptosis and the inflammatory response, so that it can protect the impaired brain caused by ischemia-reperfusion.
8. The research reveals the partial mechanism of eye-acupuncture in treating ischemia brain vascular disease. Also it offers the molecular biology evidence for the clinical treatment of the brain vascular disease by eye-acupuncture.
方法:分组方法:将健康雄性SD大鼠240只随机分为假手术组66只,模型组和眼针组各87只。每组又分成脑缺血再灌3h, 24h,72h等时间点。
模型制作方法:参照Longa改良的线栓方法制作大鼠局灶性脑缺血再灌注模型。
试验方法:眼针组取穴:上焦区、下焦区、肝区、肾区。刺法:用35x25mm毫针在相应眼穴区距眶内缘2mm处,平刺,由该区始点向该区终点方向,刺入0.3cm,行捻转手法,留针30分钟,留针15分钟时行针一次,持续时间1分钟。针刺时机:再灌注即刻对动物神经功能缺损评分,随机分到模型组与眼针组,眼针组各时间点在缺血再灌注即刻及处死动物前针刺,动物存活期间每12小时针刺一次。假手术组及模型组不做任何处理,常规喂养。
检测方法及指标:参照Longa 5分制评分标准评价神经功能缺损症状评分;采用TTC染色测量梗死体积;光镜及电镜观察缺血半暗带脑组织病理改变及超微结构;应用TUNEL法观察缺血半暗区脑组织神经元凋亡;采用免疫组化法检测缺血半暗区脑组织Fax,Faxl表达;采用RT一PCR法检测缺血半暗区脑组织caspase-3,caspase-8 mRNA蛋白表达;采用Elisa方法检测缺血半暗区脑组织及血清肿瘤坏死因子-α(TNF-α)含量。
数据统计方法:所有实验数据以均值土标准差( x土s)表示。所得数据用SPSS 10.0软件进行分析,组间比较采用单因素方差分析。
结果
1.眼针对神经功能缺损症状评分的影响
假手术组大鼠手术完成后,无神经缺损体征出现。眼针组、模型组再灌注3h出现明显神经功能缺损。3h时间点,眼针组与模型组两组间比较无显著差异; 24h、72h时间点眼针组与模型组两组间比较有极显著差异。
2、眼针对脑梗死体积的影响
再灌注24h,假手术组未发现有梗死病灶,模型组和眼针组梗死病灶主要位于右侧额、颞、顶叶皮质及尾状核、壳核区域。眼针组梗死体积显著小于模型组。
3、眼针对缺血半暗带区神经元病理改变及超微结构的影响
眼针治疗组与模型组比较,脑组织形态有明显改善。肿胀神经元少见,细胞边界清晰,核仁明显。
电镜观察:眼针组半暗区神经元细胞可见较多线粒体、粗面内质网、核糖体;与模型组比较明显改善,与假手术组比较有轻微改变。神经胶质细胞较假手术组比较可见少量高尔基体、粗面内质网、线粒体、核糖体,胶质细胞基本正常。毛细血管内皮光滑,基本正常。
4、眼针对缺血半暗区神经元细胞凋亡的影响
模型组及眼针组凋亡细胞于脑缺血再灌注后3h开始增多,模型组、眼针组各时间点凋亡细胞与假手术组相比均有显著性差异;眼针组与模型组比较,3h时间点,无显著性差异,24h、72h时间点凋亡细胞数明显减少,有显著性差异。
5、眼针对缺血半暗区神经元Fax Faxl表达的影响
免疫组化染色显示:模型组、眼针组阳性目标表达均于脑缺血再灌注后3h开始增加,各时间点模型组、眼针组与假手术组比较阳性细胞表达均有显著性差异;眼针组与模型组比较,3h时间点阳性目标表达无显著性差异;24h、72h时间点阳性目标表达显著减少,有显著性差异。
6、眼针对缺血半暗带区Caspase-3, Caspase-8 mRNA表达的影响
RT-PCR结果显示各时间点假手术组Caspase-3, Caspase-8 mRNA仅见微弱表达;各时间点模型组、眼针组与假手术组比较Caspase-3,Caspase-8 mRNA表达有显著差异;眼针组与模型组比较,3小时无显著性差异;24小时、72小时有非常显著性差异。
7、眼针对缺血半暗区脑组织及血液肿瘤坏死因子(TNF-α)含量的影响
缺血半暗带区皮层脑组织中TNF-α含量,各时间点模型组、眼针组与假手术组比含量有非常显著性差异。3小时时间点眼针组与模型组比较,无显著性差异;24小时时间点有非常显著性差异;72小时有显著性差异。血液中TNF-α含量与脑组织中的含量趋势基本相同。
结论
1.改良线拴法制作脑缺血再灌注大鼠模型组出现神经功能缺损症状及脑梗死病灶,证明模型成功。
2.中风病的病位在脑,与肝肾关系密切;眼针可改善脑缺血再灌注大鼠神经功能缺损.提示眼针可醒脑开窍,调脏腑虚实、通经络阻滞,因此提出:“眼络于脑,通调脏腑”假说。
3.眼针可降低脑缺血再灌注大鼠神经功能缺损症状评分,缩小脑梗死体积,改善缺血半暗带区神经元受损程度。
4.脑缺血再灌注大鼠模型缺血半暗区细胞凋亡严重,眼针可抑制缺血半暗带区细胞凋亡。
5.脑缺血再灌注大鼠模型缺血半暗区Fax,Faxl表达明显增高;眼针可下调缺血半暗带区Fax,Faxl表达。
6.脑缺血再灌注大鼠模型缺血半暗区Caspase-3, Caspase-8 mRNA表达明显增高;眼针可下调缺血半暗带区Caspase-3, Caspase-8 mRNA表达。
7.脑缺血再灌注大鼠模型缺血半暗区脑组织及血液中肿瘤坏死因子(TNF-α)含量明显升高,眼针可使其表达下调。
8.本课题揭示了眼针治疗缺血性脑血管病可能是通过抑制凋亡信号传导,从而抑制细胞凋亡,挽救缺血半暗区,从而对脑缺血再灌注损伤起保护作用。
Objective and background:
The key to the treatment of Ischemic Cerebrovascular Disease is to rescue the dying neurons in ischemic penumbra and promote the recovery of injured nerve function. Cerebral ischemia-reperfusion injury is the pathological phenomenon that after a short time of ischemia, the brain tissue restores the blood supply while to make the ischemic injury more serious. The mechanism of cerebral ischemic injury is much complicated. The death of the neurons in ischemic penumbra of cerebral ischemia-reperfusion injury takes the apoptosis as the main form for dying. Eye-acupuncture therapy is effective to cure stroke. During that, a lot of experiences are accumulated. But the function mechanism is still in study. The subject is to probe the protective mechanism of eye-acupuncture in the treatment of cerebral ischemia-reperfusion injury by restricting neuron apoptosis and regulating the death receptor signal path of neuron apoptosis. At the same time, find the function mechanism of eye-acupuncture treatment to Ischemic Stroke, in order to provide scientific evidence for the clinical experience.
Method:
Group division: Divided the healthy male SD rats into 3 groups, that is, the sham-operated group, model group and eye-acupuncture group. Each group was divided into groups with different test period like 3h, 24h, or 72h etc. for the cerebral ischemia-reperfusion.
Model Making: Make the model rat of focal cerebral ischemia-reperfusion according to the improved method of Longa.
Experimental method: The points searching of eye-acupuncture group: Shangjiao area, Xiajiao area, liver area, kidney area . Method of acupuncturing: Acupuncture the point area levelly with 2mm distance from the internal orbital margin with the 0.25mmx40mm tiny needles, from starting to the ending in the point area. Until 0.3cm into the area, change to take the twisting practices for 30 minutes. During that time, the needle should be practiced for 1 minute every 15 minutes. Acupuncturing timing: Give the assessment to neurological function deficits at the starting moment of reperfusion. Give the assessment to neurological function deficits at the starting moment of reperfusion. The rats are divided into every group at random. The test hours for acupuncture should be chosen at the moment of reperfusion and before death. Give the acupuncture every 12 hours in the period of living. The rats of sham-operated and model group are fed as usual without any treatment.
Method of testing and indicators:Make the scores of neurological function deficits according to Longa 5-score standards; adopt the TTC staining to measure the infarct volume; observe the pathological changes and the ultra-structure of the brain tissue in ischemic penumbra by micro-spectroscope and electron microscope; detect neuronal apoptosis of the brain tissue in ischemic penumbra by the TUNEL method; test the expression of Fax/Faxl of the brain tissue in ischemic penumbra by immunohistochemistry; test the protein expression of caspase-3mRNA and caspase-8mRNA by RT-PCR of the brain tissue in ischemic penumbra by RT-PCR methods; test the content of TNF-αin ischemic penumbra of the brain tissue and in serum by using the Elisa method.
Method of data statistic: All the data, in the expression form of±SD, is analyzed by spss10.0 software. And the group comparison adopts the single factor variance analysis.
Results:
1.The effect of eye-acupuncture on neurological function deficits
No neurological function deficits appear after the operation in sham-operated group. At the point of 3h is chemia-reperfusion in model group and eye-acupuncture group,The symptom of neurological function deficits is the most serious one in the 24h and 72h ischemia-reperfusion in model group, so the difference is remarkable between eye-acupuncture group and model group.
2. The effect of eye-acupuncture on Infarct volumes
No infarct lesion is found in sham-operated group after 24h following ischemia-reperfusion. The infarct lesion in model and eye-acupuncture group lies in the right part of forehead, temporal, parietal cortex, caudate nucleus and Putamen region. The Infarct volume in eye-acupuncture group is obviously less than that in model group.
3.The effect of eye-acupuncture on the pathological changes and the ultra-structure of the neurons in ischemic penumbra
The model group shows the typical infarct changes. The necrotic foci can be seen clearly in the parietal part of the ischemic side, the lateral striatum and the prefrontal. Fewer cells exist in the necrotic foci.
Observation by micro-spectroscope:There are rich organelles in neuron cytoplasm in penumbra of the sham-operated group. The nucleus is big and elliptic, with the clear and smooth structure of two membranes. The chromatin in the nuclear has uniform distribution.
4.The effect of eye-acupuncture on neuron apoptosis in ischemia penumbra
Apoptotic cells of model group and eye-acupuncture group just begin to increase after 3h following ischemia-reperfusion. It shows no statistically significant difference at 3h in eye-acupuncture group, compared with the model group. The number of apoptotic cells is obviously less at 24h and 72h in eye-acupuncture group, compared with the model group. There is significant difference by statistics .
5. The effect of eye-acupuncture on the expression of Fax/Faxl of the neurons in ischemicpenumbra
The model group, eye needle group masculine goal expression After Nao ischemia pours into again 3h to start to increase, various time model group, the eye needle group and the sham-operation group compare the masculine cell expression to have the significance difference; The eye needle group and the model group compare, a 3h time masculine goal expresses the non-significance difference; 24h, the 72h time masculine goal expression remarkable reduction, has the significance difference
6.The effect of eye-acupuncture on the expression of caspase-3mRNA caspase-8mRNA in penumbra
Various time model group, the eye needle group and the sham-operation group compare Caspase-3, Caspase-8 the mRNA expression to have the remarkable difference; The eye needle group and the model group compare, 3 hour non-significance difference; 24 hour, 72 hours have the unusual significance difference.
7. The effect of eye-acupuncture on TNF-αof blood and brain tissue in penumbra
Various time model group, the eye needle group and the sham-operation group compare Caspase-3, Caspase-8 the mRNA expression to have the remarkable difference; The eye needle group and the model group compare, 3 hour non-significance difference; 24 hour, 72 hours have the unusual significance difference.
Conclusion:
1. When making the model rat of cerebral ischemia-reperfusion with the improved line-tying method, it appears the symptom of neurological deficit and the cerebral infarction lesion. It proves that the model making is successful.
2. Stroke disease lies in the brain and has close relationship with the liver and kidney. Eye- acupuncture can improve the scores of neurological function deficit of the cerebral ischemia-reperfusion rats and many molecular biology dictators. It proves that eye-acupuncture can regulate the actual situation of viscera and clear the block of the meridian. Thus, we have the belief that“The eye connects with the brain with the meridian and regulates the viscera.”
3. Eye- acupuncture can reduce the scores of neurological function deficit of the cerebral ischemia-reperfusion rats, decrease the infarct volume, improve the injury of impaired neuron in penumbra and at the same time protect the impaired brain caused by ischemia-reperfusion.
4. Apoptosis cells have serious condition in penumbra of the cerebral ischemia-reperfusion rats in model group. It shows that can exist in the cerebral ischemia-reperfusion injury, while eye- acupuncture can restrict the apoptosis in the penumbra to protect the impaired brain caused by ischemia-reperfusion.
5. The expression of Fax and Faxl in the ischemic penumbra of the cerebral ischemia-reperfusion rats in model group increases obviously, which proves that the high expression of the promoted apoptosis signals exist in the cerebral ischemia-reperfusion injury. Eye- acupuncture may decrease the expression of Fax and Faxl in the ischemic penumbra to restrict the conduct of the apoptosis signals, so that it can protect the impaired brain caused by ischemia-reperfusion.
6. The expression of Caspase-3 mRNA and Caspase-8mRNA in the ischemic penumbra of the cerebral ischemia-reperfusion rats in model group increases remarkably, which proves that the high expression of the promoted apoptosis signals exist in the cerebral ischemia-reperfusion injury. Eye- acupuncture may decrease the expression of Caspase-3 mRNA and Caspase-8mRNA in the ischemic penumbra to restrict the conduct of the apoptosis signals, so that it can protect the impaired brain caused by ischemia-reperfusion.
7. The content of TNF-αof blood and brain tissue in the ischemic penumbra of the cerebral ischemia-reperfusion rats in model group increases remarkably, which proves that the content of TNF-αexisting in the cerebral ischemia-reperfusion injury increase obviously. Eye-acupuncture can decrease the expression. Eye- acupuncture may decrease the content of TNF-αof blood and brain tissue in the ischemic penumbra of the cerebral ischemia-reperfusion to restrict the apoptosis and the inflammatory response, so that it can protect the impaired brain caused by ischemia-reperfusion.
8. The research reveals the partial mechanism of eye-acupuncture in treating ischemia brain vascular disease. Also it offers the molecular biology evidence for the clinical treatment of the brain vascular disease by eye-acupuncture.
引文
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