慢性酒精中毒脑组织形态学变化及Tau蛋白、β-APP表达与TSAH死亡机制关系的研究
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摘要
背景与目的
酗酒后轻微外力引起的致死性外伤性蛛网膜下腔出血(traumatic subarachnoid haemorrhage,TSAH)时有发生,TSAH常与酗酒有关,国内外学者对此已有共识,但其发生及死亡机制尚未完全清楚。本课题组前期已建立了稳定的大鼠灌酒后TSAH模型,结果显示,慢性灌酒组大鼠TSAH发生率及死亡率分别高达81%和76%,远高于急性灌酒组的27%和4.8%(P<0.01)。综合相关文献及前期实验结果,我们提出长期酗酒对中枢神经系统(central nerves systerm, CNS)代谢、功能和形态各方面的累积性毒理作用,可能构成协同脑震荡性损伤导致TSAH死亡的神经病理学基础的假设。本研究通过观察慢性灌酒中毒的CNS形态学变化,以及与神经纤维轴突运输、信号转导密切相关的Tau蛋白、β-APP表达状况,探讨酗酒与TSAH死亡机制的相关性。
材料与方法
1动物模型及分组
CL级雄性SD大鼠,体重300±30g,常规分笼饲养,随机分:灌水组10只、灌水打击组10只、灌酒组10只、灌酒打击组29只、灌酒停酒组6只。连续灌胃食用水或白酒(52%v/v北京红星二锅头)4周,剂量:前2周8 ml/kg/次、后2周12 ml/kg/次,每天9:00和16:00间隔7小时两次灌胃。
2方法
灌水组和灌酒组最后一次灌胃2h后,戊巴比妥腹腔注射麻醉后股动脉放血处死;灌水打击和灌酒打击组用自制单摆式打击装置给予脑震荡性打击,同时监测心电15min,存活大鼠打击后观测2h,戊巴比妥腹腔注射麻醉后股动脉放血处死,灌酒打击组中死亡的大鼠归为死亡组,各组均立即剖胸主动脉插管,含钙2%多聚甲醛-2.5%戊二醛固定液灌注固定30min,开颅提取全脑,置于4%中性多聚甲醛液固定6h,旁正中矢状切取脑组织,常规石蜡连续切片,分别HE、Bielschowsky’s镀银染色及Tau蛋白、β-APP免疫组织化学染色,光镜观察组织病理形态,图像采集系统观测、采图,Image-Pro Plus 6.0图像分析软件累积积分光密度(IOD sum)等量化病理学测量。
同时,备制透射电镜脑组织样本立即置入2.5%戊二醛中,按常规方法清洗、固定、包埋、切片、染色,在JEM-1400透射电子显微镜下观察。
3统计分析
所有实验数据均采用均数±标准差表示,通过SPSS 17.0及Excel 2003分析处理数据,检验方法采用方差分析(ANOVA)、独立样本T检验、卡方检验。统计学检验所得值差异显著性以P<0.05、P<0.01表示。
结果
1实验大鼠一般情况观察
灌水组饮食、活动状况良好,体重持续增加,第2w始体重增加明显(P<0.01)。灌酒组毛发粗糙、无光泽,精神状况差,并随着灌酒时间延长,逐渐出现体形消瘦、营养不良、进食量、活动减少、肢体瘫软等慢性酒精中毒改变。灌酒组大鼠灌酒后体重逐渐下降,第1w、第3w、第4w体重均明显下降,较灌酒前均差异显著(P<0.01)。
灌水打击组心电图呈一过性心率减慢、QRS波群波幅增大,0.3±0.16s后恢复;灌酒打击组大鼠QRS波群波幅明显增大,恢复时间1.7±0.36s,较灌水打击组明显延长(P<0.05);打击死亡组大鼠QRS波幅明显增大,10s-1min后心率进行性减慢后呈直线。
灌酒打击组TSAH发生率和死亡率分别为79.3%,51.7%,显著高于灌水打击组(P<0.01,P<0.05),根据Fisher分级法,灌酒打击组大鼠TSAH级别高于灌水打击组。
2常规大体和组织病理学观察
灌水组神经元、胶质细胞形态和分布正常,神经纤维粗细染色均匀、排列平直紧密。灌水打击组全脑轻度淤血,散在局限性小灶性出血,神经元、胶质细胞轻度水变性,尼氏体浅淡、数目减少,神经纤维不规则扭曲增粗、断裂、周隙扩大,排列疏松。
灌酒组全脑表面轻度淤血,神经元排列紊乱,形态不一,散在固缩深染1型变和肿胀淡染2型变神经元,胞体肿大变圆,核增大、偏位,中央性尼氏体溶解、消失;胶质细胞增生;小脑蒲肯野细胞固缩、数目减少、疏密不均;神经纤维排列稍疏松、间隙增大,轻度不规则增粗、变形。
灌酒打击组全脑表面弥漫淤血,脑干周围多见厚层TSAH,未见明显脑挫裂伤,神经元及神经胶质细胞高度水变性、周隙扩大,多见核固缩、溶解、消失,中央尼氏体不同程度边集、溶解、消失,多见噬神经及卫星现象;散在神经元脱失,胶质细胞增生;小脑蒲肯野细胞不规则减少,染色、疏密不均、核固缩;延脑多见红色神经元及暗神经元;神经纤维疏松水肿、周隙增宽,可见明显增粗、扭曲、断裂,局部肿胀明显。灌酒停酒组大鼠较灌酒组神经元及神经纤维病变有所缓解。
3透射电镜观察
灌水组脑干组织致密,神经纤维轴索粗细均匀、形态规整,电子密度较高,髓鞘深色、厚薄均匀,轴膜和髓鞘结合紧密,神经骨架纤维粗细均匀;神经元核、线粒体、高尔基复合体、内质网丰富,大小形态、排列规整,多见核糖体。灌水打击组组织较疏松水肿,电子密度较低,神经纤轴索粗细、深浅不均,髓鞘厚薄不一、散在裂隙分层;神经骨架纤维水肿、排列松散;神经元线粒体疏松肿胀、嵴扩张,内质网轻度扩大,突触间隙增宽、致密斑密度降低。
灌酒组脑干组织疏松水肿,神经轴索弥漫粗细、形态不均、排列紊乱,髓鞘不规则分层、厚薄不均,神经纤维轴浆疏密不均,神经骨架纤维粗细不一、部分溶解,髓鞘脂质散在裂隙分层,神经元线粒体明显疏松肿胀、嵴长短粗细不一、外膜不完整,可见胞体、核固缩神经元,核膜皱折、异染色质增多、聚集,核仁固缩;胶质细胞增多,间质疏松水肿。灌酒打击组脑干神经纤维髓鞘高度疏松水肿,可见板层分离、褶叠和内陷,絮状物附着,部分区域形成囊泡样突起突入周围间隙中;神经骨架纤维崩解,局部被絮状沉淀物和膜性结构所代替,周边可见膜性细胞器的积聚,可见空化的线粒体;突触数量减少,突触小泡堆积;胶质细胞增多,间质水肿;在轴膜和髓鞘改变区,轴浆和细胞器逐渐积聚,呈反应性轴突肿胀;神经元体积明显缩小,胞浆及核电子密度增高、核膜皱折,异染色质团块状聚集,核仁消失或变小,核浆比例增大,胞周缝隙增宽,胞膜不完整,线粒体和内质网明显疏松肿胀。灌酒停酒组大鼠较灌酒组神经元及神经纤维病变有所缓解,水肿减轻。
4免疫组化观察
4.1 Tau蛋白IOD sum值
正常组脑组织Tau蛋白主要在神经元的轴突和胞浆阳性表达。灌酒组脑干、小脑、海马、额叶等脑区Tau IOD sum值显著减小(P<0.05)。灌水打击组额叶脑区Tau IOD sum值显著增高(P<0.05),脑干、小脑、海马等部位Tau IOD sum值变化差异不显著(P>0.05)。慢性灌酒打击组脑干、小脑、海马、额叶等部位Tau IOD sum值显著增高(P<0.05)。慢性灌酒打击死亡组脑干、海马、额叶等部位Tau IOD sum值均高于慢性灌酒打击存活组(P<0.05)。
4.2β-APP IOD sum值
正常组β-APP主要在神经元胞浆阳性表达。灌酒组脑干、小脑、海马等脑区IOD sum值显著增高(P<0.05),额叶变化不显著(P>0.05)。灌水打击组脑干、海马等脑区β-APP IODsum值显著增高(P<0.05),小脑、额叶等部位差异不显著(P>0.05)。慢性灌酒打击组脑干、小脑、海马、额叶等部位β-APP IOD sum值显著减小(P<0.05)。慢性灌酒打击死亡组小脑、海马等部位β-APP IOD sum值均显著高于慢性灌酒打击存活组(P<0.05)。
结论
1、慢性酒精中毒可引起CNS神经纤维细胞骨架的形态结构破坏,降低传导功能的代偿性,导致易损性增加,提示其可能为慢性酗酒后,在脑震荡性打击下高死亡率发生的病理形态基础。
2、大鼠脑干、小脑、海马、额叶等脑区Tau蛋白、β-APP的表达和分布显示,慢性酒精中毒显著降低CNS骨架蛋白Tau表达、上调CNSβ-APP的表达,分析认为上述变化可能是长期慢性酗酒脑白质萎缩、神经突起数量减少的机制之一,参与了慢性酒精中毒TSAH死亡的病理代谢基础。
3、结合本课题组前期工作结果,认为酗酒相关的TSAH中,慢性酒精累积性CNS毒理学效应构成了神经损伤的重要病理学基础,可能是协同轻微的脑震荡性打击促进死亡的机制之一。
Background and objective
In recent years, alcoholism with a slight external force, causing traumatic subarachnoid hemorrhage (TSAH) to death shows a rising trend. TSAH was often reported alcohol-related, but its mechanism of occurrence and death has not yet been entirely clear. Our team has established a stable TSAH model after alcoholism in rats, with the incidence and mortality rate of TSAH 81% and 76%, in the chronic group, much higher than 27% and 4.8% in the acute group (P<0.01). Therefore, we proposed that the long-term cumulative toxicity of alcoholism on the central nervous system (CNS), especially that in the brain stem central life on the metabolism, function and morphology, commonly cause death with the synergistic effect of TSAH and concussion injury. This study is based on previous work, by observing neuronal axonal transport, signal transduction that is closely related to the Tau protein,β-APP expression and distribution, to further discuss the correlation of Tau Protein,β-APP and the mechanism of death associated with TSAH in cases of chronic alcoholism in rat brains.
Materials and Methods
1 Animals model and groups
CL-class male SD rats weighing 300±30 g, sub-conventional breeding were randomly divided: chronic water group(n=10), chronic water group with strike(n=10), chronic alcoholism group(n=10), chronic alcoholism group with strike(n=29), chronic alcoholism group stop drinking 1 month(n=6). Intragastric administration with drinking water or white wine (Beijing Red Star Erguotou, 52% v / v) for 4 weeks, dose: 2 weeks before 8 ml / kg / time, 2 weeks after 12 ml / kg / time, 9:00 and 16: 00 interval 7 hours twice one day ig.
2 Methods
Disposal of the rats after 4 w: chronic water group and chronic alcoholism group were killed by femoral artery bleeding after intraperitoneal injection of pentobarbital 2 h after the last gavage, chronic water group with strike and chronic alcoholism group with strike were given a concussion blow by a home-made device with pendulum, with a simultaneous monitor of ECG for 15 min. Two hours after strike the rats were killed using the above method. The dead rats in chronic alcoholism group with strike were classified as dead group. Immediately after death, the rats were fixed by perfusion of fixative (2.5% glutaraldehyde, 2% paraformaldehyde) for 30 min, using aortic cannulation. The whole brains were extracted and fixed in 4% neutral paraformaldehyde for 6 h. Sagittal sections of the brain were prepared for routine paraffin serial sections, HE, Bielschowsky's silver staining and Tau protein,β-APP immunohistochemical staining. The pathological changes were observed with light microscopy, image acquisition system, mining plans, Image-Pro Plus 6.0 image analysis software, cumulative integral optical density (IOD sum) and other quantitative pathological measurements. Brain tissues for electron microscopy were immediately placed in 2.5% glutaraldehyde, washed, fixed, embedded, sectioned and stained by conventional methods, and were observed using the JEM-1400 transmission electron microscope.
3 Statistical Analysis
All experimental data analysis was performed using the softs of SPSS 17.0 and Excel 2003, including analysis of variance (ANOVA) test, independent sample T test, chi-square test. P<0.05 or P<0.01is regarded as significant.
Results
1 General observation
For rats in chronic water group, diet and spirit were kept in good condition, and the weight increased continuously, especially in the second week, it had a significant increase (P<0.01). For rats in chronic alcoholism group, the hair was rough and dull, the mental state was poor. With the increase of alcoholism, gradually, the rats showed the loss of body weight, malnutrition, decrease of food intake and activity, body limp and other changes of chronic alcoholism. Chronic alcoholism rats decreased gradually in weight, with the weight after 1w, 3w, 4w was significantly lower than that before alcoholism (P<0.05).
There were no significant changes in the heart rate before and after strike for rats in chronic water group and chronic alcoholism group (P>0.05). For rats in chronic water group with strike, the electrocardiogram transiently showed slow heart rate, increased QRS amplitude waves for 0.3±0.16 s; for rats in chronic alcoholism group, heart rate slowed down, QRS wave amplitude significantly increased and recoveried after 1.7±0.36 s. The recovery time was significantly longer than that in the chronic water group with strike (P<0.05); dead rats significantly increased QRS amplitude, the heart rate slowed down after 10 s-1 min,then became a straight line.
TSAH morbidity and mortality in alcoholism group with strike were 79.3%, 51.7%, significantly higher than that in the water with strike group (P<0.05). According to Fisher grading, the grade of TSAH alcoholism with strike group was higher than that in water with strike group.
2 General observation and histopathological observation
In chronic water group, the brain surface was smooth without vascular texture. By HE staining, the neurons and glial cell were found to be normal in morphology and distribution. Nerve fibers arranged regularly and closely with uniform size and staining. Bielschowsky's silver staining showed that brain stem nerve fibers were straight and arranged closely. The brains of chronic water group with strike showed mild cerebral congestion and scattered and limited hemorrhage. Neurons and glial cells had mild edema, and Nissl bodies were stained more lightly with the number reduced. Nerve fibers were irregularly thicken, broken, had expanded perivitelline and arranged loosely, with no TSAH, contusion or other lesions.
Chronic alcoholism rats showed mild cerebral congestion on brain surface with vascular texture. Neurons arranged in disorder, reduced in number. Some of them sporadically had type 1 (pyknotic darkly stained) or type 2 (swelling lightly stained) changes. The cells were swollen and round, the nucleus increased in size and displaced. Central nissl bodies dissolved and disappeared; glial cells proliferated; cerebellar Purkinje cell shrinked, reduced in number, with uneven spacing. Nerve fibers were arranged slightly loosely, with increased gap, as well as mild irregular thickening and deformation, but there were no TSAH, contusion and other changes found. In chronic alcoholism group with strike, congestion was found in brain surface, with thick patchy TSAH found mainly in ventral brain stem, and no cerebral contusion found. Neurons and glial cells were in a high degree of hydropic degeneration, with the perivitelline space expanded, and more nucleus pyknosis, dissolved, disappeared. Central Nissl bodies displaced peripherally in the cell, dissolved, disappeared at different levels, neuronophagia and satellitosis were commonly found; neurons sporadically disappeared, gliosis proliferated; cerebellar Purkinje cells were unregularly reduced in number with uneven staining, density and pyknosis; red and dark neurons were found more commonly in medulla; nerve fibers were loose and swollen, cell gap was widened with irregular thickening, distort, break, and local swelling. Chronic alcoholism group with a stop of 1 month had milder pathological changs in neurons and nerve fibers than chronic alcoholism group.
3 TEM changes
In chronic water group, the brain stem was dense, nerve fiber axonal had uniform thickness, regular shape and high electron density. The myelin was dark with uniform thickness, and was tightly shafted with the membrane. Axoplasm and myelin of nerve fiber were dense and uniform. Nerve skeleton fiber had uniform thickness. Neuronal nuclei had rich mitochondria, Golgi complex, endoplasmic reticulum, which were regular in size, shape and arrangement, with ribosome commonly found. For chronic water group with strike, the brain tissue was loose, loose and swollen and lower electron density. Thickness and coloration of axonal nerve fiber and thickness of myelin were uneven. Skeleton of nerve fibers were loose and swollen. The myelin sporadically had layered fracture. Mitochondria of neurons were loose and swelled with its cristae expanded. Endoplasmic reticulum expanded mildly, and synaptic cleft widened mildly, and density of dense plaque was decreased.
For chronic alcoholism rats, the brain stem was loose and swollen, axonal nerve fibers were widespreadly uneven in thickness and shape, disorganized with the myelin layer irregular and uneven in thickness and density. Nerve fiber skeleton had uneven thickness with some of them dissolved. The myelin sporadically had layered fracture. Mitochondria of neurons was obviously swelling and loose, with various crest length and thickness, and the outer membrane disintegrated. Neurons with the pyknosis of cell body and nuclear were found, with the nuclear membrane folded, heterochromatin increased and clustered, nucleolar condensated; Glial cells increased in number and the interstitial was loose and swollen.
In chronic alcoholism with strike group, the myelin sheath of nerve fibers was highly loose with serious edema in the brain stem. Lamellar separation, folding, and retraction were found in the myelin sheath with floc attachment. Vesicle-like protrusions formed in part of the region and broke into the surrounding gap. The microtubules and microfilaments collapsed with some of them replaced by membranous structures and some membranous organelles (including cavitation mitochondria) clustering around them. The number of synapses reduced, synaptic vesicles accumulated; glial cells increased, interstitial occurred edema. Axoplasm and organelles accumulated gradually in the changed membranes and myelin-axis and resulted in reactive axonal swelling.
Size of neurons decreased, electron density of cytoplasm and nucleus increased, nuclear membrane folded, heterochromatin clustered, nucleolus disappeared or became smaller, the nuclear cytoplasm ratio increased, the extracellular gap was widened, the membrane was disintegrated; loose and swollen, mitochondria and endoplasmic reticulum loose swelling.
4 The expression of Tau,β-APP
4.1 The IOD sum of Tau
In normal rats, Tau protein was expressed mainly in the axons and the cytoplasm. In the chronic alcoholism group, the Tau IOD sum of brainstem, cerebellum, hippocampus, frontal lobe and other brain regions reduced significantly (P<0.05); In the chronic water group with strike, Tau IOD sum of the frontal lobe and other brain regions was significantly higher (P<0.05), but that of brain stem, cerebellum, hippocampus and other parts had no significant change (P>0.05); In the chronic alcoholism and strike group, the brainstem, cerebellum, hippocampus, frontal lobe and other parts of Tau IOD sum value was significantly higher (P<0.05). In the chronic alcoholism group with strike, Tau IOD sum of the brain stem, hippocampus, frontal lobe and other parts were significantly higher in the death rats than in the survival rats (P<0.05).
4.2 The IOD sum ofβ-APP
In normal rats,β-APP was mainly expressed in the neurons cytoplasm. In chronic alcoholism rats, the brain stem, cerebellum, hippocampus and other brain regions had significantly higher IOD sum (P<0.05), but that of frontal lobe not (P>0.05). In the chronic water group with strike,β-APP IOD sum was significantly higher in the brain stem, hippocampus and other brain regions (P<0.05), but not in cerebellum, frontal lobe and other parts (P>0.05). In the chronic alcoholism and strike group,β-APP IOD sum was significantly reduced in the rat brain stem, cerebellum, hippocampus, frontal lobe and other parts (P<0.05), but that in the dead rats were significantly higher than that in the survival (P<0.05).
Conclusions
1. Chronic alcoholism damaged the cytoskeleton structure of nerve fibers in CNS, might degrade the compensation of conduction, and lead to the increase of vulnerability, suggesting that it may be the pathological basis of the high mortality in concussion after chronic alcoholism.
2. Expression and distribution of Tau protein,β-APP in rat brain stem, cerebellum, hippocampus, frontal lobe suggested that chronic alcoholism significantly degraded the expression of Tau protein and caused overexpression ofβ-APP in CNS, as might be one of the mechanisms of white matter atrophy and reduction of neurite number, contribute the pathological basis in death of TSAH in chronic alcoholism.
3. Combined with the results of previous work in our team, consider that the toxicological cumulative effects of chronic alcoholism in CNS constitute an important pathological basis in nerve injury in TSAH of the alcoholism-related, possibly, one of the mechanisms to promote the death with mild concussion.
酗酒后轻微外力引起的致死性外伤性蛛网膜下腔出血(traumatic subarachnoid haemorrhage,TSAH)时有发生,TSAH常与酗酒有关,国内外学者对此已有共识,但其发生及死亡机制尚未完全清楚。本课题组前期已建立了稳定的大鼠灌酒后TSAH模型,结果显示,慢性灌酒组大鼠TSAH发生率及死亡率分别高达81%和76%,远高于急性灌酒组的27%和4.8%(P<0.01)。综合相关文献及前期实验结果,我们提出长期酗酒对中枢神经系统(central nerves systerm, CNS)代谢、功能和形态各方面的累积性毒理作用,可能构成协同脑震荡性损伤导致TSAH死亡的神经病理学基础的假设。本研究通过观察慢性灌酒中毒的CNS形态学变化,以及与神经纤维轴突运输、信号转导密切相关的Tau蛋白、β-APP表达状况,探讨酗酒与TSAH死亡机制的相关性。
材料与方法
1动物模型及分组
CL级雄性SD大鼠,体重300±30g,常规分笼饲养,随机分:灌水组10只、灌水打击组10只、灌酒组10只、灌酒打击组29只、灌酒停酒组6只。连续灌胃食用水或白酒(52%v/v北京红星二锅头)4周,剂量:前2周8 ml/kg/次、后2周12 ml/kg/次,每天9:00和16:00间隔7小时两次灌胃。
2方法
灌水组和灌酒组最后一次灌胃2h后,戊巴比妥腹腔注射麻醉后股动脉放血处死;灌水打击和灌酒打击组用自制单摆式打击装置给予脑震荡性打击,同时监测心电15min,存活大鼠打击后观测2h,戊巴比妥腹腔注射麻醉后股动脉放血处死,灌酒打击组中死亡的大鼠归为死亡组,各组均立即剖胸主动脉插管,含钙2%多聚甲醛-2.5%戊二醛固定液灌注固定30min,开颅提取全脑,置于4%中性多聚甲醛液固定6h,旁正中矢状切取脑组织,常规石蜡连续切片,分别HE、Bielschowsky’s镀银染色及Tau蛋白、β-APP免疫组织化学染色,光镜观察组织病理形态,图像采集系统观测、采图,Image-Pro Plus 6.0图像分析软件累积积分光密度(IOD sum)等量化病理学测量。
同时,备制透射电镜脑组织样本立即置入2.5%戊二醛中,按常规方法清洗、固定、包埋、切片、染色,在JEM-1400透射电子显微镜下观察。
3统计分析
所有实验数据均采用均数±标准差表示,通过SPSS 17.0及Excel 2003分析处理数据,检验方法采用方差分析(ANOVA)、独立样本T检验、卡方检验。统计学检验所得值差异显著性以P<0.05、P<0.01表示。
结果
1实验大鼠一般情况观察
灌水组饮食、活动状况良好,体重持续增加,第2w始体重增加明显(P<0.01)。灌酒组毛发粗糙、无光泽,精神状况差,并随着灌酒时间延长,逐渐出现体形消瘦、营养不良、进食量、活动减少、肢体瘫软等慢性酒精中毒改变。灌酒组大鼠灌酒后体重逐渐下降,第1w、第3w、第4w体重均明显下降,较灌酒前均差异显著(P<0.01)。
灌水打击组心电图呈一过性心率减慢、QRS波群波幅增大,0.3±0.16s后恢复;灌酒打击组大鼠QRS波群波幅明显增大,恢复时间1.7±0.36s,较灌水打击组明显延长(P<0.05);打击死亡组大鼠QRS波幅明显增大,10s-1min后心率进行性减慢后呈直线。
灌酒打击组TSAH发生率和死亡率分别为79.3%,51.7%,显著高于灌水打击组(P<0.01,P<0.05),根据Fisher分级法,灌酒打击组大鼠TSAH级别高于灌水打击组。
2常规大体和组织病理学观察
灌水组神经元、胶质细胞形态和分布正常,神经纤维粗细染色均匀、排列平直紧密。灌水打击组全脑轻度淤血,散在局限性小灶性出血,神经元、胶质细胞轻度水变性,尼氏体浅淡、数目减少,神经纤维不规则扭曲增粗、断裂、周隙扩大,排列疏松。
灌酒组全脑表面轻度淤血,神经元排列紊乱,形态不一,散在固缩深染1型变和肿胀淡染2型变神经元,胞体肿大变圆,核增大、偏位,中央性尼氏体溶解、消失;胶质细胞增生;小脑蒲肯野细胞固缩、数目减少、疏密不均;神经纤维排列稍疏松、间隙增大,轻度不规则增粗、变形。
灌酒打击组全脑表面弥漫淤血,脑干周围多见厚层TSAH,未见明显脑挫裂伤,神经元及神经胶质细胞高度水变性、周隙扩大,多见核固缩、溶解、消失,中央尼氏体不同程度边集、溶解、消失,多见噬神经及卫星现象;散在神经元脱失,胶质细胞增生;小脑蒲肯野细胞不规则减少,染色、疏密不均、核固缩;延脑多见红色神经元及暗神经元;神经纤维疏松水肿、周隙增宽,可见明显增粗、扭曲、断裂,局部肿胀明显。灌酒停酒组大鼠较灌酒组神经元及神经纤维病变有所缓解。
3透射电镜观察
灌水组脑干组织致密,神经纤维轴索粗细均匀、形态规整,电子密度较高,髓鞘深色、厚薄均匀,轴膜和髓鞘结合紧密,神经骨架纤维粗细均匀;神经元核、线粒体、高尔基复合体、内质网丰富,大小形态、排列规整,多见核糖体。灌水打击组组织较疏松水肿,电子密度较低,神经纤轴索粗细、深浅不均,髓鞘厚薄不一、散在裂隙分层;神经骨架纤维水肿、排列松散;神经元线粒体疏松肿胀、嵴扩张,内质网轻度扩大,突触间隙增宽、致密斑密度降低。
灌酒组脑干组织疏松水肿,神经轴索弥漫粗细、形态不均、排列紊乱,髓鞘不规则分层、厚薄不均,神经纤维轴浆疏密不均,神经骨架纤维粗细不一、部分溶解,髓鞘脂质散在裂隙分层,神经元线粒体明显疏松肿胀、嵴长短粗细不一、外膜不完整,可见胞体、核固缩神经元,核膜皱折、异染色质增多、聚集,核仁固缩;胶质细胞增多,间质疏松水肿。灌酒打击组脑干神经纤维髓鞘高度疏松水肿,可见板层分离、褶叠和内陷,絮状物附着,部分区域形成囊泡样突起突入周围间隙中;神经骨架纤维崩解,局部被絮状沉淀物和膜性结构所代替,周边可见膜性细胞器的积聚,可见空化的线粒体;突触数量减少,突触小泡堆积;胶质细胞增多,间质水肿;在轴膜和髓鞘改变区,轴浆和细胞器逐渐积聚,呈反应性轴突肿胀;神经元体积明显缩小,胞浆及核电子密度增高、核膜皱折,异染色质团块状聚集,核仁消失或变小,核浆比例增大,胞周缝隙增宽,胞膜不完整,线粒体和内质网明显疏松肿胀。灌酒停酒组大鼠较灌酒组神经元及神经纤维病变有所缓解,水肿减轻。
4免疫组化观察
4.1 Tau蛋白IOD sum值
正常组脑组织Tau蛋白主要在神经元的轴突和胞浆阳性表达。灌酒组脑干、小脑、海马、额叶等脑区Tau IOD sum值显著减小(P<0.05)。灌水打击组额叶脑区Tau IOD sum值显著增高(P<0.05),脑干、小脑、海马等部位Tau IOD sum值变化差异不显著(P>0.05)。慢性灌酒打击组脑干、小脑、海马、额叶等部位Tau IOD sum值显著增高(P<0.05)。慢性灌酒打击死亡组脑干、海马、额叶等部位Tau IOD sum值均高于慢性灌酒打击存活组(P<0.05)。
4.2β-APP IOD sum值
正常组β-APP主要在神经元胞浆阳性表达。灌酒组脑干、小脑、海马等脑区IOD sum值显著增高(P<0.05),额叶变化不显著(P>0.05)。灌水打击组脑干、海马等脑区β-APP IODsum值显著增高(P<0.05),小脑、额叶等部位差异不显著(P>0.05)。慢性灌酒打击组脑干、小脑、海马、额叶等部位β-APP IOD sum值显著减小(P<0.05)。慢性灌酒打击死亡组小脑、海马等部位β-APP IOD sum值均显著高于慢性灌酒打击存活组(P<0.05)。
结论
1、慢性酒精中毒可引起CNS神经纤维细胞骨架的形态结构破坏,降低传导功能的代偿性,导致易损性增加,提示其可能为慢性酗酒后,在脑震荡性打击下高死亡率发生的病理形态基础。
2、大鼠脑干、小脑、海马、额叶等脑区Tau蛋白、β-APP的表达和分布显示,慢性酒精中毒显著降低CNS骨架蛋白Tau表达、上调CNSβ-APP的表达,分析认为上述变化可能是长期慢性酗酒脑白质萎缩、神经突起数量减少的机制之一,参与了慢性酒精中毒TSAH死亡的病理代谢基础。
3、结合本课题组前期工作结果,认为酗酒相关的TSAH中,慢性酒精累积性CNS毒理学效应构成了神经损伤的重要病理学基础,可能是协同轻微的脑震荡性打击促进死亡的机制之一。
Background and objective
In recent years, alcoholism with a slight external force, causing traumatic subarachnoid hemorrhage (TSAH) to death shows a rising trend. TSAH was often reported alcohol-related, but its mechanism of occurrence and death has not yet been entirely clear. Our team has established a stable TSAH model after alcoholism in rats, with the incidence and mortality rate of TSAH 81% and 76%, in the chronic group, much higher than 27% and 4.8% in the acute group (P<0.01). Therefore, we proposed that the long-term cumulative toxicity of alcoholism on the central nervous system (CNS), especially that in the brain stem central life on the metabolism, function and morphology, commonly cause death with the synergistic effect of TSAH and concussion injury. This study is based on previous work, by observing neuronal axonal transport, signal transduction that is closely related to the Tau protein,β-APP expression and distribution, to further discuss the correlation of Tau Protein,β-APP and the mechanism of death associated with TSAH in cases of chronic alcoholism in rat brains.
Materials and Methods
1 Animals model and groups
CL-class male SD rats weighing 300±30 g, sub-conventional breeding were randomly divided: chronic water group(n=10), chronic water group with strike(n=10), chronic alcoholism group(n=10), chronic alcoholism group with strike(n=29), chronic alcoholism group stop drinking 1 month(n=6). Intragastric administration with drinking water or white wine (Beijing Red Star Erguotou, 52% v / v) for 4 weeks, dose: 2 weeks before 8 ml / kg / time, 2 weeks after 12 ml / kg / time, 9:00 and 16: 00 interval 7 hours twice one day ig.
2 Methods
Disposal of the rats after 4 w: chronic water group and chronic alcoholism group were killed by femoral artery bleeding after intraperitoneal injection of pentobarbital 2 h after the last gavage, chronic water group with strike and chronic alcoholism group with strike were given a concussion blow by a home-made device with pendulum, with a simultaneous monitor of ECG for 15 min. Two hours after strike the rats were killed using the above method. The dead rats in chronic alcoholism group with strike were classified as dead group. Immediately after death, the rats were fixed by perfusion of fixative (2.5% glutaraldehyde, 2% paraformaldehyde) for 30 min, using aortic cannulation. The whole brains were extracted and fixed in 4% neutral paraformaldehyde for 6 h. Sagittal sections of the brain were prepared for routine paraffin serial sections, HE, Bielschowsky's silver staining and Tau protein,β-APP immunohistochemical staining. The pathological changes were observed with light microscopy, image acquisition system, mining plans, Image-Pro Plus 6.0 image analysis software, cumulative integral optical density (IOD sum) and other quantitative pathological measurements. Brain tissues for electron microscopy were immediately placed in 2.5% glutaraldehyde, washed, fixed, embedded, sectioned and stained by conventional methods, and were observed using the JEM-1400 transmission electron microscope.
3 Statistical Analysis
All experimental data analysis was performed using the softs of SPSS 17.0 and Excel 2003, including analysis of variance (ANOVA) test, independent sample T test, chi-square test. P<0.05 or P<0.01is regarded as significant.
Results
1 General observation
For rats in chronic water group, diet and spirit were kept in good condition, and the weight increased continuously, especially in the second week, it had a significant increase (P<0.01). For rats in chronic alcoholism group, the hair was rough and dull, the mental state was poor. With the increase of alcoholism, gradually, the rats showed the loss of body weight, malnutrition, decrease of food intake and activity, body limp and other changes of chronic alcoholism. Chronic alcoholism rats decreased gradually in weight, with the weight after 1w, 3w, 4w was significantly lower than that before alcoholism (P<0.05).
There were no significant changes in the heart rate before and after strike for rats in chronic water group and chronic alcoholism group (P>0.05). For rats in chronic water group with strike, the electrocardiogram transiently showed slow heart rate, increased QRS amplitude waves for 0.3±0.16 s; for rats in chronic alcoholism group, heart rate slowed down, QRS wave amplitude significantly increased and recoveried after 1.7±0.36 s. The recovery time was significantly longer than that in the chronic water group with strike (P<0.05); dead rats significantly increased QRS amplitude, the heart rate slowed down after 10 s-1 min,then became a straight line.
TSAH morbidity and mortality in alcoholism group with strike were 79.3%, 51.7%, significantly higher than that in the water with strike group (P<0.05). According to Fisher grading, the grade of TSAH alcoholism with strike group was higher than that in water with strike group.
2 General observation and histopathological observation
In chronic water group, the brain surface was smooth without vascular texture. By HE staining, the neurons and glial cell were found to be normal in morphology and distribution. Nerve fibers arranged regularly and closely with uniform size and staining. Bielschowsky's silver staining showed that brain stem nerve fibers were straight and arranged closely. The brains of chronic water group with strike showed mild cerebral congestion and scattered and limited hemorrhage. Neurons and glial cells had mild edema, and Nissl bodies were stained more lightly with the number reduced. Nerve fibers were irregularly thicken, broken, had expanded perivitelline and arranged loosely, with no TSAH, contusion or other lesions.
Chronic alcoholism rats showed mild cerebral congestion on brain surface with vascular texture. Neurons arranged in disorder, reduced in number. Some of them sporadically had type 1 (pyknotic darkly stained) or type 2 (swelling lightly stained) changes. The cells were swollen and round, the nucleus increased in size and displaced. Central nissl bodies dissolved and disappeared; glial cells proliferated; cerebellar Purkinje cell shrinked, reduced in number, with uneven spacing. Nerve fibers were arranged slightly loosely, with increased gap, as well as mild irregular thickening and deformation, but there were no TSAH, contusion and other changes found. In chronic alcoholism group with strike, congestion was found in brain surface, with thick patchy TSAH found mainly in ventral brain stem, and no cerebral contusion found. Neurons and glial cells were in a high degree of hydropic degeneration, with the perivitelline space expanded, and more nucleus pyknosis, dissolved, disappeared. Central Nissl bodies displaced peripherally in the cell, dissolved, disappeared at different levels, neuronophagia and satellitosis were commonly found; neurons sporadically disappeared, gliosis proliferated; cerebellar Purkinje cells were unregularly reduced in number with uneven staining, density and pyknosis; red and dark neurons were found more commonly in medulla; nerve fibers were loose and swollen, cell gap was widened with irregular thickening, distort, break, and local swelling. Chronic alcoholism group with a stop of 1 month had milder pathological changs in neurons and nerve fibers than chronic alcoholism group.
3 TEM changes
In chronic water group, the brain stem was dense, nerve fiber axonal had uniform thickness, regular shape and high electron density. The myelin was dark with uniform thickness, and was tightly shafted with the membrane. Axoplasm and myelin of nerve fiber were dense and uniform. Nerve skeleton fiber had uniform thickness. Neuronal nuclei had rich mitochondria, Golgi complex, endoplasmic reticulum, which were regular in size, shape and arrangement, with ribosome commonly found. For chronic water group with strike, the brain tissue was loose, loose and swollen and lower electron density. Thickness and coloration of axonal nerve fiber and thickness of myelin were uneven. Skeleton of nerve fibers were loose and swollen. The myelin sporadically had layered fracture. Mitochondria of neurons were loose and swelled with its cristae expanded. Endoplasmic reticulum expanded mildly, and synaptic cleft widened mildly, and density of dense plaque was decreased.
For chronic alcoholism rats, the brain stem was loose and swollen, axonal nerve fibers were widespreadly uneven in thickness and shape, disorganized with the myelin layer irregular and uneven in thickness and density. Nerve fiber skeleton had uneven thickness with some of them dissolved. The myelin sporadically had layered fracture. Mitochondria of neurons was obviously swelling and loose, with various crest length and thickness, and the outer membrane disintegrated. Neurons with the pyknosis of cell body and nuclear were found, with the nuclear membrane folded, heterochromatin increased and clustered, nucleolar condensated; Glial cells increased in number and the interstitial was loose and swollen.
In chronic alcoholism with strike group, the myelin sheath of nerve fibers was highly loose with serious edema in the brain stem. Lamellar separation, folding, and retraction were found in the myelin sheath with floc attachment. Vesicle-like protrusions formed in part of the region and broke into the surrounding gap. The microtubules and microfilaments collapsed with some of them replaced by membranous structures and some membranous organelles (including cavitation mitochondria) clustering around them. The number of synapses reduced, synaptic vesicles accumulated; glial cells increased, interstitial occurred edema. Axoplasm and organelles accumulated gradually in the changed membranes and myelin-axis and resulted in reactive axonal swelling.
Size of neurons decreased, electron density of cytoplasm and nucleus increased, nuclear membrane folded, heterochromatin clustered, nucleolus disappeared or became smaller, the nuclear cytoplasm ratio increased, the extracellular gap was widened, the membrane was disintegrated; loose and swollen, mitochondria and endoplasmic reticulum loose swelling.
4 The expression of Tau,β-APP
4.1 The IOD sum of Tau
In normal rats, Tau protein was expressed mainly in the axons and the cytoplasm. In the chronic alcoholism group, the Tau IOD sum of brainstem, cerebellum, hippocampus, frontal lobe and other brain regions reduced significantly (P<0.05); In the chronic water group with strike, Tau IOD sum of the frontal lobe and other brain regions was significantly higher (P<0.05), but that of brain stem, cerebellum, hippocampus and other parts had no significant change (P>0.05); In the chronic alcoholism and strike group, the brainstem, cerebellum, hippocampus, frontal lobe and other parts of Tau IOD sum value was significantly higher (P<0.05). In the chronic alcoholism group with strike, Tau IOD sum of the brain stem, hippocampus, frontal lobe and other parts were significantly higher in the death rats than in the survival rats (P<0.05).
4.2 The IOD sum ofβ-APP
In normal rats,β-APP was mainly expressed in the neurons cytoplasm. In chronic alcoholism rats, the brain stem, cerebellum, hippocampus and other brain regions had significantly higher IOD sum (P<0.05), but that of frontal lobe not (P>0.05). In the chronic water group with strike,β-APP IOD sum was significantly higher in the brain stem, hippocampus and other brain regions (P<0.05), but not in cerebellum, frontal lobe and other parts (P>0.05). In the chronic alcoholism and strike group,β-APP IOD sum was significantly reduced in the rat brain stem, cerebellum, hippocampus, frontal lobe and other parts (P<0.05), but that in the dead rats were significantly higher than that in the survival (P<0.05).
Conclusions
1. Chronic alcoholism damaged the cytoskeleton structure of nerve fibers in CNS, might degrade the compensation of conduction, and lead to the increase of vulnerability, suggesting that it may be the pathological basis of the high mortality in concussion after chronic alcoholism.
2. Expression and distribution of Tau protein,β-APP in rat brain stem, cerebellum, hippocampus, frontal lobe suggested that chronic alcoholism significantly degraded the expression of Tau protein and caused overexpression ofβ-APP in CNS, as might be one of the mechanisms of white matter atrophy and reduction of neurite number, contribute the pathological basis in death of TSAH in chronic alcoholism.
3. Combined with the results of previous work in our team, consider that the toxicological cumulative effects of chronic alcoholism in CNS constitute an important pathological basis in nerve injury in TSAH of the alcoholism-related, possibly, one of the mechanisms to promote the death with mild concussion.
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