慢性脑低灌注状态大鼠脑组织线粒体结构和功能改变及丁苯酞注射液对其影响
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摘要
目的:血管性痴呆(vascular dementia VD)已经成为中老年人群中的常见病、多发病。VD突出的临床特点是学习、记忆等认知功能下降,伴或不伴神经功能缺损的症状和体征。目前,血管性痴呆的发病机制尚未完全明确,治疗也仍以控制病情、对症治疗、加强护理为主,尚无特殊有效的根治方法。因此,探索VD的发病机理,制定合理的治疗方案,是医学领域研究的重要课题。VD病因之一可能与慢性低灌注状态所造成的脑缺血缺氧有关。所谓慢性脑低灌注状态是高血压、脑动脉硬化引起的脑动脉狭窄所致的脑组织长期低于生理阈值而出现的慢性缺血性神经系统损害的一种病理状态,是VD、AD(Alzheimer's disease AD)和Binswanger病等多种疾病发展过程中的一个共同病理过程。
大量研究表明,在低灌注状态及低代谢状态下认知功能可发生减退,而脑部自由基代谢的紊乱是低灌注时产生脑实质和脑微血管系统损害的重要的可能机制。线粒体既是机体产生氧自由基的主要细胞器,又是细胞进行能量代谢的主要场所。线粒体内部结构是氧自由基损伤的首要目标。因此,线粒体结构和功能的异常可能在VD的发病过程中起关键性作用。
近年来,临床上有关伎用丁基苯酞对脑缺血具有神经保护作用,已引起了人们广泛关注。丁基苯(dl-n-butylphthalidedl-NBP)是源于天然的化学合成药物,药理学实验表明有良好的抗脑缺血和脑保护作用,尤其是对急性局灶性缺血后脑神经元线粒体形态和功能具有保护作用。目前,有关丁基苯酞神经保护作用的大部分研究都集中在急性缺血模型,有关作用于慢性脑低灌注模型的实验研究报道尚缺乏。本实验通过能较好模拟人类慢性脑低灌注的结扎大鼠双侧颈总动脉的动物模型,通过水迷宫试验测试大鼠学习记忆受损情况,行透射电镜观察线粒体形态变化,用分光光度法测定大鼠脑线粒体ATP酶活性,超氧化物岐化酶(superoxide dismutaseSOD)活性,丙二醛(malondialdehyde MDA)含量,并探讨丁苯酞注射对其影响的药理机制,以期能为临床治疗血管性认知功能障碍提供一个新思路。
方法:用雄性SD大鼠55只,月龄3～4个月,起始体重280～340g,随机分为假手术对照组(n=10),生理盐水对照组(n=15),丁苯酞注射液高剂量组(n=15),丁苯酞注射液低剂量组(n=15)。每组大鼠行双侧颈总动脉永久结扎术,假手术组大鼠除不结扎双侧颈总动脉外,余处理同生理盐水组和丁苯酞注射液组。丁苯酞注射液高剂量组手术第4周后腹腔注射丁苯酞注射液,(剂量6mg/kg),每日二次。丁苯酞注射液低剂量组手术第4周后腹腔注射丁苯酞注射液,(剂量2mg/kg),每日二次。生理盐水组腹腔注射等剂量生理盐水,假手术组不给药,连续给予四周。各组大鼠在水迷宫测试后,随机各取2只,1%的戊巴比妥钠麻醉后,立即断头在冰盘上快速取脑,,取一侧颞叶皮层约1mm×1mm×2 mm,放入磷酸缓冲的4%戊二醛溶液固定,1%锇酸中后固定,脱水透明,环氧树脂包埋,超薄切片,电镜观察、照相。末次给药后,动物均禁食12h,断头,在冰盘上快速取脑,提取线粒体,并悬浮于EDTA-蔗糖T ris-HCl缓冲液中。考马斯亮蓝法测定蛋白浓度。调节蛋白含量至1mg/ml。分别测定Na~+-K~+-ATP酶活力、SOD活力和MDA含量。运用SPSS13.0统计软件进行单因素方差分析,采用LSD-t检验进行各组间的两两比较,P＜0.05表示有统计学意义。
结果:(1)各组大鼠存活情况:假手术组死亡0只,生理盐水组死亡5只,死亡率为33.3%。丁苯酞注射液低剂量组、丁苯酞注射液高剂量组分别死亡4只、5只,死亡率分别为26.7%和33.3%。各组存活大鼠作为研究对象。(2)水迷宫试验:生理盐水组大鼠学习、记忆成绩显著下降,表现为寻找平台潜伏期时间延长,明显高于假手术组(p＜0.05)。丁苯酞注射液低剂量组、丁苯酞注射液高剂量组分别与生理盐水组比较,学习、记忆成绩均显著改善(p＜0.05)。丁苯酞注射液低剂量组、丁苯酞注射液高剂量组分别与与假手术组比较,学习、记忆成绩均有所下降,但无显著性差异(P＞0.05)。丁苯酞注射液高剂量组与丁苯酞注射液低剂量组相比学习、记忆成绩无明显差别(p＞0.05)。(3)线粒体超微结构改变:假手术组线粒体超微结构清楚,无明显异常改变。生理盐水组线粒体大部分嵴融合消失,线粒体水肿,有的线粒体双层单位膜消失,粗面内质网脱颗粒,神经元胞质水肿,细胞器明显减少。丁苯酞注射液高剂量组线粒体接近于正常,未见线粒体嵴断裂、空泡增多等,有的出现粗面内质网腔扩大、线粒体肥大等可逆性损伤。丁苯酞注射液低剂量组线粒体形态基本规则,大多数嵴完整,排列紧密规律。(4)线粒体ATP酶活性测定结果情况:与假手术组相比,生理盐水组线粒体ATP酶活性明显下降(p＜0.05)。与生理盐组相比,丁苯酞注射液低剂量组线粒体ATP酶活性显著增加(p＜0.05)。与生理盐水组相比,丁苯酞注射液高剂量组线粒体ATP酶活性亦显著增加(p＜0.05)。丁苯酞注射液低剂量组与丁苯酞注射液高剂量组相比ATP酶活性无明显差异(p＞0.05)。(5)SOD、MDA测定结果情况:与假手术组相比,生理盐水组线粒体SOD活力下降,MDA含量增加(p＜0.05)。与生理盐水组相比丁苯酞注射液低剂量组SOD活力有所增加,MDA含量有所下降(p＜0.05)。与生理盐水组相比,丁苯酞注射液高剂量组SOD活力明显增加,MDA含量明显下降(p＜0.01)。与丁苯酞注射液低剂量组相比,丁苯酞注射液高剂量组SOD活力,MDA含量均无明显差异(p＞0.05)。
结论:(1)本实验采用双侧颈总动脉永久性结扎成功复制慢性脑低灌注大鼠模型(occlusion of bilateral 2-VO),水迷宫试验证实了模型大鼠学习和记忆能力下降,进一步证实模型可靠,透射电镜观察可见大鼠神经元线粒体超微结构有明显改变。(2)慢性脑低灌注大鼠线粒体SOD活性下降,MDA含量增高,提示线粒体氧化损伤可能是VD的发病机制之一。(3)慢性脑低灌注大鼠线粒体ATP酶活性下降。提示能量代谢障碍可能是VD的发病机制之一。(4)通过丁苯酞注射液治疗,慢性脑低灌注大鼠学习记忆能力明显高于对照组,病理学进一步证实可减轻线粒体超微结构损伤程度。与对照组比较,经丁苯酞注射液治疗后慢性脑低灌注大鼠海马线粒体SOD活性增高,MDA含量降低,ATP酶活性增高。提示丁苯酞注射液对慢性脑低灌注状态大鼠脑组织线粒体结构和功能具有保护作用。为临床治疗血管性痴呆提供一个新思路。
Objectives:Vascular dementia(VD) has become a common and frequent disease in middle- aged and aged people.The prominent clinical characteristics of VD are that learning, memory and other cognitive function decline,with or without symptoms and sign of neurologic impairment.At present,the pathogenesis of VD has not been illustrated clearly,and has not been effectively dealed with,either.Therefore,it's an important subject in medicine field to explore the pathogenesis of VD and make a reasonable therapeutic regimen.One of the VD etiopathogenisis may be related to the ischemia and hypoxia caused by Chronic cerebral hypoperfusion.The so-called Chronic cerebral hypoperfusion is a pathologic status of pathological status of chronic ischemic nerve system injury with long term low perfusion brain that lower than threshold caused by hypertension and cerebral artery sclerosis.Chronic cerebral hypoperfusion is a common pathological course of the developing of vascular dementia,Alzheimer's disease, Bingswanger's disease,and so all.
A large number of studies have shown that cognitive fuction can be impaired under hypoperfusion and hypometabolism.And the disorder of free radical metabolism in brain may be one of the major possible mechanism,which results in impairment of cerebral texture and microvascular system.Mitochondria is the main organelle which not only produce oxygen free radicals but also are the main sites of energy metabolism.So the internal structure of mitochondrial are the primary objective impaired by oxygen free radicals. Therefore,the abnormality of mitochondrial structure and function may play a key role in the pathogenesis of VD.
In recent years,which has attracted extensive attention, the clinical use of dl-n-butylphthalide(dl-NBP) on cerebral ischemia has a neuroprotective effect.DL-NBP are synthetic drugs derived from natural chemicals.Pharmacological experiments show that there is good anti-cerebral ischemia and cerebral protection,especially for acute focal ischemic neuronal mitochondrial structure and function,which play a protective role.But most studies about dl-NBP's neuroprotective effect have only focused on acute cerebral ischemia model at home and abroad,while the researches about its effect on chronic cerebral hypoperfusion model have still lacked.But most studies about dl-NBP's neuroprotective effect have only focused on acute cerebral ischemia model at home and abroad,while the researches about its effect on chronic cerebral hypoperfusion model have still lacked.This experiment mimicried human chronic cerebral hypoperfusion by the ligation of bilateral carotid arteries in Sprague -Dawley male rats,to observe their changes of learning and memory by the water maze test,to observe morphological changes of mitochondria by transmission electron microscope to observe ATPase activity,SOD activity and MDA content in mitochondria by spectrophotometric determination,and at the same time to explore the effect of dl-NBP,a new type of neuroprotective medicamentum,on them. We provided a new evidence for clinic treating vascular cognitive disorder as well as revealed the pathogenesis of VD.
Methods:55 Sprague-Dawlay rats,3～4months old,280～340 g in weight,were randomly divided into sham group(n=10), saline group(n=15),dl-NBP low dose treatment group(n=15) and dl-NBP high dose treatment group(n=15).Saline group and treatment group were followed permanent occlusion of bilateral common carotid arteries(2-VO) accoding to de la Torre's method.Sham group was handled as Saline and treatment group except not ligating bilateral common carotid arteries.After 4 weeks following the operation,dl-NBP low dose treatment group were injected dl-NBP(4mg/kg/d) through abdomen cavity for 4 weeks.And dl-NBP high dose treatment group were injected dl-NBP(12mg/kg/d) through abdomen cavity for 4 weeks too.Saline group were injected isodose saline.Congnitive ability of all Survival rats was evaluated by water maze test after 8 weeks.After the last water maze test,brain tissue in temporal lobe cortex of 2 rats of every group were taken out,put into solution of 4%glutaral,after a series treatment,observed and photoed under transmission electron microscope.The remaining animals were fasted 12 h,decapitated,taken out brain tissue on ice,extracted mitochondria,and suspended them in EDTA sucrose- Tris-HCl buffer solution.Protein level was measured by Coomassie brilliant blue method.Protein concentration adjust to 1mg/ml.ATPase activity,SOD activity and MDA content in mitochondria were measured separately.One-way analysis of variance was carried out with SPSS 13.0 statistics software,LSD-t test was used to compare the differences among the groups.P＜0.05 meant statistical significance.
Results:(1):The survival condition of each group:0 died in the sham group.5 rats died in the saline group and 8 weeks group.The death rate was 33.3%.4 and 5 rats died respectively in dl-NBP low dose treatment group and dl-NBP high dose treatment group.The death rates were 26.7%and 33.3% respectively.All survival rats of each group were as study objects.(2):Water maze test:Learning and memory performances of rats decreased obviously in saline group,showing escape latency(EL) extended which were clearly higher than sham group(p＜0.05).Learning and memory performances in dl-NBP low dose treatment group and dl-NBP high dose treatment group improved significantly(p＜0.05), compared with saline group.Learning and memory performances in dl-NBP low dose treatment group and dl-NBP high dose treatment group still decreased but not notablely,if compared with sham group(p＞0.05).Learning and memory performances in dl-NBP high dose treatment group didn't improve notablely,compared with dl-NBP low dose treatment group.(3):the changes of mitochondria ultrastructure:In sham group mitochondrial ultrastructure is observed clearly,and there is no obvious abnormalities.In saline group the majority of mitochondrial cristae disappearing,mitochondrion edema,the double-unit membrane of some mitochondrium disappearing, rough endoplasmic reticulum degranulation,neuronal cytoplasmic edema,Organelle decreased significantly.In dl-NBP high dose treatment group mitochondria are close to normal.In this group lysis of mitochondrial cristae and vacuolization of mitochondria were not observed but in some of mitochondria there were some reversible injury,such as rough endoplasmic reticulum cavity to expand and mitochondrial hypertrophy.In dl-NBP low dose treatment group most of mitochondrial morphology are regular,the mitochondrial cristae are complete.(4) The results of ATPase activity in mitochondria: Compared with sham group,ATPase activity in mitochondria significantly decreased in saline group(p＜0.05).Compared with saline group,ATPase activity in mitochondria increased in dl-NBP low dose treatment group(p＜0.05).Compared with saline group,ATPase activity in mitochondria significantly increased in dl-NBP high dose treatment group(p＜0.05).After dl-NBP treatment,the ATPase activity increased.Compared with dl-NBP low dose treatment group,ATPase activty of dl-NBP high dose treatment group increased but not notablely(p＞0.05). (5) The results of SOD activity and MDA content in mitochondria:Compared with sham group,SOD activity in mitochondria decreased in saline group,and MDA content increased(p＜0.05).Compared with saline group,SOD activity in mitochondria increased in dl-NBP low dose treatment group,and MDA content decreased(p＜0.05).Compared with saline group,SOD activity in mitochondria significantly increased in dl-NBP high dose treatment group,and MDA content decreased(p＜0.05).After dl-NBP treatment,the SOD activity increased and MDA content decreased(p＜0.05). Compared with dl-NBP low dose treatment group,SOD activty of dl-NBP high dose treatment group increased more,and MDA decreased but not notablely(p＞0.05).
Conclusions:(1)This experiment successfully established chronic cerebral hypoperfusion model by ligation of bilateral common carotid arteries in rats.Water maze test verified that model rats existed learning and memory disorder,which further confirmed that the model is dependable.Mitochondria ultrastructure of the chronic cerebral hypoperfusion rats has notablely changed,which was observed by transmission electron microscope.(2) The SOD activity decreased and MDA content increased in mitochondria of the chronic cerebral hypoperfusion rats,Which shows that mitochondrial oxidative damage may be one of the pathogenesis of VD.(3) The ATPase activity increased in mitochondria of the chronic cerebral hypoperfusion rats,Which shows that energy metabolism disorder may be one of the pathogenesis of VD.(4) The mitochondria structure and function in Rats with Chronic Cerebral Hypoperfusion can be protected by dl-NBP.
大量研究表明,在低灌注状态及低代谢状态下认知功能可发生减退,而脑部自由基代谢的紊乱是低灌注时产生脑实质和脑微血管系统损害的重要的可能机制。线粒体既是机体产生氧自由基的主要细胞器,又是细胞进行能量代谢的主要场所。线粒体内部结构是氧自由基损伤的首要目标。因此,线粒体结构和功能的异常可能在VD的发病过程中起关键性作用。
近年来,临床上有关伎用丁基苯酞对脑缺血具有神经保护作用,已引起了人们广泛关注。丁基苯(dl-n-butylphthalidedl-NBP)是源于天然的化学合成药物,药理学实验表明有良好的抗脑缺血和脑保护作用,尤其是对急性局灶性缺血后脑神经元线粒体形态和功能具有保护作用。目前,有关丁基苯酞神经保护作用的大部分研究都集中在急性缺血模型,有关作用于慢性脑低灌注模型的实验研究报道尚缺乏。本实验通过能较好模拟人类慢性脑低灌注的结扎大鼠双侧颈总动脉的动物模型,通过水迷宫试验测试大鼠学习记忆受损情况,行透射电镜观察线粒体形态变化,用分光光度法测定大鼠脑线粒体ATP酶活性,超氧化物岐化酶(superoxide dismutaseSOD)活性,丙二醛(malondialdehyde MDA)含量,并探讨丁苯酞注射对其影响的药理机制,以期能为临床治疗血管性认知功能障碍提供一个新思路。
方法:用雄性SD大鼠55只,月龄3～4个月,起始体重280～340g,随机分为假手术对照组(n=10),生理盐水对照组(n=15),丁苯酞注射液高剂量组(n=15),丁苯酞注射液低剂量组(n=15)。每组大鼠行双侧颈总动脉永久结扎术,假手术组大鼠除不结扎双侧颈总动脉外,余处理同生理盐水组和丁苯酞注射液组。丁苯酞注射液高剂量组手术第4周后腹腔注射丁苯酞注射液,(剂量6mg/kg),每日二次。丁苯酞注射液低剂量组手术第4周后腹腔注射丁苯酞注射液,(剂量2mg/kg),每日二次。生理盐水组腹腔注射等剂量生理盐水,假手术组不给药,连续给予四周。各组大鼠在水迷宫测试后,随机各取2只,1%的戊巴比妥钠麻醉后,立即断头在冰盘上快速取脑,,取一侧颞叶皮层约1mm×1mm×2 mm,放入磷酸缓冲的4%戊二醛溶液固定,1%锇酸中后固定,脱水透明,环氧树脂包埋,超薄切片,电镜观察、照相。末次给药后,动物均禁食12h,断头,在冰盘上快速取脑,提取线粒体,并悬浮于EDTA-蔗糖T ris-HCl缓冲液中。考马斯亮蓝法测定蛋白浓度。调节蛋白含量至1mg/ml。分别测定Na~+-K~+-ATP酶活力、SOD活力和MDA含量。运用SPSS13.0统计软件进行单因素方差分析,采用LSD-t检验进行各组间的两两比较,P＜0.05表示有统计学意义。
结果:(1)各组大鼠存活情况:假手术组死亡0只,生理盐水组死亡5只,死亡率为33.3%。丁苯酞注射液低剂量组、丁苯酞注射液高剂量组分别死亡4只、5只,死亡率分别为26.7%和33.3%。各组存活大鼠作为研究对象。(2)水迷宫试验:生理盐水组大鼠学习、记忆成绩显著下降,表现为寻找平台潜伏期时间延长,明显高于假手术组(p＜0.05)。丁苯酞注射液低剂量组、丁苯酞注射液高剂量组分别与生理盐水组比较,学习、记忆成绩均显著改善(p＜0.05)。丁苯酞注射液低剂量组、丁苯酞注射液高剂量组分别与与假手术组比较,学习、记忆成绩均有所下降,但无显著性差异(P＞0.05)。丁苯酞注射液高剂量组与丁苯酞注射液低剂量组相比学习、记忆成绩无明显差别(p＞0.05)。(3)线粒体超微结构改变:假手术组线粒体超微结构清楚,无明显异常改变。生理盐水组线粒体大部分嵴融合消失,线粒体水肿,有的线粒体双层单位膜消失,粗面内质网脱颗粒,神经元胞质水肿,细胞器明显减少。丁苯酞注射液高剂量组线粒体接近于正常,未见线粒体嵴断裂、空泡增多等,有的出现粗面内质网腔扩大、线粒体肥大等可逆性损伤。丁苯酞注射液低剂量组线粒体形态基本规则,大多数嵴完整,排列紧密规律。(4)线粒体ATP酶活性测定结果情况:与假手术组相比,生理盐水组线粒体ATP酶活性明显下降(p＜0.05)。与生理盐组相比,丁苯酞注射液低剂量组线粒体ATP酶活性显著增加(p＜0.05)。与生理盐水组相比,丁苯酞注射液高剂量组线粒体ATP酶活性亦显著增加(p＜0.05)。丁苯酞注射液低剂量组与丁苯酞注射液高剂量组相比ATP酶活性无明显差异(p＞0.05)。(5)SOD、MDA测定结果情况:与假手术组相比,生理盐水组线粒体SOD活力下降,MDA含量增加(p＜0.05)。与生理盐水组相比丁苯酞注射液低剂量组SOD活力有所增加,MDA含量有所下降(p＜0.05)。与生理盐水组相比,丁苯酞注射液高剂量组SOD活力明显增加,MDA含量明显下降(p＜0.01)。与丁苯酞注射液低剂量组相比,丁苯酞注射液高剂量组SOD活力,MDA含量均无明显差异(p＞0.05)。
结论:(1)本实验采用双侧颈总动脉永久性结扎成功复制慢性脑低灌注大鼠模型(occlusion of bilateral 2-VO),水迷宫试验证实了模型大鼠学习和记忆能力下降,进一步证实模型可靠,透射电镜观察可见大鼠神经元线粒体超微结构有明显改变。(2)慢性脑低灌注大鼠线粒体SOD活性下降,MDA含量增高,提示线粒体氧化损伤可能是VD的发病机制之一。(3)慢性脑低灌注大鼠线粒体ATP酶活性下降。提示能量代谢障碍可能是VD的发病机制之一。(4)通过丁苯酞注射液治疗,慢性脑低灌注大鼠学习记忆能力明显高于对照组,病理学进一步证实可减轻线粒体超微结构损伤程度。与对照组比较,经丁苯酞注射液治疗后慢性脑低灌注大鼠海马线粒体SOD活性增高,MDA含量降低,ATP酶活性增高。提示丁苯酞注射液对慢性脑低灌注状态大鼠脑组织线粒体结构和功能具有保护作用。为临床治疗血管性痴呆提供一个新思路。
Objectives:Vascular dementia(VD) has become a common and frequent disease in middle- aged and aged people.The prominent clinical characteristics of VD are that learning, memory and other cognitive function decline,with or without symptoms and sign of neurologic impairment.At present,the pathogenesis of VD has not been illustrated clearly,and has not been effectively dealed with,either.Therefore,it's an important subject in medicine field to explore the pathogenesis of VD and make a reasonable therapeutic regimen.One of the VD etiopathogenisis may be related to the ischemia and hypoxia caused by Chronic cerebral hypoperfusion.The so-called Chronic cerebral hypoperfusion is a pathologic status of pathological status of chronic ischemic nerve system injury with long term low perfusion brain that lower than threshold caused by hypertension and cerebral artery sclerosis.Chronic cerebral hypoperfusion is a common pathological course of the developing of vascular dementia,Alzheimer's disease, Bingswanger's disease,and so all.
A large number of studies have shown that cognitive fuction can be impaired under hypoperfusion and hypometabolism.And the disorder of free radical metabolism in brain may be one of the major possible mechanism,which results in impairment of cerebral texture and microvascular system.Mitochondria is the main organelle which not only produce oxygen free radicals but also are the main sites of energy metabolism.So the internal structure of mitochondrial are the primary objective impaired by oxygen free radicals. Therefore,the abnormality of mitochondrial structure and function may play a key role in the pathogenesis of VD.
In recent years,which has attracted extensive attention, the clinical use of dl-n-butylphthalide(dl-NBP) on cerebral ischemia has a neuroprotective effect.DL-NBP are synthetic drugs derived from natural chemicals.Pharmacological experiments show that there is good anti-cerebral ischemia and cerebral protection,especially for acute focal ischemic neuronal mitochondrial structure and function,which play a protective role.But most studies about dl-NBP's neuroprotective effect have only focused on acute cerebral ischemia model at home and abroad,while the researches about its effect on chronic cerebral hypoperfusion model have still lacked.But most studies about dl-NBP's neuroprotective effect have only focused on acute cerebral ischemia model at home and abroad,while the researches about its effect on chronic cerebral hypoperfusion model have still lacked.This experiment mimicried human chronic cerebral hypoperfusion by the ligation of bilateral carotid arteries in Sprague -Dawley male rats,to observe their changes of learning and memory by the water maze test,to observe morphological changes of mitochondria by transmission electron microscope to observe ATPase activity,SOD activity and MDA content in mitochondria by spectrophotometric determination,and at the same time to explore the effect of dl-NBP,a new type of neuroprotective medicamentum,on them. We provided a new evidence for clinic treating vascular cognitive disorder as well as revealed the pathogenesis of VD.
Methods:55 Sprague-Dawlay rats,3～4months old,280～340 g in weight,were randomly divided into sham group(n=10), saline group(n=15),dl-NBP low dose treatment group(n=15) and dl-NBP high dose treatment group(n=15).Saline group and treatment group were followed permanent occlusion of bilateral common carotid arteries(2-VO) accoding to de la Torre's method.Sham group was handled as Saline and treatment group except not ligating bilateral common carotid arteries.After 4 weeks following the operation,dl-NBP low dose treatment group were injected dl-NBP(4mg/kg/d) through abdomen cavity for 4 weeks.And dl-NBP high dose treatment group were injected dl-NBP(12mg/kg/d) through abdomen cavity for 4 weeks too.Saline group were injected isodose saline.Congnitive ability of all Survival rats was evaluated by water maze test after 8 weeks.After the last water maze test,brain tissue in temporal lobe cortex of 2 rats of every group were taken out,put into solution of 4%glutaral,after a series treatment,observed and photoed under transmission electron microscope.The remaining animals were fasted 12 h,decapitated,taken out brain tissue on ice,extracted mitochondria,and suspended them in EDTA sucrose- Tris-HCl buffer solution.Protein level was measured by Coomassie brilliant blue method.Protein concentration adjust to 1mg/ml.ATPase activity,SOD activity and MDA content in mitochondria were measured separately.One-way analysis of variance was carried out with SPSS 13.0 statistics software,LSD-t test was used to compare the differences among the groups.P＜0.05 meant statistical significance.
Results:(1):The survival condition of each group:0 died in the sham group.5 rats died in the saline group and 8 weeks group.The death rate was 33.3%.4 and 5 rats died respectively in dl-NBP low dose treatment group and dl-NBP high dose treatment group.The death rates were 26.7%and 33.3% respectively.All survival rats of each group were as study objects.(2):Water maze test:Learning and memory performances of rats decreased obviously in saline group,showing escape latency(EL) extended which were clearly higher than sham group(p＜0.05).Learning and memory performances in dl-NBP low dose treatment group and dl-NBP high dose treatment group improved significantly(p＜0.05), compared with saline group.Learning and memory performances in dl-NBP low dose treatment group and dl-NBP high dose treatment group still decreased but not notablely,if compared with sham group(p＞0.05).Learning and memory performances in dl-NBP high dose treatment group didn't improve notablely,compared with dl-NBP low dose treatment group.(3):the changes of mitochondria ultrastructure:In sham group mitochondrial ultrastructure is observed clearly,and there is no obvious abnormalities.In saline group the majority of mitochondrial cristae disappearing,mitochondrion edema,the double-unit membrane of some mitochondrium disappearing, rough endoplasmic reticulum degranulation,neuronal cytoplasmic edema,Organelle decreased significantly.In dl-NBP high dose treatment group mitochondria are close to normal.In this group lysis of mitochondrial cristae and vacuolization of mitochondria were not observed but in some of mitochondria there were some reversible injury,such as rough endoplasmic reticulum cavity to expand and mitochondrial hypertrophy.In dl-NBP low dose treatment group most of mitochondrial morphology are regular,the mitochondrial cristae are complete.(4) The results of ATPase activity in mitochondria: Compared with sham group,ATPase activity in mitochondria significantly decreased in saline group(p＜0.05).Compared with saline group,ATPase activity in mitochondria increased in dl-NBP low dose treatment group(p＜0.05).Compared with saline group,ATPase activity in mitochondria significantly increased in dl-NBP high dose treatment group(p＜0.05).After dl-NBP treatment,the ATPase activity increased.Compared with dl-NBP low dose treatment group,ATPase activty of dl-NBP high dose treatment group increased but not notablely(p＞0.05). (5) The results of SOD activity and MDA content in mitochondria:Compared with sham group,SOD activity in mitochondria decreased in saline group,and MDA content increased(p＜0.05).Compared with saline group,SOD activity in mitochondria increased in dl-NBP low dose treatment group,and MDA content decreased(p＜0.05).Compared with saline group,SOD activity in mitochondria significantly increased in dl-NBP high dose treatment group,and MDA content decreased(p＜0.05).After dl-NBP treatment,the SOD activity increased and MDA content decreased(p＜0.05). Compared with dl-NBP low dose treatment group,SOD activty of dl-NBP high dose treatment group increased more,and MDA decreased but not notablely(p＞0.05).
Conclusions:(1)This experiment successfully established chronic cerebral hypoperfusion model by ligation of bilateral common carotid arteries in rats.Water maze test verified that model rats existed learning and memory disorder,which further confirmed that the model is dependable.Mitochondria ultrastructure of the chronic cerebral hypoperfusion rats has notablely changed,which was observed by transmission electron microscope.(2) The SOD activity decreased and MDA content increased in mitochondria of the chronic cerebral hypoperfusion rats,Which shows that mitochondrial oxidative damage may be one of the pathogenesis of VD.(3) The ATPase activity increased in mitochondria of the chronic cerebral hypoperfusion rats,Which shows that energy metabolism disorder may be one of the pathogenesis of VD.(4) The mitochondria structure and function in Rats with Chronic Cerebral Hypoperfusion can be protected by dl-NBP.
引文
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2 Deven BD,Goad EH,Petri HL.Dissociation of hippocampal and striatal contributions to spatial navigation in the water maze[J].Neurobiol Learn Mem,1996,66(3):305-323
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4 De Jong Gl,Farkas E,Stienst ra CM,et al.Cerebral hypoperfusion yields capillary damage in the hippocampal CA1 area that correlates with spatial memory impairment[J].Neuroscience,1999,91(1):203-210
5 DE Jong GI,Vos RA,Steur EN,etal.Cerebrovascular hypoper-fusioma risk factor for Alzheimer's disease?Animal model and postmortem human studies[J].Ann NY Acad Sci.1997,826:56-74
6 Ohta H,Nishikawa H,Kimura H,et al.Chronic cerebral hypoperfusion by permanent internal carotid ligation produces learning impairment without brain damage in rats.Neuroscience,1997,79:1039-1050
7 Pulsinelli WA,Brierley JB.A new model of bilateral hemispheric ischemia in the unanesthetized rat[J].Stroke,1997,10:267-272
8 Sarti C,Pantoni L,Bartolini L,et al.Cognitive impairment and chronic cerebral hypoperfusion:What can be learned from experimental models.J Neurol Sci 2002;203-204:263-266
9 HaiJ,Ding MX,Guo ZL,etal.A new rat model of chroniccere-bral hypoperfusion associated with arteriovenous malformations[J].J Neurosurg,2002,97:1198-1202
10 Halliwell B,Gulleridge JMC.Antioxidants of human extracellular fluids.Arch Biochem Biophys,1990,280(1):l-8
11 Christen Y.Oxidative stress and Alzheimer's disease.Am J Clin Nutr,2000,71(2):621—629
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