脑出血灶周与脑梗死半暗带细胞凋亡、Bcl-2、细胞骨架、COX-2的对比研究
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摘要
本实验选取相同条件下的同种系的清洁级大鼠,在完全相同的实验条件下、相同的实验试剂进行实验,首次以脑梗死做对照,观察脑梗死缺血半暗带标志物在急性脑出血后出血灶周的变化,探讨脑出血灶周是否存在类似脑梗死缺血半暗带的区域。本实验用TUNEL法及免疫组化方法观察比较脑出血侧组和脑梗死侧组各时间点凋亡细胞、Bcl-2、MAP-2、GFAP、COX-2的阳性表达。结果显示出血灶与正常脑组织间有一过渡带,此区改变与梗死半暗带病理改变类似。脑出血灶周Bcl-2在3 h可见阳性表达,72h内持续升高,出血侧组与梗死侧组峰值比较无统计学差异。凋亡细胞分布区域与Bcl-2阳性表达类似。MAP-2和GFAP阳性表达在脑梗死半暗带区和脑出血灶周曲线图表达趋势基本一致。脑梗死缺血半暗带区和脑出血灶周MAP-2表达均较正常组明显降低,且形态异常,随时间延长MAP-2数量减少,变形加重。脑梗死后3h缺血半暗带区和脑出血后3h出血灶周GFAP数量增多,随着时间的延长GFAP阳性细胞均持续增加。COX-2阳性细胞也主要在脑梗死缺血半暗带区和脑出血灶周表达,并呈动态变化,二者曲线图均呈现先升高后降低趋势。三组实验结果表明脑出血灶周与脑梗死半暗带区细胞骨架变化和组织修复反应基本相同,72h内出血灶周与脑梗死半暗带区细胞死亡方式和程度及炎症反应的程度相同。据此可说明脑出血急性期出血灶周与梗死缺血半暗带存在相似的、动态的可逆病理变化过程,且持续时间较梗死长,本实验结果提示可达72h。本实验结果为临床治疗脑出血提供了新思路,具有重要的临床价值和社会价值。目前还未见相似的研究报道。
Intracerebral hemorrhage (ICH) account for 12-15 percent of cerebral stroke. Its mutilation rate, fatility is very high, and the ICH patients become younger and younger. It is different from cerebral ischemia, because its therapy don′t have obviously breakthrough in recent years. Clinic random comparision experimrnts have proved that clearing hemorrhage in the head counldn′t improve patients prognosis, and 1/3 patients still got worse after operation.This phenomnon hint there is secondary lesion around hemorrhage besides hemorrhage direct damage. Whether there is ischemic penumbra around hemorrhage which is similar with ischemic penumbra (IP) of cerebral infarction become a hot spot in recent years.
The conception of IP was mentioned in cerebral ischemia. It was ischemic cerebral tissue around infarction central. Its electric activity stopped, but keep ionic balance and structure completely. Its function can be reversed. Now we decide IP mainly by imageology, including SPECT, PET, function MRI and Xe-CT, etc. But experiment discovered the domain was considered as IP by imageology whin 1 hour, but its function could be reversed before 4 hours by pathology. It told us that imageology and pathology did not sychronized. So, in infarction research, someone used pathology methods to decide IP, especially suggested cystoskeleton light changes could be a marker of IP. We chose apoptosis cell, Bcl-2, MAP-2, GFAP and COX-2 as IP markers. Research had already confirmed cells mainly apptosis in IP, Bcl-2 and COX-2 became more in IP, astrocytes were activated and cystoskeleton lightly changed in IP.
90 rats were divided into 5 groups: nomal group, hemorrhage and contral hemorrhage group, ischemic and contral ischemic group .The last 4 groups were divided into 3h、6h、12h、24h、48h、72h subgroups. Self blood was injected in rats′head to make cerebral hemorrhage models, and used Longa method to block MCA to make infarction models. TUNEL method was used to detect apoptosis cells. Mimunohistochemical methods were performed to examine the expression of MAP-2, GFAP, COX-2 in rat brain. Comparing the quantity of MAP-2, GFAP, COX-2 between hemorrhage and contral hemorrhage group, ischemic and contral ischemic group hemorrhage and ischemic group.
There was a transitional zone between hematoma and normal tissue. This zone was similar with IP in pathology.They both had neuron necrosis and degenerated, astrocytes and gitter cell activated, cell and tissue edematous, and inflamation cells infiltrated.As time went on, these changes aggravated.In ischemic group, Bcl-2 sexual cells mainly located in IP, they raised up from 3h, reached peak at 24h, and then decreased. In hemorrhage group, Bcl-2 sexual cells mainly located around hemorrhage, they raised up from 3h, kept increasing until 72h. The peak numerical of two groups had no difference in statistics. The apoptosis cells mainly located in IP and aound hemorrhage. MAP-2 and GFAP curve chart had the same expressing tendency. In ischemic group and hemorrhage group, MAP-2 both decreased and its shape disfigurated. As time went on, MAP-2 disfigurated seriously and quantity decreased. In ischemic group and hemorrhage group, from 3 hours GFAP-expressing cells increased. As time went on, astrocyte swollened and its quantity increased. So as tissue damage around IP and hemorrhage became seriously (MAP-2 decreased), astrocyte absorbing and clearing necrotic tissue ability enhanced (GFAP increased). COX-2 sexual cells mainly located in IP and perihemorrhage, and two curve chart had the same expressing tendency.
The experiments got conclusion as follow: 1 In acute hemorrhage time, perihemorrhage area and IP had the similar, dynamic pathology process. They both had neuron necrosis and degenerated, astrocytes and gitter cell activated, cell and tissue edematous, and inflamation cells infiltrated. As time went on, these changes aggravated. 2 Both perihemorrhage area and IP had Bcl-2 sexual cells and apoptosis cells. The peak numerical of two groups had no difference in statistics. It illustrate IP and perihemorrhage area had similar cell dead style, that is apoptosis .3 MAP-2 curve chart had the same express tendency in ischemic group and hemorrhage group As time went on, MAP-2 disfigurated seriously and quantity decreased. In ischemic group and hemorrhage group,both GFAP-expressing cells increased. So tissue damage perihemorrhage is the same with IP, as time went on, tissue necrosis became seriously, repairment enhanced.4 COX-2 sexual cells mainly located in IP and para-hemorrhage, and two curve chart had the same express tendency.Inflamation plays an important part in both perihemorrhage and IP.5 Perihemorrhage area and IP had similar pathology change, And this time persisted longer than IP exist time. According to this research, hemorrhage penumbra could exist within 72 hours. The precisely time need further research.
This research first used cerebral ischemia as comparision, investgated IP marker (apoptosis cell, Bcl-2, MAP-2, GFAP and COX-2) expressing regular pattern. Connection with past imagology research conclusion that there were cerebral blood flow decreased in perihemorrhage area, we concluded that there was penumbra similar with cerebral infarction. This conclusion provided new thoery for hemorrhage therapy, and its significance was very important.
Intracerebral hemorrhage (ICH) account for 12-15 percent of cerebral stroke. Its mutilation rate, fatility is very high, and the ICH patients become younger and younger. It is different from cerebral ischemia, because its therapy don′t have obviously breakthrough in recent years. Clinic random comparision experimrnts have proved that clearing hemorrhage in the head counldn′t improve patients prognosis, and 1/3 patients still got worse after operation.This phenomnon hint there is secondary lesion around hemorrhage besides hemorrhage direct damage. Whether there is ischemic penumbra around hemorrhage which is similar with ischemic penumbra (IP) of cerebral infarction become a hot spot in recent years.
The conception of IP was mentioned in cerebral ischemia. It was ischemic cerebral tissue around infarction central. Its electric activity stopped, but keep ionic balance and structure completely. Its function can be reversed. Now we decide IP mainly by imageology, including SPECT, PET, function MRI and Xe-CT, etc. But experiment discovered the domain was considered as IP by imageology whin 1 hour, but its function could be reversed before 4 hours by pathology. It told us that imageology and pathology did not sychronized. So, in infarction research, someone used pathology methods to decide IP, especially suggested cystoskeleton light changes could be a marker of IP. We chose apoptosis cell, Bcl-2, MAP-2, GFAP and COX-2 as IP markers. Research had already confirmed cells mainly apptosis in IP, Bcl-2 and COX-2 became more in IP, astrocytes were activated and cystoskeleton lightly changed in IP.
90 rats were divided into 5 groups: nomal group, hemorrhage and contral hemorrhage group, ischemic and contral ischemic group .The last 4 groups were divided into 3h、6h、12h、24h、48h、72h subgroups. Self blood was injected in rats′head to make cerebral hemorrhage models, and used Longa method to block MCA to make infarction models. TUNEL method was used to detect apoptosis cells. Mimunohistochemical methods were performed to examine the expression of MAP-2, GFAP, COX-2 in rat brain. Comparing the quantity of MAP-2, GFAP, COX-2 between hemorrhage and contral hemorrhage group, ischemic and contral ischemic group hemorrhage and ischemic group.
There was a transitional zone between hematoma and normal tissue. This zone was similar with IP in pathology.They both had neuron necrosis and degenerated, astrocytes and gitter cell activated, cell and tissue edematous, and inflamation cells infiltrated.As time went on, these changes aggravated.In ischemic group, Bcl-2 sexual cells mainly located in IP, they raised up from 3h, reached peak at 24h, and then decreased. In hemorrhage group, Bcl-2 sexual cells mainly located around hemorrhage, they raised up from 3h, kept increasing until 72h. The peak numerical of two groups had no difference in statistics. The apoptosis cells mainly located in IP and aound hemorrhage. MAP-2 and GFAP curve chart had the same expressing tendency. In ischemic group and hemorrhage group, MAP-2 both decreased and its shape disfigurated. As time went on, MAP-2 disfigurated seriously and quantity decreased. In ischemic group and hemorrhage group, from 3 hours GFAP-expressing cells increased. As time went on, astrocyte swollened and its quantity increased. So as tissue damage around IP and hemorrhage became seriously (MAP-2 decreased), astrocyte absorbing and clearing necrotic tissue ability enhanced (GFAP increased). COX-2 sexual cells mainly located in IP and perihemorrhage, and two curve chart had the same expressing tendency.
The experiments got conclusion as follow: 1 In acute hemorrhage time, perihemorrhage area and IP had the similar, dynamic pathology process. They both had neuron necrosis and degenerated, astrocytes and gitter cell activated, cell and tissue edematous, and inflamation cells infiltrated. As time went on, these changes aggravated. 2 Both perihemorrhage area and IP had Bcl-2 sexual cells and apoptosis cells. The peak numerical of two groups had no difference in statistics. It illustrate IP and perihemorrhage area had similar cell dead style, that is apoptosis .3 MAP-2 curve chart had the same express tendency in ischemic group and hemorrhage group As time went on, MAP-2 disfigurated seriously and quantity decreased. In ischemic group and hemorrhage group,both GFAP-expressing cells increased. So tissue damage perihemorrhage is the same with IP, as time went on, tissue necrosis became seriously, repairment enhanced.4 COX-2 sexual cells mainly located in IP and para-hemorrhage, and two curve chart had the same express tendency.Inflamation plays an important part in both perihemorrhage and IP.5 Perihemorrhage area and IP had similar pathology change, And this time persisted longer than IP exist time. According to this research, hemorrhage penumbra could exist within 72 hours. The precisely time need further research.
This research first used cerebral ischemia as comparision, investgated IP marker (apoptosis cell, Bcl-2, MAP-2, GFAP and COX-2) expressing regular pattern. Connection with past imagology research conclusion that there were cerebral blood flow decreased in perihemorrhage area, we concluded that there was penumbra similar with cerebral infarction. This conclusion provided new thoery for hemorrhage therapy, and its significance was very important.
引文
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