丁基苯酞对慢性脑缺血老龄大鼠的VEGF和HO-1的影响
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摘要
近年来随着脑血管疾病发病率和死亡率的不断上升，人们对它的研究日益重视，但人们的研究多集中在急性脑缺血上，对慢性脑缺血的研究很少。随着人口的老龄化，痴呆困扰着越来越多的老年人，带来了严重的社会、经济和和家庭问题，目前对慢性脑缺血的研究变得尤其重要。慢性脑缺血是各类痴呆的共同病理表现，常伴发于血管性痴呆、阿尔茨海默病、Binswanger病和动静脉畸形等多种脑血管疾病的病理过程中，由于其发病的时间长，脑的损伤和修复过程交织在一起，使损伤的机制更为复杂，发病早期主要以认知功能损害为主要表现，最终可导致持久或进展性的认知和神经功能损害。研究表明，及早恢复血流供应是改善脑组织损伤的关键，人们已发现慢性脑缺血后脑组织血管密度增高与病人预后改善有关，因此研究促进血管生长的因素对慢性脑缺血患者的恢复有非常重要的意义。
血管内皮生长因子(vascular endothelial growth factor，VEGF)是一种高度特异的血管内皮细胞有丝分裂原和血管源性因子，可特异性地作用与血管内皮细胞，诱导内皮细胞增殖及毛细血管瓣生成，促进新生血管生成，对生理性血管生成和病理性血管再生都有重要的调节作用，VEGF还具有增加血管的通透性以及神经营养和神经保护作用，组织缺血、缺氧是诱导VEGF表达的强刺激因素，慢性脑缺血时VEGF表达增多对慢性脑缺血后的神经细胞恢复起重要作用。内源性一氧化碳(carbon monoxide，CO)可激活鸟苷酸环化酶，调节环磷酸鸟苷(CAMP)的生成，是一种具有许多生理功能的细胞信使，近年来发现CO有与一氧化氮(nitrogenmonoxidum，NO)相类似的内皮依赖性血管舒张作用，而且具有增加VEGF的合成能力。血红素氧化酶(hemeoxygenase，HO)是CO产生的关键酶，在HO的作用下，血红素氧化分解为螯合铁、CO和胆绿素；HO-1是诱导型HO，可被氧应激、缺血、金属、热休克等应激诱导表达。以前人们的兴趣集中在HO-1对血红素的降解能力上，最新研究发现HO-1具有抗氧化、抗炎、抑制血小板凝集、细胞保护、调节血管张力等许多重要生物学作用，HO／CO系统近年已逐渐成为研究的热点。丁基苯酞(dl-butyphthalide，NBP)是从芹菜籽中提取出来的，后经人工合成为消旋体，为治疗脑血管病的一种新药，在急性脑缺血的研究中，它可改善小鼠全脑缺血脑能量代谢，可以增加局部脑缺血大鼠缺血区的脑血流，能缩小大鼠大脑中动脉结扎后(MCAO)梗塞的面积和改善神经功能缺失，能改善MCAO后大鼠记忆功能障碍，但在慢性脑缺血中有关NBP的报道却很少。本文主要通过建立大鼠慢性脑缺血模型，运用免疫组化技术和计算机图像分析技术观察丁基苯酞对VEGF和HO-1在慢性脑缺血大鼠海马和皮层表达的影响，进一步探讨丁基苯酞对慢性脑缺血大鼠血管再通、再生及对脑细胞的保护作用。
材料与方法
1．实验动物与分组
健康wistar大鼠80只，雌雄不拘，12～14月龄，体重450～550g，由河南省郑州大学实验动物中心提供，随机分为4组，每组20只，分为A组：对照组(假手术+溶剂)；B组：单纯缺血组(手术+溶剂)；C组：缺血低剂量治疗组(手术+低剂量NBP+溶剂)；D组：缺血高剂量治疗组(手术+高剂量NBP+溶剂)。B、C、D组参照ohta等永久性结扎双侧颈总动脉制作慢性脑缺血模型，对照组除不结扎双侧颈总动脉外，其他处理同模型组。C组和D组造模2月后每日分别按60mg／kg、120mg／kg给予丁基苯酞灌胃，丁基苯酞用花生油稀释至2ml；A组和B组每日仅给予花生油2ml；各组皆连续灌胃一个月。模型成功标准：大鼠在继续饲养过程中，行动活泼，正常吃食饮水，眼部无分泌物，毛色光洁，体重增加。凡2VO 3个月后死亡的大鼠，不记在内。
2．模型的制作
将各组大鼠以10％的水合氯醛(0.3ml／100g)腹腔注射麻醉，作颈部正中切口，分离双侧颈总动脉，双重丝线结扎其近心端及远心端，间断缝合皮肤，放回原条件下饲养。对照组只分离颈总动脉但不结扎，其他处理同其余各组。
3．标本的制作
术后3个月，以10％的水合氯醛腹腔注射麻醉各组大鼠，经心脏灌注后，断头取左脑，放入4％的多聚甲醛中固定，固定好的脑组织经梯度酒精脱水，二甲苯透明、浸蜡、包埋，制作脑冠状切片，以备HE染色和免疫组化染色。
4．观察内容
(1)采用HE染色观察各组大鼠皮层和海马的神经元细胞变化。
(2)免疫组化观察各组大鼠皮层和海马的VEGF和HO-1的表达。
5．统计方法
数据以(?)±s表示，采用spss12.0软件进行数据处理，多组间比较采用单因素方差分析，用LSD法进行两两比较，检验水准α=0.05。
结果
1．实验动物行为观察
各组大鼠在手术前行为正常，术后各组大鼠均出现精神萎靡，反应迟钝，神情呆滞，少食，少饮。术后1天，除A组精神恢复外，其余各组行为改善不明显。3～5天B、C、D大鼠逐渐恢复，一周时基本正常，眼睛明亮，无分泌物，行动灵活，吃食、饮水情况良好。经Morris水迷宫实验证实，造模成功。
2．HE染色
A组：皮质和海马偶有神经元细胞变性、死亡，大部分细胞形态正常。B组：皮质和海马大量神经元细胞缺血性固缩、退变、死亡，正常神经细胞数较A组明显减少。C组：皮质和海马神经元变性、死亡较B组减轻，正常神经细胞数较B组明显增多。D组：皮质和海马神经元变性、死亡较C组明显减轻，正常神经细胞数较C组明显增多。
3．VEGF免疫组化染色
各组取皮层和海马进行观察，并统计VEGF蛋白表达的阳性细胞数，VEGF蛋白阳性表达主要见于皮质和海马的神经元、神经胶质细胞、血管内皮细胞，胞浆呈棕黄色。A组VEGF蛋白呈少量表达，B组VEGF蛋白表达明显多于A组，差异具有统计学意义(α＜0.05)，C组和D组VEGF蛋白表达均明显多于B组，差异具有统计学意义(α＜0.05)，且D组VEGF蛋白表达明显多于C组，差异具有统计学意义(α＜0.05)。
4．HO-1免疫组化染色
各组取皮层和海马进行观察并统计HO-1蛋白表达的阳性细胞数，HO-1蛋白阳性表达主要见于皮质和海马的神经元、神经胶质细胞，胞浆呈棕黄色。A组HO-1蛋白呈少量表达，B组HO-1蛋白表达明显多于A组，差异具有统计学意义(α＜0.05)，C组和D组HO-1蛋白表达均明显多于B组，差异具有统计学意义(α＜0.05)，且D组HO-1蛋白表达明显多于C组，差异具有统计学意义(α＜0.05)。
结论
1．永久性结扎大鼠双侧颈总动脉(2VO)模型是一种比较成熟的普遍认可的慢性脑缺血动物模型，实验操作简便，重复性好。
2．大鼠慢性脑缺血3个月后，皮层和海马神经元细胞大量变性、死亡，血管内皮生长因子和血红素氧化酶—1表达少量增多。
3．丁基苯酞(NBP)可减轻皮层和海马神经元变性、死亡，同时可增加血管内皮生长因子和血红素氧化酶—1在慢性脑缺血大鼠皮层和海马的表达。
Background
Because the morbidity and mortality of cerebrovascular is rising continually lately, humans attach more importance to study cerebrovascular. But many studies have focused on acute ischemic brain impairments is insufficiency to chronic cerebral hypoperfusion.But, with the ratio of senile increasing in country, More and more people get into trouble with dementia, which brings social economic and family problem. Now,the study of the chronic cerebral ischemia becomes especially importance.Chronic ischemic brain impairment is a common pathological state ,it complicated in many diseases such as Vascular dementia ,Alzheimer disease ,Binswanger disease ,Arterivenous malformation and so on .Because it is chronic ,the impairment and repairment is more complicated.In the early stages,its cardinal manifestation is cognitive disorder.At last,it can cause eternal or progressive impairment of cognitive and neurological. It is criticality for improving brain impairment to recover bloodstream provision early. Studies have proved that the increasing of blood vessel density after chronic cerebral hypoperfusion is related to amendent and prognosis of patient.So it is very important for the recovery of patient to study the factor of vascular growth .
vascular endothlial growth factor(VEGF) is a very specific factor to promote vascular growth. It plays an important regulating effection in physiology vascular generation an pathological vascular generation.it can promote vascular regeneration,increase vascular permeability and protect the neuron. The ischemia and hypoxia of tissue is a stimulant factor for the expression of VEGF,The VEGF plays an imoortant role in the cellur recovery after chronic cerebral ischemia. The endogenous carbon monoxide(CO)can activate guanylate cyclase,regulate the cyclic guanosine,it is a cell messenger which have a great deal of biological functions .Recently ,many studies discover that carbon monoxide(CO) have the effect of endothelial dependence vasodilation to be similar to nitrogen monoxidum (NO),and have the capability of promoting composition of VEGF.Hemeoxygenase(HO) is the key enzyme to creat CO.Till now ,people have discovered three kinds of isoenzyme of H0:H0-l is type induction,HO-2 and HO-3 are type architecture.HO-1 is also called heart shock protein(HSP) and it can be inducted by multiple stress,for example,Heme hypoxia ischemia and injurment. Many studies focused on the its capability of degredating to heme in previous years,the latest study have found that HO-1 have many biological functions in antioxygen antiinflammatory inhibiting platelet aggregation cytoprotection accommodating angiotasis and so on. Recently,the study of the HO/CO system has become a hot topic. Dl-butyphthalide(NBP) was isolated from the seeds of of celery,then was synthesized racemic artificially.NBP is a new drug to cure cerebrovascular disease .In the studyof acute brain ischemic ,NBP was shown to improve brain energy metabolism in mice with complete cerebral ischemic ,and to enhance the rCBF of rats with regional ischemic,and to decrease cerebral infarct volume of rat with MCAO, and to protect rats from ischemic neurological damage,and to improve impairment of learning and memory.But there is very few reports about NBPin the chronic cerebral ischemic. Studies hare proved that chromic cerebral ischemia which is established by occlusiving bilateral carotid artery is am effectual model to study vascular cognitive disorder an estimate the drays of dementia. In this article , by establishing 2V0 model of chronic cerebral hypoperfusion and observing the expressions of VEGF and HO-1 by immunohistochemically,we explore the effection of NBP on revasculari -zation regeneration blocking cell apoptosis and the protection of neurocyte after chronic cerebral ischemia.
Materials and methods
1. Experiment animal and grouping
Selected 80 healthy wistar rats and distributed to 4 groups randomly,regardless of male or female,the age from 12 to 14 months and the weight from 450-550g,Offered by the center of experimental animal of Zhengzhou university henna,20 rats each group. Group A:control group (sham operation plus);group B:ischemia group;group C:ischemic and low dose treatment group;group D:ischemic and high dose treatment group. Cronic cerebral ischemic rat models were established of B C D by permanent occlusiving and snipping bilateral carotid artery .The treatment of the control group is the same to the model groups,but without the snip of bilateral carotid artery. The rats of groups Cand D began to receive daily oral dose of 60mg/kg and 120mg/kg NBP(dissolved into 2ml oil) from the 2 months after the operation,which will last a month.The rats of groups Aand Breceived the same volume of oil,which will last the same time .The standards of successed model are:in the process of the next time ,active ,without secretion of eyes,normal eating and so on,except the rats died after three months.
2. manufacturation of model
The rats were perfused with 10% chloral hydrate for anesthesia,split the center of the neck ,isolate and permanent occlusion and snip of bilateral common carotid arteries,suture skin and breed the rats in virgin circumstance.In rats of control group, bilateral common carotid arteriesonly is only isolated but not be ligated and snipped,other treatments are the same to the rest groups.
3. manufacturation of specimen
Three months after 2VO,the rats were perfused with 10% chloral hydrate for anesthesia. Cut the head and take out of left brain tissue after perfusing from the heart,then put brain tissue to 4% paraformaldehyde for fixation. Brain tissue is manufactured coronal section for HE and immunohistochemistry staining after dehydration clearing dipping candle and embedding.
4. The content of observation
(1)The changes of neuron in cortex and hippocampus were observed by HE.
(2)The expressions of vascular endothliat growth factor(VEGF) and Hemeoxygenase-1(HO-1) were observed by immunohistochemically.
5.Statistical method
The data was handled with SPSS12.0 static software. The diferece of every two groups was compared with one-way analysis of variance and LSD method,significant level is a=0.05.
Result
1.The conditions of experiment animal All the rats were normality before operation. But all the rats were depressed reacted slowly ate little after operation.The next day, the rats of control group recovered significantly, the rats of other groups did not recover obviously .However the operation groups recovered slowly until 3-5 days.A week later,they were normal.
2.The results of pathological section with HE staining Only a fewdegenerated and dead neurons were found in cortex and hippocampus,while most neurons were normal in group A.The number of degenerated and dead neurons significantly decreased in group B compared with group A.The number of degenerated and dead neurons was less in group C compared with group B,the normal neuron cells in group C are more than group B. The number of degenerated and dead neurons was less in group D.compared with group C, the normal neuron cells in group D are more than group C.
3.The results of immunohistochemical for VEGF The expression of VEGF protein positive mainly in cortex and hippocapus neuron glial cell vascular endothelial cell,endochylema assumes buffy.The number of VEGF protein immunopositive cells were markly increased in group B compairing with group A.The difference between group B and group A was significant (p<0.05).The number of VEGF protein immunopositive cells was markly increased in group C and group D comparing with group B.The difference between group C,D and group B was significant(p<0.005). The number of VEGF protein immunopositive cells was markly increased in group D comparing with group C. The difference between group D and group C was significant(p<0.005).
4.The results of immunohistochemical for HO-1 The HO-1 protein mainlyappears in cortex and hippocampus.The HO-1 protein immunopositive cells were markly increased in group B compairing with group A. The difference between group B and group A was significant (p<0.05). .The number of HO-1 protein immunopositive cells was markly increased in group C and group D comparing with group B.The difference between group C,D and group B was significant(p<0.005). The number of HO-1 protein immunopositive cells was markly increased in group D comparing with group C. The difference between group D and group C was significant(p<0.005).
Conclusion
1.Chronic cerebral ischemia rat model was established by permanent occulusion and snip of bilateral common carotid arteries.The progress of the model and chronic cerebral ischemia is almost same .Its operation is simple and its reproducibility is good.
2.Three months after chronic cerebral ischemic ,neurons of cortex and hippocampus degenerated and died ,the number of neurocyte decreasd.
3.NBP can lessen the degeneration death of neurons of cortex and hippocampus,at the same ,it can also promote the expression of VEGF and HO-1 in cortex and hippocampus after Chronic cerebral ischemia.
血管内皮生长因子(vascular endothelial growth factor，VEGF)是一种高度特异的血管内皮细胞有丝分裂原和血管源性因子，可特异性地作用与血管内皮细胞，诱导内皮细胞增殖及毛细血管瓣生成，促进新生血管生成，对生理性血管生成和病理性血管再生都有重要的调节作用，VEGF还具有增加血管的通透性以及神经营养和神经保护作用，组织缺血、缺氧是诱导VEGF表达的强刺激因素，慢性脑缺血时VEGF表达增多对慢性脑缺血后的神经细胞恢复起重要作用。内源性一氧化碳(carbon monoxide，CO)可激活鸟苷酸环化酶，调节环磷酸鸟苷(CAMP)的生成，是一种具有许多生理功能的细胞信使，近年来发现CO有与一氧化氮(nitrogenmonoxidum，NO)相类似的内皮依赖性血管舒张作用，而且具有增加VEGF的合成能力。血红素氧化酶(hemeoxygenase，HO)是CO产生的关键酶，在HO的作用下，血红素氧化分解为螯合铁、CO和胆绿素；HO-1是诱导型HO，可被氧应激、缺血、金属、热休克等应激诱导表达。以前人们的兴趣集中在HO-1对血红素的降解能力上，最新研究发现HO-1具有抗氧化、抗炎、抑制血小板凝集、细胞保护、调节血管张力等许多重要生物学作用，HO／CO系统近年已逐渐成为研究的热点。丁基苯酞(dl-butyphthalide，NBP)是从芹菜籽中提取出来的，后经人工合成为消旋体，为治疗脑血管病的一种新药，在急性脑缺血的研究中，它可改善小鼠全脑缺血脑能量代谢，可以增加局部脑缺血大鼠缺血区的脑血流，能缩小大鼠大脑中动脉结扎后(MCAO)梗塞的面积和改善神经功能缺失，能改善MCAO后大鼠记忆功能障碍，但在慢性脑缺血中有关NBP的报道却很少。本文主要通过建立大鼠慢性脑缺血模型，运用免疫组化技术和计算机图像分析技术观察丁基苯酞对VEGF和HO-1在慢性脑缺血大鼠海马和皮层表达的影响，进一步探讨丁基苯酞对慢性脑缺血大鼠血管再通、再生及对脑细胞的保护作用。
材料与方法
1．实验动物与分组
健康wistar大鼠80只，雌雄不拘，12～14月龄，体重450～550g，由河南省郑州大学实验动物中心提供，随机分为4组，每组20只，分为A组：对照组(假手术+溶剂)；B组：单纯缺血组(手术+溶剂)；C组：缺血低剂量治疗组(手术+低剂量NBP+溶剂)；D组：缺血高剂量治疗组(手术+高剂量NBP+溶剂)。B、C、D组参照ohta等永久性结扎双侧颈总动脉制作慢性脑缺血模型，对照组除不结扎双侧颈总动脉外，其他处理同模型组。C组和D组造模2月后每日分别按60mg／kg、120mg／kg给予丁基苯酞灌胃，丁基苯酞用花生油稀释至2ml；A组和B组每日仅给予花生油2ml；各组皆连续灌胃一个月。模型成功标准：大鼠在继续饲养过程中，行动活泼，正常吃食饮水，眼部无分泌物，毛色光洁，体重增加。凡2VO 3个月后死亡的大鼠，不记在内。
2．模型的制作
将各组大鼠以10％的水合氯醛(0.3ml／100g)腹腔注射麻醉，作颈部正中切口，分离双侧颈总动脉，双重丝线结扎其近心端及远心端，间断缝合皮肤，放回原条件下饲养。对照组只分离颈总动脉但不结扎，其他处理同其余各组。
3．标本的制作
术后3个月，以10％的水合氯醛腹腔注射麻醉各组大鼠，经心脏灌注后，断头取左脑，放入4％的多聚甲醛中固定，固定好的脑组织经梯度酒精脱水，二甲苯透明、浸蜡、包埋，制作脑冠状切片，以备HE染色和免疫组化染色。
4．观察内容
(1)采用HE染色观察各组大鼠皮层和海马的神经元细胞变化。
(2)免疫组化观察各组大鼠皮层和海马的VEGF和HO-1的表达。
5．统计方法
数据以(?)±s表示，采用spss12.0软件进行数据处理，多组间比较采用单因素方差分析，用LSD法进行两两比较，检验水准α=0.05。
结果
1．实验动物行为观察
各组大鼠在手术前行为正常，术后各组大鼠均出现精神萎靡，反应迟钝，神情呆滞，少食，少饮。术后1天，除A组精神恢复外，其余各组行为改善不明显。3～5天B、C、D大鼠逐渐恢复，一周时基本正常，眼睛明亮，无分泌物，行动灵活，吃食、饮水情况良好。经Morris水迷宫实验证实，造模成功。
2．HE染色
A组：皮质和海马偶有神经元细胞变性、死亡，大部分细胞形态正常。B组：皮质和海马大量神经元细胞缺血性固缩、退变、死亡，正常神经细胞数较A组明显减少。C组：皮质和海马神经元变性、死亡较B组减轻，正常神经细胞数较B组明显增多。D组：皮质和海马神经元变性、死亡较C组明显减轻，正常神经细胞数较C组明显增多。
3．VEGF免疫组化染色
各组取皮层和海马进行观察，并统计VEGF蛋白表达的阳性细胞数，VEGF蛋白阳性表达主要见于皮质和海马的神经元、神经胶质细胞、血管内皮细胞，胞浆呈棕黄色。A组VEGF蛋白呈少量表达，B组VEGF蛋白表达明显多于A组，差异具有统计学意义(α＜0.05)，C组和D组VEGF蛋白表达均明显多于B组，差异具有统计学意义(α＜0.05)，且D组VEGF蛋白表达明显多于C组，差异具有统计学意义(α＜0.05)。
4．HO-1免疫组化染色
各组取皮层和海马进行观察并统计HO-1蛋白表达的阳性细胞数，HO-1蛋白阳性表达主要见于皮质和海马的神经元、神经胶质细胞，胞浆呈棕黄色。A组HO-1蛋白呈少量表达，B组HO-1蛋白表达明显多于A组，差异具有统计学意义(α＜0.05)，C组和D组HO-1蛋白表达均明显多于B组，差异具有统计学意义(α＜0.05)，且D组HO-1蛋白表达明显多于C组，差异具有统计学意义(α＜0.05)。
结论
1．永久性结扎大鼠双侧颈总动脉(2VO)模型是一种比较成熟的普遍认可的慢性脑缺血动物模型，实验操作简便，重复性好。
2．大鼠慢性脑缺血3个月后，皮层和海马神经元细胞大量变性、死亡，血管内皮生长因子和血红素氧化酶—1表达少量增多。
3．丁基苯酞(NBP)可减轻皮层和海马神经元变性、死亡，同时可增加血管内皮生长因子和血红素氧化酶—1在慢性脑缺血大鼠皮层和海马的表达。
Background
Because the morbidity and mortality of cerebrovascular is rising continually lately, humans attach more importance to study cerebrovascular. But many studies have focused on acute ischemic brain impairments is insufficiency to chronic cerebral hypoperfusion.But, with the ratio of senile increasing in country, More and more people get into trouble with dementia, which brings social economic and family problem. Now,the study of the chronic cerebral ischemia becomes especially importance.Chronic ischemic brain impairment is a common pathological state ,it complicated in many diseases such as Vascular dementia ,Alzheimer disease ,Binswanger disease ,Arterivenous malformation and so on .Because it is chronic ,the impairment and repairment is more complicated.In the early stages,its cardinal manifestation is cognitive disorder.At last,it can cause eternal or progressive impairment of cognitive and neurological. It is criticality for improving brain impairment to recover bloodstream provision early. Studies have proved that the increasing of blood vessel density after chronic cerebral hypoperfusion is related to amendent and prognosis of patient.So it is very important for the recovery of patient to study the factor of vascular growth .
vascular endothlial growth factor(VEGF) is a very specific factor to promote vascular growth. It plays an important regulating effection in physiology vascular generation an pathological vascular generation.it can promote vascular regeneration,increase vascular permeability and protect the neuron. The ischemia and hypoxia of tissue is a stimulant factor for the expression of VEGF,The VEGF plays an imoortant role in the cellur recovery after chronic cerebral ischemia. The endogenous carbon monoxide(CO)can activate guanylate cyclase,regulate the cyclic guanosine,it is a cell messenger which have a great deal of biological functions .Recently ,many studies discover that carbon monoxide(CO) have the effect of endothelial dependence vasodilation to be similar to nitrogen monoxidum (NO),and have the capability of promoting composition of VEGF.Hemeoxygenase(HO) is the key enzyme to creat CO.Till now ,people have discovered three kinds of isoenzyme of H0:H0-l is type induction,HO-2 and HO-3 are type architecture.HO-1 is also called heart shock protein(HSP) and it can be inducted by multiple stress,for example,Heme hypoxia ischemia and injurment. Many studies focused on the its capability of degredating to heme in previous years,the latest study have found that HO-1 have many biological functions in antioxygen antiinflammatory inhibiting platelet aggregation cytoprotection accommodating angiotasis and so on. Recently,the study of the HO/CO system has become a hot topic. Dl-butyphthalide(NBP) was isolated from the seeds of of celery,then was synthesized racemic artificially.NBP is a new drug to cure cerebrovascular disease .In the studyof acute brain ischemic ,NBP was shown to improve brain energy metabolism in mice with complete cerebral ischemic ,and to enhance the rCBF of rats with regional ischemic,and to decrease cerebral infarct volume of rat with MCAO, and to protect rats from ischemic neurological damage,and to improve impairment of learning and memory.But there is very few reports about NBPin the chronic cerebral ischemic. Studies hare proved that chromic cerebral ischemia which is established by occlusiving bilateral carotid artery is am effectual model to study vascular cognitive disorder an estimate the drays of dementia. In this article , by establishing 2V0 model of chronic cerebral hypoperfusion and observing the expressions of VEGF and HO-1 by immunohistochemically,we explore the effection of NBP on revasculari -zation regeneration blocking cell apoptosis and the protection of neurocyte after chronic cerebral ischemia.
Materials and methods
1. Experiment animal and grouping
Selected 80 healthy wistar rats and distributed to 4 groups randomly,regardless of male or female,the age from 12 to 14 months and the weight from 450-550g,Offered by the center of experimental animal of Zhengzhou university henna,20 rats each group. Group A:control group (sham operation plus);group B:ischemia group;group C:ischemic and low dose treatment group;group D:ischemic and high dose treatment group. Cronic cerebral ischemic rat models were established of B C D by permanent occlusiving and snipping bilateral carotid artery .The treatment of the control group is the same to the model groups,but without the snip of bilateral carotid artery. The rats of groups Cand D began to receive daily oral dose of 60mg/kg and 120mg/kg NBP(dissolved into 2ml oil) from the 2 months after the operation,which will last a month.The rats of groups Aand Breceived the same volume of oil,which will last the same time .The standards of successed model are:in the process of the next time ,active ,without secretion of eyes,normal eating and so on,except the rats died after three months.
2. manufacturation of model
The rats were perfused with 10% chloral hydrate for anesthesia,split the center of the neck ,isolate and permanent occlusion and snip of bilateral common carotid arteries,suture skin and breed the rats in virgin circumstance.In rats of control group, bilateral common carotid arteriesonly is only isolated but not be ligated and snipped,other treatments are the same to the rest groups.
3. manufacturation of specimen
Three months after 2VO,the rats were perfused with 10% chloral hydrate for anesthesia. Cut the head and take out of left brain tissue after perfusing from the heart,then put brain tissue to 4% paraformaldehyde for fixation. Brain tissue is manufactured coronal section for HE and immunohistochemistry staining after dehydration clearing dipping candle and embedding.
4. The content of observation
(1)The changes of neuron in cortex and hippocampus were observed by HE.
(2)The expressions of vascular endothliat growth factor(VEGF) and Hemeoxygenase-1(HO-1) were observed by immunohistochemically.
5.Statistical method
The data was handled with SPSS12.0 static software. The diferece of every two groups was compared with one-way analysis of variance and LSD method,significant level is a=0.05.
Result
1.The conditions of experiment animal All the rats were normality before operation. But all the rats were depressed reacted slowly ate little after operation.The next day, the rats of control group recovered significantly, the rats of other groups did not recover obviously .However the operation groups recovered slowly until 3-5 days.A week later,they were normal.
2.The results of pathological section with HE staining Only a fewdegenerated and dead neurons were found in cortex and hippocampus,while most neurons were normal in group A.The number of degenerated and dead neurons significantly decreased in group B compared with group A.The number of degenerated and dead neurons was less in group C compared with group B,the normal neuron cells in group C are more than group B. The number of degenerated and dead neurons was less in group D.compared with group C, the normal neuron cells in group D are more than group C.
3.The results of immunohistochemical for VEGF The expression of VEGF protein positive mainly in cortex and hippocapus neuron glial cell vascular endothelial cell,endochylema assumes buffy.The number of VEGF protein immunopositive cells were markly increased in group B compairing with group A.The difference between group B and group A was significant (p<0.05).The number of VEGF protein immunopositive cells was markly increased in group C and group D comparing with group B.The difference between group C,D and group B was significant(p<0.005). The number of VEGF protein immunopositive cells was markly increased in group D comparing with group C. The difference between group D and group C was significant(p<0.005).
4.The results of immunohistochemical for HO-1 The HO-1 protein mainlyappears in cortex and hippocampus.The HO-1 protein immunopositive cells were markly increased in group B compairing with group A. The difference between group B and group A was significant (p<0.05). .The number of HO-1 protein immunopositive cells was markly increased in group C and group D comparing with group B.The difference between group C,D and group B was significant(p<0.005). The number of HO-1 protein immunopositive cells was markly increased in group D comparing with group C. The difference between group D and group C was significant(p<0.005).
Conclusion
1.Chronic cerebral ischemia rat model was established by permanent occulusion and snip of bilateral common carotid arteries.The progress of the model and chronic cerebral ischemia is almost same .Its operation is simple and its reproducibility is good.
2.Three months after chronic cerebral ischemic ,neurons of cortex and hippocampus degenerated and died ,the number of neurocyte decreasd.
3.NBP can lessen the degeneration death of neurons of cortex and hippocampus,at the same ,it can also promote the expression of VEGF and HO-1 in cortex and hippocampus after Chronic cerebral ischemia.
引文
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