急慢性酒精中毒大鼠脑组织Cyt-C、Caspase-9、Caspase-3的表达与TSAH死亡机制的研究
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摘要
背景与目的外伤性蛛网膜下腔出血(traumatic subarachnoid haemorrhage,TSAH)是一种不同于与脑
挫伤所致的蛛网膜下腔出血和病理性蛛网膜下腔出血的独立脑蛛网膜下腔出血,与外伤性硬脑膜外出血、硬脑膜下腔出血并列为外伤性颅内三大腔隙出血。国内外学者报道的TSAH常与酗酒有关。本课题组的前期工作已建立了急慢性灌酒后大鼠TSAH模型,实验结果显示:灌酒后大鼠TSAH出血率:急性组28.6%、慢性组82.4%,死亡率:急性组0、慢性组58.8%,提示慢性灌酒TSAH死亡率远大于急性灌酒组,从脑组织Ngb、Hif-1α、Na+,K+-ATPase等氧和能量代谢、tPA和MMP-9等血管壁和神经细胞毒性、血管形态结构及其生物力学三个方面,探讨了饮酒促发TSAH的发生机制,特别是发现了暗神经元细胞。但是,尚不清楚慢性灌酒大鼠TSAH高死亡率,是否与这些暗暗神经元细胞有关及其与死亡机制的关系。而这正是TSAH死亡案件的法医学鉴定和死因分析,及其司法审判量刑的一个重要而迫切的问题之一。
由于Cyt-C→Caspase-9→Caspase-3三者是一条重要凋亡通路,与脑功能状态密切相关,因此,本研究旨在前期建立的大鼠灌酒后TSAH模型基础上,进一步观察脑组织Cyt-C、Caspase-9、Caspase-3表达及其与脑干神经核暗细胞的关系,从分子生物学、毒理学及形态学角度探讨慢性灌酒后大鼠TSAH死亡机制。
材料与方法
1动物及分组:雄性SD大鼠80只,体重300~400g,常规分笼饲养,随机分组。
1.1急性灌酒模型:40只大鼠随机分灌酒组,一次性胃内灌酒(北京红星酒厂二锅头,52%v/v)15 ml/ kg;灌水组灌胃同等计量的食用自来水。再分别于灌酒或灌水后2 h,给予脑震荡性打击,建立灌酒打击组和灌水打击组模型,每组各10只。
1.2慢性灌酒模型:40只大鼠随机分灌酒组和灌水组,给予胃内灌酒(北京红星酒厂二锅头,52%v/v)或灌水四周,均每天9:00和15:00间隔6小时灌两次,前2周每次灌8 ml/kg、后2周每次灌12 ml/kg。再分别于最后一次灌酒或灌水后2 h,给予脑震荡性打击,建立灌酒打击组和灌水打击组明显,每组各10只。2方法各灌酒或灌水打击组大鼠,均于打击后0.5 h,开胸暴露心脏,经左心室-主动脉插管,灌注4%中性多聚甲醛液处死兼固定,剪开右心耳放血。约0.5 h后开颅取全脑,水冲洗后,置于4%中性多聚甲醛固定6小时,脱水、浸蜡、包埋,常规制作石蜡组织切片厚约5 um,分别HE染色观察脑组织形态、暗神经元数量变化;Cyt-C、Caspase-9、Caspase-3免疫组织化学染色,观察3种因子表达情况。真彩色病理图像分析系统(版本4.0)进行体视量化病理组织学观测。实验数据用SPSS13.0统计软件进行分析处理(Independent-samples T-test and One-way ANONA)。
结果
1血酒精浓度变化和行为学表现急性灌酒后2 h时大鼠血酒精浓度114.67±67.37 mg/dL,灌酒后短时间内,大鼠兴奋性较强、步态不稳,不停靠墙走动;0.5 h后逐渐出现侧卧、翻正反射消失、昏睡、呼吸缓慢等急性酒精中毒表现;2 h后呼吸频率63.5±9.4次/分和血压106.1±4.6 mmHg均较灌酒前85.1±9.3次/分和133.8±5.5 mmHg显著下降(P<0.01)。单纯灌水组灌水前后血压、呼吸无明显变化。急性灌酒打击组和灌水打击组大鼠,打击后0.5 h血压121.1±17.6 mmHg较打击前106.5±10.1 mmHg明显升高(P<0.05),呼吸频率未见明显改变。
慢性灌酒组大鼠饲养期间逐渐出现毛发粗糙发黄、无光泽、精神萎靡、两眼无神、进食量减少、基础血压升高、体重增长缓慢或下降等。2周后体重为372.8±22.2 g较灌酒前的389.7±31.7 g明显下降(P<0.05);灌水组大鼠第4周体重为405.1±20.8 g较灌水前的390.1±24.7 g增长明显(P<0.05);大鼠灌酒后第四周基础血压为152.3±13.5 mmHg较灌酒前的135.0±11.2 mmHg上升(P<0.05),最后一次灌酒后血压为140.3±10.4 mmHg较灌酒前的152.3±13.5 mmHg低(P<0.05),打击后升高为151.2±10.6 mmHg (P<0.05)。灌水打击组打击后血压为148.1±13.5 mmHg较打击前137.1±12.6 mmHg高(P<0.05)。单纯灌水组大鼠基础血压未见明显改变。灌酒和灌水组大鼠基础呼吸频率均未发生变化,仅每次灌酒后出现呼吸频率降低(P<0.05),打击后呼吸频率未见明显改变(P>0.05)。
2 TSAH、SAH发生率、死亡率及病理学观察急性灌酒打击组大鼠TSAH发生率20%,无死亡。慢性灌酒打击组的TSAH发生率50%,死亡率约40%。急慢性灌水打击组均未发现TSAH。急性单纯灌酒对照组1只出现自发性SAH。
急性单纯灌水组大鼠脑表面光泽,表面散在细小血管纹理,未见暗神经元细胞。急性灌水打击组大鼠脑表面较苍白,细小血管纹理细少或消失,未见暗神经元细胞。急性单纯灌酒组脑表面光泽,斑片状淤血,发生自发性SAH的脑干腹侧薄层斑片状蛛网膜下腔出血。急性灌酒打击组大鼠TSAH呈薄层斑片状出血,主要分布在脑干腹侧面,脑组织神经元轻度水变性、细胞周隙略扩大,偶见固缩深染的暗神经元细胞、尼氏小体消失。慢性单纯灌酒组大鼠脑表面光泽,散在淤血,可见散在暗神经元细胞,呈暗紫红色均质化、胞体皱缩、胞浆及胞核浓缩,核不清或消失,轴突不规则扭曲,多见嗜神经现象。慢性灌酒打击组大鼠发生TSAH的脑表面大片较厚层蛛网膜下腔出血,主要分布在脑干腹侧面,神经元及胶质细胞中度水变性、细胞周隙明显扩大、尼氏小体消失,多见散在暗神经元细胞。
3 Cyt-C、Caspase-9、Caspase-3的表达
急性灌水打击组大鼠神经元胞浆Cyt-C、Caspase-9、Caspase-3均表达较弱,阳性细胞数分别为7.0±1.83、6.8±1.54、6.7±1.13,各脑区之间无差异(P>0.05)。急性灌酒打击组Cyt-C、Caspase-9、Caspase-3的IOD值3550.6±285.69、3061.5±365.14、2950.5±266.76高于急性灌水打击组的1875.7±297.05、1606.6±225.18、1675.7±295.82 (P<0.05)。急性单纯灌水组和急性灌水打击组之间以及急性单纯灌酒组和急性灌酒打击组之间阳性细胞数和IOD值无差异(P>0.05)。慢性单纯灌酒组大鼠神经元Cyt-C、Caspase-9、Caspase-3阳性细胞数20.58±5.26、15.2±3.61、15.8±3.80和IOD值3871.9±248.82、3397.3±373.22、3401.7±305.57均高于慢性单纯灌水组的6.7±1.85、6.8±1.55、6.3±0.91和1838.9±238.71、1692.4±273.98、1674.7±241.04(P<0.01)和急性单纯灌酒组的12.4±3.31、9.0±2.15、10.9±2.48和3448.3±297.48、2920.6±230.96、2844.8±240.61(P<0.05);慢性灌酒打击组大鼠神经元Cyt-C、Caspase-9、Caspase-3阳性细胞数22.78±5.63、17.3±3.56、18.1±3.99和IOD值4182.4±289.90、3826.3±378.04、3728.6±300.77均高于慢性单纯灌酒组的20.58±5.26、15.2±3.61、15.8±3.80和3871.9±248.82、3397.3±373.22、3401.7±305.57(P<0.05)和慢性灌水打击组的8.1±2.0、6.7±1.62、6.8±1.40和1891.43±300.62、1681.6±224.36、1686.5±280.03(P<0.01)。
4暗神经元的表达
急性灌酒组大鼠脑组织偶见暗神经元,灌水组未见暗神经元;慢性灌酒打击组大鼠脑组织暗神经元数量为10.5±2.24多于慢性单纯灌酒组的9.0±2.29 (P<0.05)。暗神经元的面积1204.8±390.82 um2小于正常神经元的1905.8±913.71 um2 (P<0.05),周长和平均直径分别为138.02±19.36和41.76±6.06均较正常神经元的167.07±41.03和50.56±12.10短(P>0.05)。
结论
1、急慢性酒精中毒可致明显外伤性蛛网膜下腔出血的发生,尤其是慢性酗酒的TSAH发生率及死亡率均高于急性醉酒的,提示酗酒者TSAH案件的法医学分析,应考虑慢性酗酒神经中枢毒理性因素,酗酒与轻微头颈部外力打击应属于协同死因。
2、急慢性酒精中毒可引起脑组织Cyt-C、Caspase-9、Caspase-3表达增强,与暗神经元的发生之间存在关联性,特别是慢性酒精中毒脑组织暗神经元数量明显增多,提示酒精及其代谢产物可激活Cyt-C→Caspase-9→Caspase-3凋亡途径,参与CNS暗神经元的形成,及酒精性脑萎缩、酒精性脑病等形态和功能障碍。
3、从原因、形态和机制各方面表明中枢神经系统的暗神经元不同于一般的组织细胞萎缩性病变,可能为细胞凋亡的早期过渡状态,为一种可逆性病理变化,可能构成慢性酒精中毒及其TSAH高死亡率的神经病理学基础,进而参与其死亡机制。
Background and objective
Traumatic subarachnoid haemorrhage(TSAH) is an independent brain damage, and it differs from pathological subarachnoid haemorrhage and haemorrhage caused by cerebral contusion. TSAH makes up the big three cavity haemorrhage with traumatic haemorrhage of cerebral dura mater and cerebral dura mater hypostegal cavity. It is reported TSAH is related with insobriety by foreigners. After slight beating on the brains of fuddled rats, the chronic fuddled group had a higher death rate(58.8% to 0, to the acute group) and rate of incidence (82.4% to 28.6%, to the acute group), and the results indicated that the oxygen and energy metabolism(Ngb、Hif-1α、Na+,K+-ATPase), vessel wall and cytotoxicity(tPA and MMP-9), blood vessel vitodynamics participate the TSAH mechanism, especially we saw the dark neuron in the chronic group, but we didn’t know wether the dark neuron was related with the TSAH mechanism. So we established TSAH model in fuddled rats, to observe the expression of Cyt-C、Caspase-9、Caspase-3 and the quantity of dark neuron, the effect of drinking upon apoptosis, function and energy metabolism of neurons, and we expect to explain the possible death mechanism of TSAH from molecular biology, toxicology and morphology.
The important apoptosis pathway Cyt-C→Caspase-9→Caspase-3 is related with the functional status of brain, so we detect the expression of Cyt-C、Caspase-9、Caspase-3 and dark neuron in the basic of TSAH model to approach the TSAH mechanism from molecular biology, toxicology, morphous. Materials and method
80 Male SD(Sprague-Dawley) rats, 300~400g, randomization. Acute fuddled model: the fuddled group 20 Male SD rats, the edible alcohol of Erguotou of Peking(52%v/v)was injected into rats’stomach through esophagus, 15ml/kg for one time. The control group 20 Male SD rats, Used same amount of tap water in the control group. 2h later gave them cerebral concussion beat. Chronic fuddled model: the fuddled group 20 Male SD rats, the edible alcohol of Erguotou of Peking(52%v/v)was injected into rats’stomach through esophagus, 2 times per day at 6 hours’intervals, 8ml/kg each time in the first 2 weeks and 12ml/kg each time in the next 2 weeks for four weeks in total. Used same amount of tap water in the control group. 2h later at the last time of fuddled gave them cerebral concussion beat. group n dosage liquid Chronic fuddled group 10 8;12ml/kg edible wine Chronic fuddled beaten group 10 8;12ml/kg edible wine Chronic tap water control group 10 8;12ml/kg tap water Chronic tap water beaten control group 10 8;12ml/kg tap water
The beaten group was exposed heart after 0.5h, aortic cannula,injected 4% neutral paraform, at the same time cut the auricle of right atrium. About 0.5h later, open the skull to acquire the brain, flash it with the tap water, then put it into the 4% neutral Polyoxymethylene, to fix 6h, anhydration, to soak cere, embed, make parraffin section(5um) routinely.The changes of the expression of Cyt-C, Caspase-9 and Caspase-3 and the quantity of dark neuron, histomorphology were observed by immunohistochemistry and HE, and the data was tested with SPSS13.0 statistic software(Independent-samples T-test and One-way ANONA).
Result
1. BAC and animal’s behavior The BAC was 114.67±67.37 mg/dL after 2h of gavaging wine. The rats excited a short time after drinking: moving continuously and unstably. About 0.5h later, they had some clinical manifestation of acute alcoholism such as lying on the side, hypnody, body-righting reflex disappeared, the respiration torpidity. 2h later, the breathing frequency(63.5±9.4 f/min to 85.1±9.3 f/min) and blood pressure(106.1±4.6 mmHg to 133.8±5.5 mmHg ) decreased(P<0.01) compared with before, but the control group had no change; 0.5h after being beaten, the blood pressure of acute fuddled group increased(121.1±17.6 mmHg to 106.5±10.1 mmHg)(P<0.05), but the breathing frequency had no change. In the chronic group, the hair became rough and dim, the spirit became frustrated, rats were weightless and decreased food-intake, the basic blood pressure increased, body weight decreased(372.8±22.2 g to 389.7±31.7 g) after 2 weeks (P<0.05), the body weight of control group increased(405.1±20.8 g to 390.1±24.7 g) after 4 weeks(P<0.05); 4 weeks later, the blood pressure of chronic fuddled group(152.3±13.5 mmHg to 135.0±11.2 mmHg)rised(P<0.05), after the last gavaging wine, the blood pressure(140.3±10.4 mmHg to 152.3±13.5 mmHg)decreased(P<0.05), and rised(151.2±10.6 mmHg) after being beaten(P<0.05), the control group also rised(148.1±13.5 mmHg to 137.1±12.6 mmHg) after being beaten(P<0.05), but the basic blood pressure of control group didn’t change; the breathing frequency of fuddled group and control group also didn’t change, just decreased after gavaging wine(P<0.05).
2. Incidence rate and death rate of TSAH and SAH and histomorphology
The incidence rate and death rate of TSAH are 20% and 0 in acute group, but 50% and 40% in chronic group, The incidence rate of spontaneous SAH are 10% in acute group and 0 in chronic group, death rate are both 0.
Minute blood vessel could be seen in the surface of brain in the control group, but it was pale after being beaten, while it was a little red in the acute fuddled group. In the acute fuddled group the neuron was slightly hydropic degeneration, the gap of cell was enlarged, and dark neurons could be seen by chance, nissl's body was disappeared. Lamellar and patching blood could be seen in the TSAH rats’brain, especially in the ventri-brainstem. Thick layer blood could be seen in the surface brain of TSAH in the chronic fuddled beaten group. The neuron and gliacyte were midrange hydropic degeneration, the gap of cell was enlarged, nissl's body disappeared in the chronic fuddle beaten group, and dark neurons could be seen, it was prunosus, cell body shrinkage, clarifixation, sometimes the nucelus disappeared, axis cylinder irregular twist, neuronophagia could be seen, the quantity of neuron decreased, the gliacyte hyperplasy.
3. The expression changes of Cyt-C、Caspase-9 and Caspase-3
The expression of Cyt-C、Caspase-9 and Caspase-3 in brain tissue’s neuron of normal rats was feeble, and a little neuron express in the acute group, There was no difference of the positive cells and IOD of Cyt-C、Caspase-9 and Caspase-3 in the brain between the acute fuddled group and fuddled beaten group(P>0.05), and so was the acute tap water group and the acute tap water beaten group, but the IOD of acute fuddled beaten group(3550.6±285.69,3061.5±365.14, 2950.5±266.76) is more than the control group(1875.7±297.05,1606.6±225.18,1675.7±295.82) (P<0.05). In chronic fuddled group, the positive cells and the IOD of Cyt-C、Caspase-9 and Caspase-3(20.58±5.26,15.2±3.61,15.8±3.80 and 3871.9±248.82,3397.3±373.22,3401.7±305.57) were more than those in the acute fuddled group(12.4±3.31,9.0±2.15,10.9±2.48 and 3448.3±297.48,2920.6±230.96,2844.8±240.61)(P<0.05) and the control group(6.7±1.85, 6.8±1.55,6.3±0.91 and 1838.9±238.71,1692.4±273.98,1674.7±241.04)(P<0.01), In chronic fuddled beaten group, the positive cells and IOD of Cyt-C、Caspase-9 and Caspase-3 (22.78±5.63,17.3±3.56,18.1±3.99 and 4182.4±289.90,3826.3±378.04,3728.6±300.77) was higher than the chronic fuddled group(P<0.05) and the control group(8.1±2.0,6.7±1.62, 6.8±1.40 and 1891.43±300.62,1681.6±224.36,1686.5±280.03)(P<0.01).
4. The expression changes of dark neuron
It could be seen dark neurons in the acute fuddled group by chance, and no dark neurons were seen in the control group. While there many dark neurons can be seen in the chronic group, and quantity of the chronic fuddled beaten group(10.9±1.52,9.7±1.77,9.1±1.85) was more than the chronic fuddled group(9.2±1.75,7.8±1.69,7.1±1.64)(P<0.05) except the frontal(12.8±1.99 to 11.6±1.43). The area of dark neuron(1204.8±390.82 um2) was smaller than the normal neuron(1905.8±913.71 um2)(P<0.05), the perimeter(138.02±19.36 to 167.07±41.03) and the average diameter(41.76±6.06 to 50.56±12.10) were both shorter than the normal neuron(P>0.05).
Conclusion
1. There were closely correlations between slight violent beaten after fuddled and the higher incidence rate and death rate of TSAH. Especially the incidence rate and the death rate of chronic fuddled group was higher than the acute group, which indicated that the drinking should be set into consideration sufficiently while analyzing these cases of TSAH after drinking in forensic medicine. The slight external force and insobriety were synergism cause of death.
2. Alcoholism could make high expression of Cyt-C, Caspase-9 and Caspase-3 which was related with the dark neuron, especially in the chronic alcoholism brain there were many dark neurons that indicated alcohol and its metabolic product could activate the apoptosis pathway Cyt-C→Caspase-9→Caspase-3, which may participated the formation of dark neuron and play the roles on atrophy of brain, alcoholic encephalopathy and functional disturbance.
3. The dark neuron was not the same as atrophy cell from morphology, cause and mechanism, maybe it was a kind of transient status, it could bccome apoptosis cell or recovery or necrosis. So the dark neuron may participate the TSAH death mechanism as the base of neuropathology.
挫伤所致的蛛网膜下腔出血和病理性蛛网膜下腔出血的独立脑蛛网膜下腔出血,与外伤性硬脑膜外出血、硬脑膜下腔出血并列为外伤性颅内三大腔隙出血。国内外学者报道的TSAH常与酗酒有关。本课题组的前期工作已建立了急慢性灌酒后大鼠TSAH模型,实验结果显示:灌酒后大鼠TSAH出血率:急性组28.6%、慢性组82.4%,死亡率:急性组0、慢性组58.8%,提示慢性灌酒TSAH死亡率远大于急性灌酒组,从脑组织Ngb、Hif-1α、Na+,K+-ATPase等氧和能量代谢、tPA和MMP-9等血管壁和神经细胞毒性、血管形态结构及其生物力学三个方面,探讨了饮酒促发TSAH的发生机制,特别是发现了暗神经元细胞。但是,尚不清楚慢性灌酒大鼠TSAH高死亡率,是否与这些暗暗神经元细胞有关及其与死亡机制的关系。而这正是TSAH死亡案件的法医学鉴定和死因分析,及其司法审判量刑的一个重要而迫切的问题之一。
由于Cyt-C→Caspase-9→Caspase-3三者是一条重要凋亡通路,与脑功能状态密切相关,因此,本研究旨在前期建立的大鼠灌酒后TSAH模型基础上,进一步观察脑组织Cyt-C、Caspase-9、Caspase-3表达及其与脑干神经核暗细胞的关系,从分子生物学、毒理学及形态学角度探讨慢性灌酒后大鼠TSAH死亡机制。
材料与方法
1动物及分组:雄性SD大鼠80只,体重300~400g,常规分笼饲养,随机分组。
1.1急性灌酒模型:40只大鼠随机分灌酒组,一次性胃内灌酒(北京红星酒厂二锅头,52%v/v)15 ml/ kg;灌水组灌胃同等计量的食用自来水。再分别于灌酒或灌水后2 h,给予脑震荡性打击,建立灌酒打击组和灌水打击组模型,每组各10只。
1.2慢性灌酒模型:40只大鼠随机分灌酒组和灌水组,给予胃内灌酒(北京红星酒厂二锅头,52%v/v)或灌水四周,均每天9:00和15:00间隔6小时灌两次,前2周每次灌8 ml/kg、后2周每次灌12 ml/kg。再分别于最后一次灌酒或灌水后2 h,给予脑震荡性打击,建立灌酒打击组和灌水打击组明显,每组各10只。2方法各灌酒或灌水打击组大鼠,均于打击后0.5 h,开胸暴露心脏,经左心室-主动脉插管,灌注4%中性多聚甲醛液处死兼固定,剪开右心耳放血。约0.5 h后开颅取全脑,水冲洗后,置于4%中性多聚甲醛固定6小时,脱水、浸蜡、包埋,常规制作石蜡组织切片厚约5 um,分别HE染色观察脑组织形态、暗神经元数量变化;Cyt-C、Caspase-9、Caspase-3免疫组织化学染色,观察3种因子表达情况。真彩色病理图像分析系统(版本4.0)进行体视量化病理组织学观测。实验数据用SPSS13.0统计软件进行分析处理(Independent-samples T-test and One-way ANONA)。
结果
1血酒精浓度变化和行为学表现急性灌酒后2 h时大鼠血酒精浓度114.67±67.37 mg/dL,灌酒后短时间内,大鼠兴奋性较强、步态不稳,不停靠墙走动;0.5 h后逐渐出现侧卧、翻正反射消失、昏睡、呼吸缓慢等急性酒精中毒表现;2 h后呼吸频率63.5±9.4次/分和血压106.1±4.6 mmHg均较灌酒前85.1±9.3次/分和133.8±5.5 mmHg显著下降(P<0.01)。单纯灌水组灌水前后血压、呼吸无明显变化。急性灌酒打击组和灌水打击组大鼠,打击后0.5 h血压121.1±17.6 mmHg较打击前106.5±10.1 mmHg明显升高(P<0.05),呼吸频率未见明显改变。
慢性灌酒组大鼠饲养期间逐渐出现毛发粗糙发黄、无光泽、精神萎靡、两眼无神、进食量减少、基础血压升高、体重增长缓慢或下降等。2周后体重为372.8±22.2 g较灌酒前的389.7±31.7 g明显下降(P<0.05);灌水组大鼠第4周体重为405.1±20.8 g较灌水前的390.1±24.7 g增长明显(P<0.05);大鼠灌酒后第四周基础血压为152.3±13.5 mmHg较灌酒前的135.0±11.2 mmHg上升(P<0.05),最后一次灌酒后血压为140.3±10.4 mmHg较灌酒前的152.3±13.5 mmHg低(P<0.05),打击后升高为151.2±10.6 mmHg (P<0.05)。灌水打击组打击后血压为148.1±13.5 mmHg较打击前137.1±12.6 mmHg高(P<0.05)。单纯灌水组大鼠基础血压未见明显改变。灌酒和灌水组大鼠基础呼吸频率均未发生变化,仅每次灌酒后出现呼吸频率降低(P<0.05),打击后呼吸频率未见明显改变(P>0.05)。
2 TSAH、SAH发生率、死亡率及病理学观察急性灌酒打击组大鼠TSAH发生率20%,无死亡。慢性灌酒打击组的TSAH发生率50%,死亡率约40%。急慢性灌水打击组均未发现TSAH。急性单纯灌酒对照组1只出现自发性SAH。
急性单纯灌水组大鼠脑表面光泽,表面散在细小血管纹理,未见暗神经元细胞。急性灌水打击组大鼠脑表面较苍白,细小血管纹理细少或消失,未见暗神经元细胞。急性单纯灌酒组脑表面光泽,斑片状淤血,发生自发性SAH的脑干腹侧薄层斑片状蛛网膜下腔出血。急性灌酒打击组大鼠TSAH呈薄层斑片状出血,主要分布在脑干腹侧面,脑组织神经元轻度水变性、细胞周隙略扩大,偶见固缩深染的暗神经元细胞、尼氏小体消失。慢性单纯灌酒组大鼠脑表面光泽,散在淤血,可见散在暗神经元细胞,呈暗紫红色均质化、胞体皱缩、胞浆及胞核浓缩,核不清或消失,轴突不规则扭曲,多见嗜神经现象。慢性灌酒打击组大鼠发生TSAH的脑表面大片较厚层蛛网膜下腔出血,主要分布在脑干腹侧面,神经元及胶质细胞中度水变性、细胞周隙明显扩大、尼氏小体消失,多见散在暗神经元细胞。
3 Cyt-C、Caspase-9、Caspase-3的表达
急性灌水打击组大鼠神经元胞浆Cyt-C、Caspase-9、Caspase-3均表达较弱,阳性细胞数分别为7.0±1.83、6.8±1.54、6.7±1.13,各脑区之间无差异(P>0.05)。急性灌酒打击组Cyt-C、Caspase-9、Caspase-3的IOD值3550.6±285.69、3061.5±365.14、2950.5±266.76高于急性灌水打击组的1875.7±297.05、1606.6±225.18、1675.7±295.82 (P<0.05)。急性单纯灌水组和急性灌水打击组之间以及急性单纯灌酒组和急性灌酒打击组之间阳性细胞数和IOD值无差异(P>0.05)。慢性单纯灌酒组大鼠神经元Cyt-C、Caspase-9、Caspase-3阳性细胞数20.58±5.26、15.2±3.61、15.8±3.80和IOD值3871.9±248.82、3397.3±373.22、3401.7±305.57均高于慢性单纯灌水组的6.7±1.85、6.8±1.55、6.3±0.91和1838.9±238.71、1692.4±273.98、1674.7±241.04(P<0.01)和急性单纯灌酒组的12.4±3.31、9.0±2.15、10.9±2.48和3448.3±297.48、2920.6±230.96、2844.8±240.61(P<0.05);慢性灌酒打击组大鼠神经元Cyt-C、Caspase-9、Caspase-3阳性细胞数22.78±5.63、17.3±3.56、18.1±3.99和IOD值4182.4±289.90、3826.3±378.04、3728.6±300.77均高于慢性单纯灌酒组的20.58±5.26、15.2±3.61、15.8±3.80和3871.9±248.82、3397.3±373.22、3401.7±305.57(P<0.05)和慢性灌水打击组的8.1±2.0、6.7±1.62、6.8±1.40和1891.43±300.62、1681.6±224.36、1686.5±280.03(P<0.01)。
4暗神经元的表达
急性灌酒组大鼠脑组织偶见暗神经元,灌水组未见暗神经元;慢性灌酒打击组大鼠脑组织暗神经元数量为10.5±2.24多于慢性单纯灌酒组的9.0±2.29 (P<0.05)。暗神经元的面积1204.8±390.82 um2小于正常神经元的1905.8±913.71 um2 (P<0.05),周长和平均直径分别为138.02±19.36和41.76±6.06均较正常神经元的167.07±41.03和50.56±12.10短(P>0.05)。
结论
1、急慢性酒精中毒可致明显外伤性蛛网膜下腔出血的发生,尤其是慢性酗酒的TSAH发生率及死亡率均高于急性醉酒的,提示酗酒者TSAH案件的法医学分析,应考虑慢性酗酒神经中枢毒理性因素,酗酒与轻微头颈部外力打击应属于协同死因。
2、急慢性酒精中毒可引起脑组织Cyt-C、Caspase-9、Caspase-3表达增强,与暗神经元的发生之间存在关联性,特别是慢性酒精中毒脑组织暗神经元数量明显增多,提示酒精及其代谢产物可激活Cyt-C→Caspase-9→Caspase-3凋亡途径,参与CNS暗神经元的形成,及酒精性脑萎缩、酒精性脑病等形态和功能障碍。
3、从原因、形态和机制各方面表明中枢神经系统的暗神经元不同于一般的组织细胞萎缩性病变,可能为细胞凋亡的早期过渡状态,为一种可逆性病理变化,可能构成慢性酒精中毒及其TSAH高死亡率的神经病理学基础,进而参与其死亡机制。
Background and objective
Traumatic subarachnoid haemorrhage(TSAH) is an independent brain damage, and it differs from pathological subarachnoid haemorrhage and haemorrhage caused by cerebral contusion. TSAH makes up the big three cavity haemorrhage with traumatic haemorrhage of cerebral dura mater and cerebral dura mater hypostegal cavity. It is reported TSAH is related with insobriety by foreigners. After slight beating on the brains of fuddled rats, the chronic fuddled group had a higher death rate(58.8% to 0, to the acute group) and rate of incidence (82.4% to 28.6%, to the acute group), and the results indicated that the oxygen and energy metabolism(Ngb、Hif-1α、Na+,K+-ATPase), vessel wall and cytotoxicity(tPA and MMP-9), blood vessel vitodynamics participate the TSAH mechanism, especially we saw the dark neuron in the chronic group, but we didn’t know wether the dark neuron was related with the TSAH mechanism. So we established TSAH model in fuddled rats, to observe the expression of Cyt-C、Caspase-9、Caspase-3 and the quantity of dark neuron, the effect of drinking upon apoptosis, function and energy metabolism of neurons, and we expect to explain the possible death mechanism of TSAH from molecular biology, toxicology and morphology.
The important apoptosis pathway Cyt-C→Caspase-9→Caspase-3 is related with the functional status of brain, so we detect the expression of Cyt-C、Caspase-9、Caspase-3 and dark neuron in the basic of TSAH model to approach the TSAH mechanism from molecular biology, toxicology, morphous. Materials and method
80 Male SD(Sprague-Dawley) rats, 300~400g, randomization. Acute fuddled model: the fuddled group 20 Male SD rats, the edible alcohol of Erguotou of Peking(52%v/v)was injected into rats’stomach through esophagus, 15ml/kg for one time. The control group 20 Male SD rats, Used same amount of tap water in the control group. 2h later gave them cerebral concussion beat. Chronic fuddled model: the fuddled group 20 Male SD rats, the edible alcohol of Erguotou of Peking(52%v/v)was injected into rats’stomach through esophagus, 2 times per day at 6 hours’intervals, 8ml/kg each time in the first 2 weeks and 12ml/kg each time in the next 2 weeks for four weeks in total. Used same amount of tap water in the control group. 2h later at the last time of fuddled gave them cerebral concussion beat. group n dosage liquid Chronic fuddled group 10 8;12ml/kg edible wine Chronic fuddled beaten group 10 8;12ml/kg edible wine Chronic tap water control group 10 8;12ml/kg tap water Chronic tap water beaten control group 10 8;12ml/kg tap water
The beaten group was exposed heart after 0.5h, aortic cannula,injected 4% neutral paraform, at the same time cut the auricle of right atrium. About 0.5h later, open the skull to acquire the brain, flash it with the tap water, then put it into the 4% neutral Polyoxymethylene, to fix 6h, anhydration, to soak cere, embed, make parraffin section(5um) routinely.The changes of the expression of Cyt-C, Caspase-9 and Caspase-3 and the quantity of dark neuron, histomorphology were observed by immunohistochemistry and HE, and the data was tested with SPSS13.0 statistic software(Independent-samples T-test and One-way ANONA).
Result
1. BAC and animal’s behavior The BAC was 114.67±67.37 mg/dL after 2h of gavaging wine. The rats excited a short time after drinking: moving continuously and unstably. About 0.5h later, they had some clinical manifestation of acute alcoholism such as lying on the side, hypnody, body-righting reflex disappeared, the respiration torpidity. 2h later, the breathing frequency(63.5±9.4 f/min to 85.1±9.3 f/min) and blood pressure(106.1±4.6 mmHg to 133.8±5.5 mmHg ) decreased(P<0.01) compared with before, but the control group had no change; 0.5h after being beaten, the blood pressure of acute fuddled group increased(121.1±17.6 mmHg to 106.5±10.1 mmHg)(P<0.05), but the breathing frequency had no change. In the chronic group, the hair became rough and dim, the spirit became frustrated, rats were weightless and decreased food-intake, the basic blood pressure increased, body weight decreased(372.8±22.2 g to 389.7±31.7 g) after 2 weeks (P<0.05), the body weight of control group increased(405.1±20.8 g to 390.1±24.7 g) after 4 weeks(P<0.05); 4 weeks later, the blood pressure of chronic fuddled group(152.3±13.5 mmHg to 135.0±11.2 mmHg)rised(P<0.05), after the last gavaging wine, the blood pressure(140.3±10.4 mmHg to 152.3±13.5 mmHg)decreased(P<0.05), and rised(151.2±10.6 mmHg) after being beaten(P<0.05), the control group also rised(148.1±13.5 mmHg to 137.1±12.6 mmHg) after being beaten(P<0.05), but the basic blood pressure of control group didn’t change; the breathing frequency of fuddled group and control group also didn’t change, just decreased after gavaging wine(P<0.05).
2. Incidence rate and death rate of TSAH and SAH and histomorphology
The incidence rate and death rate of TSAH are 20% and 0 in acute group, but 50% and 40% in chronic group, The incidence rate of spontaneous SAH are 10% in acute group and 0 in chronic group, death rate are both 0.
Minute blood vessel could be seen in the surface of brain in the control group, but it was pale after being beaten, while it was a little red in the acute fuddled group. In the acute fuddled group the neuron was slightly hydropic degeneration, the gap of cell was enlarged, and dark neurons could be seen by chance, nissl's body was disappeared. Lamellar and patching blood could be seen in the TSAH rats’brain, especially in the ventri-brainstem. Thick layer blood could be seen in the surface brain of TSAH in the chronic fuddled beaten group. The neuron and gliacyte were midrange hydropic degeneration, the gap of cell was enlarged, nissl's body disappeared in the chronic fuddle beaten group, and dark neurons could be seen, it was prunosus, cell body shrinkage, clarifixation, sometimes the nucelus disappeared, axis cylinder irregular twist, neuronophagia could be seen, the quantity of neuron decreased, the gliacyte hyperplasy.
3. The expression changes of Cyt-C、Caspase-9 and Caspase-3
The expression of Cyt-C、Caspase-9 and Caspase-3 in brain tissue’s neuron of normal rats was feeble, and a little neuron express in the acute group, There was no difference of the positive cells and IOD of Cyt-C、Caspase-9 and Caspase-3 in the brain between the acute fuddled group and fuddled beaten group(P>0.05), and so was the acute tap water group and the acute tap water beaten group, but the IOD of acute fuddled beaten group(3550.6±285.69,3061.5±365.14, 2950.5±266.76) is more than the control group(1875.7±297.05,1606.6±225.18,1675.7±295.82) (P<0.05). In chronic fuddled group, the positive cells and the IOD of Cyt-C、Caspase-9 and Caspase-3(20.58±5.26,15.2±3.61,15.8±3.80 and 3871.9±248.82,3397.3±373.22,3401.7±305.57) were more than those in the acute fuddled group(12.4±3.31,9.0±2.15,10.9±2.48 and 3448.3±297.48,2920.6±230.96,2844.8±240.61)(P<0.05) and the control group(6.7±1.85, 6.8±1.55,6.3±0.91 and 1838.9±238.71,1692.4±273.98,1674.7±241.04)(P<0.01), In chronic fuddled beaten group, the positive cells and IOD of Cyt-C、Caspase-9 and Caspase-3 (22.78±5.63,17.3±3.56,18.1±3.99 and 4182.4±289.90,3826.3±378.04,3728.6±300.77) was higher than the chronic fuddled group(P<0.05) and the control group(8.1±2.0,6.7±1.62, 6.8±1.40 and 1891.43±300.62,1681.6±224.36,1686.5±280.03)(P<0.01).
4. The expression changes of dark neuron
It could be seen dark neurons in the acute fuddled group by chance, and no dark neurons were seen in the control group. While there many dark neurons can be seen in the chronic group, and quantity of the chronic fuddled beaten group(10.9±1.52,9.7±1.77,9.1±1.85) was more than the chronic fuddled group(9.2±1.75,7.8±1.69,7.1±1.64)(P<0.05) except the frontal(12.8±1.99 to 11.6±1.43). The area of dark neuron(1204.8±390.82 um2) was smaller than the normal neuron(1905.8±913.71 um2)(P<0.05), the perimeter(138.02±19.36 to 167.07±41.03) and the average diameter(41.76±6.06 to 50.56±12.10) were both shorter than the normal neuron(P>0.05).
Conclusion
1. There were closely correlations between slight violent beaten after fuddled and the higher incidence rate and death rate of TSAH. Especially the incidence rate and the death rate of chronic fuddled group was higher than the acute group, which indicated that the drinking should be set into consideration sufficiently while analyzing these cases of TSAH after drinking in forensic medicine. The slight external force and insobriety were synergism cause of death.
2. Alcoholism could make high expression of Cyt-C, Caspase-9 and Caspase-3 which was related with the dark neuron, especially in the chronic alcoholism brain there were many dark neurons that indicated alcohol and its metabolic product could activate the apoptosis pathway Cyt-C→Caspase-9→Caspase-3, which may participated the formation of dark neuron and play the roles on atrophy of brain, alcoholic encephalopathy and functional disturbance.
3. The dark neuron was not the same as atrophy cell from morphology, cause and mechanism, maybe it was a kind of transient status, it could bccome apoptosis cell or recovery or necrosis. So the dark neuron may participate the TSAH death mechanism as the base of neuropathology.
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[27] Hitoo Nishino,Czurko Andras,Michiko Kumazaki.1309 Appearance of collapsed dark neurons,immunoglobulin g and complement factor C3 in rat brain after transient focal ischemia[J].Neuroscience Research Supplements,1993,18:s136. 29
[28] Paul Barenberg,Howard Strahlendorf,Jean Strahlendorf.Hypoxia induces an excitotoxic-type of dark cell degeneration in cerebellar Purkinje neurons[J].Neuroscience Research 40(2001)245-254.
[29]高晶,郭玉璞,赵庆杰.脑梗死后细胞骨架和暗神经元的变化[J].中华神经科杂志,2000,33 (1):39-41.
[30] Obernier JA,Bouldin TW,Crews FT. Binge ethanol exposure in adult rats causes necrotic cell death[J].Alcohol Clin Exp Res.2002,26(4):547-557.
[31] K.Ishida,H.Shimizu,H.Hida,et al.Argyrophilic dark neurons represent various states of neuronal damage in brain insults: some come to die and others survive[J].Neuroscience, 2004,125(3):633-644.
[32]于晓军,赵福弟,刘卯阳,等.大鼠脑震荡模型的建立及病理学研究[J].法医学杂志, 1992,8(3):119-123.
[33] Young C,Olney JW.Neuroapoptosis in the infant mouse brain triggered by a transient small increase in blood alcohol concentration[J].Neurobiol Dis.2006,22(3):548-554.
[34] Obernier JA,Bouldin TW,Crews FT.Binge ethanol exposure in adult rats causes necrotic cell death[J].Alcohol Clin Exp Res.2002,26(4):547-557.
[35] K.Ishida,H.Shimizu,H.Hida,et al.Argyrophilic dark neurons represent various states of neuronal damage in brain insults: some come to die and others survive[J]. Neuroscience, 2004,125(3):633-644.
[36] Ramachandran V,Perez A,Chen J,et al.In utero ethanol exposure causes mitochondrial dysfunction, which can result in apoptotic cell death in fetal brain:a potential role for 4-hydroxynonenal[J].Alcohol Clin Exp Res.2001,25(6):862-871.
[37] Clive Harper,Izuru Matsumoto.Ethanol and brain damage[J].Current Opinion in Pharmacology,2005,5:73-78.
[38] Robert Pawlak,Anna Skrzypiec,Stanislaw Sulkowski,et,al.Ethanol-induced neurotoxicity is counterbalanced by increased cell proliferation in mouse dentate gyrus[J].Neuroscience Letters 327(2002)83-86.
[39]赵丽,韩微,陈嘉峰,等.酒精中毒大鼠脑血管病变及相应脑组织损伤的病理观察[J].中风与神经疾病杂志.2005,33(1):51-53.
[40] Zink BJ,Walsh RF,Fenstel PJ.Effect of ethanol on traumatic injury [J].J Neurotrauma,1993, 10:275.
[1] Flesh.M.über die Verschiedenheiten im chemischen Verhalten der Nervenzellen.Mitt. Natforsch.Ges.Bern.1887,1073–1082,192-199.
[2] Cammermeyer J(1961) The importance of avoiding“dark neurons”in experimental neuropathology[J].Acta Neuropathol:245-270.
[3] Tewari HB,Bourne GH.Histochemical studies on the "dark" and "light" cells of the cerebellum of rat. Acta Neuropathol.1963 Sep 2;3:1-15.
[4] Nemetschek-Gansler H.On the ultrastructure of dark neurons.Verh Anat Ges.1964;59:328-33.
[5] Andrea Zsombok,Zsolt T′oth,Ferenc Gallyas.Basophilia,acidophilia and argyrophilia of“dark”(compacted) neurons during their formation, recovery or death in anotherwise undamaged environment[J].Journal of Neuroscience Methods 142(2005)145-152.
[6] Jean Strahlendorf,Cathy Box,Jennifer Attridge,et al.AMPA-induced dark cell degeneration of cerebellar Purkinje neurons involves activation of caspases and apparent mitochondrial dysfunction[J].Brain Res,994 (2003) 146-159.
[7] Richard Kellermayer,Andrea Zsombok,Ferenc Gallyas,et al.Electrically induced gel-to-gel phase-transition in neurons[J].Cell Biology International 30(2006)175-182[8] FerencGallyas,Balázs Gasz,András Szigeti,et al.Pathological circumstances impair the ability of“dark”neurons to undergo spontaneous recovery[J].Brain Res,2006,1110(1):211-220.
[9] Ohtsuka A,Murakami T.Dark neurons in the mouse brain : an investigation into the possible significance of their variable appearance within a day and their relation to negatively charged cell coats[J].Arch Histol Cytol,1996,59:79-85.
[10]高晶,郭玉璞,赵庆杰.脑梗死后细胞骨架和暗神经元的变化[J].中华神经科杂志,2000,33 (1):39-41.
[11] K.Ishida,H.Shimizu,H.Hida,et al.Argyrophilic dark neurons represent various states of neuronal damage in brain insults: some come to die and others survive[J]. Neuroscience, 2004,125(3):633-644.
[12] Erzsébet K?vesdia,József Pálb,Ferenc Gallyas.The fate of“dark”neurons produced bytransient focal cerebral ischemia in a non-necrotic and non-excitotoxic environment: Neurobiological aspects[J]. Brain Res,1147(2007)272-283.
[13] Graeber M.B.,Moran L.B..Mechanism of cell death in neurodegenerative diseases: fashion, fiction and facts.Brain Pathol.2002,12:385-390.
[14] Ferenc Gallyas,Orsolya Farkas,Mária Mázló.Gel-to-gel phase transition may occur in mammalian cells:Mechanism of formation of“dark”(compacted) neurons[J].Biology of the Cell,2004,96(4):313-324.
[15] F.Gallyas,G.Zoltay,W.Dames.Formation of“dark”(argyrophilic) neuron of various origin proceeds with a common mechanism of biophysical nature (a novel hypothesis)[J].Acta Neuropathol(1992)83:504-509.
[16] Auer RN,Kalimo H,Olsson Y,et al.The temporal evolution of hypoglycemic brain damage.I. Light- and electron-microscopic findings in the rat cerebral cortex[J].Acta Neuropathol 1985,67(1-2):13-24.
[17] T.Griffiths,M.C. Evans,B.S.Meldrum.Status epilepticus:The reversibility of calcium loading and acute neuronal pathological changes in the rat hippocampus[J].Neuroscience,1984,12 (2):557-567.
[18] Ingvar M,Morgan PF,Auer RN.The nature and timing of excitotoxic neuronal necrosis in the cerebral cortex, hippocampus and thalamus due to flurothyl-induced status epilepticus[J]. Acta Neuropathol.1988,75(4):362-369.
[19] András Czurkó,Hitoo Nishino.‘Collapsed’(argyrophilic,dark) neurons in rat model of transient focal cerebral ischemia[J].Neuroscience Letters,1993,162(1-2):71-74.
[20] Hitoo Nishino,Czurko Andras,Michiko Kumazaki.1309 Appearance of collapsed dark neurons,immunoglobulin g and complement factor C3 in rat brain after transient focalischemia[J].Neuroscience Research Supplements,1993,18:s136.
[21] H. Nishino, A. Czurkó, A. Fukuda,et al.Pathophysiological process after transient ischemia of the middle cerebral artery in the rat[J].Brain Research Bulletin,1994,35(1):51-56.
[22] Hajnal A,Lénárd L,CzurkóA,et al.Distribution and time course of appearance of "dark" neurons and EEG activity after amygdaloid kainate lesion[J].Brain Res Bull,1997,43 (2):235-243.
[23] Kazuto Ishida,Chutcharin Ungsuparkorn,Kunio Ida.Argyrophilic dark neurons appear diffusely in the brain after over hours treadmill running and swimming in the rat[J]. Neuroscience Research,1998,31,Supplement 1,S347.
[24] Kazuto Ishida,Chutcharin Ungusparkorn,Hideki Hida,et al.Argyrophilic dark neuronsdistribute with a different pattern in the brain after over hours treadmill running and swimming in the rat[J].Neuroscience Letters 277(1999)149-152.
[25] Abdel-Rahman A,Shetty AK,Abou-Donia MB.Subchronic dermal application of N,N-diethyl m-toluamide (DEET) and permethrin to adult rats, alone or in combination, causes diffuse neuronal cell death and cytoskeletal abnormalities in the cerebral cortex and the hippocampus,and Purkinje neuron loss in the cerebellum.Exper Neurol 2001,172:153-71.
[26] Abdel-Rahman A,Shetty AK,Abou-Donia MB.Disruption of the blood-brain barrier and neuronal cell death in cingulate cortex,dentate gyrus,thalamus,and hypothalamus in a rat model of Gulf-War syndrome.Neurobiol Dis 2002b,10:306-26.
[27] Abdel-Rahman A,Abou-Donia S,El-Masry E,Shetty AK,Abou-Donia MB.Stress and combined exposure to low doses of pyridostigmine bromide,DEET,and permethrin produce neurochemical and neuropathological alterations in cerebral cortex,hippocampus and cerebellum.J Toxicol Environ Health Part A 2004a,67:163-92.
[28] Abdel-Rahman A,Dechkovskaia AM,Goldstein LB,Bullman SH, KhanW,ElMasry EM,et al. Neurological deficits induced by malathion,DEET,and permethrin,alone or in combination in adult rats.J Toxicol Environ Health Part A 2004b,67:331-56.
[29] Paul Barenberg,Howard Strahlendorf,Jean Strahlendorf.Hypoxia induces an excitotoxic-type of dark cell degeneration in cerebellar Purkinje neurons[J].Neuroscience Research 40 (2001) 245-254.
[30] Obernier JA,Bouldin TW,Crews FT. Binge ethanol exposure in adult rats causes necrotic cell death[J].Alcohol Clin Exp Res.2002,26(4):547-557.
[31] K. Ishida, H. Shimizu, H. Hida,et al. Argyrophilic dark neurons represent various states of neuronal damage in brain insults: some come to die and others survive [J].Neuroscience125 (2004) 633-644.
[32] Gallyas F,Zoltay G,Horváth Z,et al. An immediate morphopathologic response of neurons to electroshock; a reliable model for producing "dark" neurons in experimental neuropathology[J].Neurobiology (Bp).1993,1(2):133-46.
[33] J. M. Powers, G. T. Mann, R. Jones,et al.A reassessment of the significance of dark neurons in serotonergic cell groups[J].Neuropharmacology,1979,18(4):383-389.
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[31] K.Ishida,H.Shimizu,H.Hida,et al.Argyrophilic dark neurons represent various states of neuronal damage in brain insults: some come to die and others survive[J].Neuroscience, 2004,125(3):633-644.
[32]于晓军,赵福弟,刘卯阳,等.大鼠脑震荡模型的建立及病理学研究[J].法医学杂志, 1992,8(3):119-123.
[33] Young C,Olney JW.Neuroapoptosis in the infant mouse brain triggered by a transient small increase in blood alcohol concentration[J].Neurobiol Dis.2006,22(3):548-554.
[34] Obernier JA,Bouldin TW,Crews FT.Binge ethanol exposure in adult rats causes necrotic cell death[J].Alcohol Clin Exp Res.2002,26(4):547-557.
[35] K.Ishida,H.Shimizu,H.Hida,et al.Argyrophilic dark neurons represent various states of neuronal damage in brain insults: some come to die and others survive[J]. Neuroscience, 2004,125(3):633-644.
[36] Ramachandran V,Perez A,Chen J,et al.In utero ethanol exposure causes mitochondrial dysfunction, which can result in apoptotic cell death in fetal brain:a potential role for 4-hydroxynonenal[J].Alcohol Clin Exp Res.2001,25(6):862-871.
[37] Clive Harper,Izuru Matsumoto.Ethanol and brain damage[J].Current Opinion in Pharmacology,2005,5:73-78.
[38] Robert Pawlak,Anna Skrzypiec,Stanislaw Sulkowski,et,al.Ethanol-induced neurotoxicity is counterbalanced by increased cell proliferation in mouse dentate gyrus[J].Neuroscience Letters 327(2002)83-86.
[39]赵丽,韩微,陈嘉峰,等.酒精中毒大鼠脑血管病变及相应脑组织损伤的病理观察[J].中风与神经疾病杂志.2005,33(1):51-53.
[40] Zink BJ,Walsh RF,Fenstel PJ.Effect of ethanol on traumatic injury [J].J Neurotrauma,1993, 10:275.
[1] Flesh.M.über die Verschiedenheiten im chemischen Verhalten der Nervenzellen.Mitt. Natforsch.Ges.Bern.1887,1073–1082,192-199.
[2] Cammermeyer J(1961) The importance of avoiding“dark neurons”in experimental neuropathology[J].Acta Neuropathol:245-270.
[3] Tewari HB,Bourne GH.Histochemical studies on the "dark" and "light" cells of the cerebellum of rat. Acta Neuropathol.1963 Sep 2;3:1-15.
[4] Nemetschek-Gansler H.On the ultrastructure of dark neurons.Verh Anat Ges.1964;59:328-33.
[5] Andrea Zsombok,Zsolt T′oth,Ferenc Gallyas.Basophilia,acidophilia and argyrophilia of“dark”(compacted) neurons during their formation, recovery or death in anotherwise undamaged environment[J].Journal of Neuroscience Methods 142(2005)145-152.
[6] Jean Strahlendorf,Cathy Box,Jennifer Attridge,et al.AMPA-induced dark cell degeneration of cerebellar Purkinje neurons involves activation of caspases and apparent mitochondrial dysfunction[J].Brain Res,994 (2003) 146-159.
[7] Richard Kellermayer,Andrea Zsombok,Ferenc Gallyas,et al.Electrically induced gel-to-gel phase-transition in neurons[J].Cell Biology International 30(2006)175-182[8] FerencGallyas,Balázs Gasz,András Szigeti,et al.Pathological circumstances impair the ability of“dark”neurons to undergo spontaneous recovery[J].Brain Res,2006,1110(1):211-220.
[9] Ohtsuka A,Murakami T.Dark neurons in the mouse brain : an investigation into the possible significance of their variable appearance within a day and their relation to negatively charged cell coats[J].Arch Histol Cytol,1996,59:79-85.
[10]高晶,郭玉璞,赵庆杰.脑梗死后细胞骨架和暗神经元的变化[J].中华神经科杂志,2000,33 (1):39-41.
[11] K.Ishida,H.Shimizu,H.Hida,et al.Argyrophilic dark neurons represent various states of neuronal damage in brain insults: some come to die and others survive[J]. Neuroscience, 2004,125(3):633-644.
[12] Erzsébet K?vesdia,József Pálb,Ferenc Gallyas.The fate of“dark”neurons produced bytransient focal cerebral ischemia in a non-necrotic and non-excitotoxic environment: Neurobiological aspects[J]. Brain Res,1147(2007)272-283.
[13] Graeber M.B.,Moran L.B..Mechanism of cell death in neurodegenerative diseases: fashion, fiction and facts.Brain Pathol.2002,12:385-390.
[14] Ferenc Gallyas,Orsolya Farkas,Mária Mázló.Gel-to-gel phase transition may occur in mammalian cells:Mechanism of formation of“dark”(compacted) neurons[J].Biology of the Cell,2004,96(4):313-324.
[15] F.Gallyas,G.Zoltay,W.Dames.Formation of“dark”(argyrophilic) neuron of various origin proceeds with a common mechanism of biophysical nature (a novel hypothesis)[J].Acta Neuropathol(1992)83:504-509.
[16] Auer RN,Kalimo H,Olsson Y,et al.The temporal evolution of hypoglycemic brain damage.I. Light- and electron-microscopic findings in the rat cerebral cortex[J].Acta Neuropathol 1985,67(1-2):13-24.
[17] T.Griffiths,M.C. Evans,B.S.Meldrum.Status epilepticus:The reversibility of calcium loading and acute neuronal pathological changes in the rat hippocampus[J].Neuroscience,1984,12 (2):557-567.
[18] Ingvar M,Morgan PF,Auer RN.The nature and timing of excitotoxic neuronal necrosis in the cerebral cortex, hippocampus and thalamus due to flurothyl-induced status epilepticus[J]. Acta Neuropathol.1988,75(4):362-369.
[19] András Czurkó,Hitoo Nishino.‘Collapsed’(argyrophilic,dark) neurons in rat model of transient focal cerebral ischemia[J].Neuroscience Letters,1993,162(1-2):71-74.
[20] Hitoo Nishino,Czurko Andras,Michiko Kumazaki.1309 Appearance of collapsed dark neurons,immunoglobulin g and complement factor C3 in rat brain after transient focalischemia[J].Neuroscience Research Supplements,1993,18:s136.
[21] H. Nishino, A. Czurkó, A. Fukuda,et al.Pathophysiological process after transient ischemia of the middle cerebral artery in the rat[J].Brain Research Bulletin,1994,35(1):51-56.
[22] Hajnal A,Lénárd L,CzurkóA,et al.Distribution and time course of appearance of "dark" neurons and EEG activity after amygdaloid kainate lesion[J].Brain Res Bull,1997,43 (2):235-243.
[23] Kazuto Ishida,Chutcharin Ungsuparkorn,Kunio Ida.Argyrophilic dark neurons appear diffusely in the brain after over hours treadmill running and swimming in the rat[J]. Neuroscience Research,1998,31,Supplement 1,S347.
[24] Kazuto Ishida,Chutcharin Ungusparkorn,Hideki Hida,et al.Argyrophilic dark neuronsdistribute with a different pattern in the brain after over hours treadmill running and swimming in the rat[J].Neuroscience Letters 277(1999)149-152.
[25] Abdel-Rahman A,Shetty AK,Abou-Donia MB.Subchronic dermal application of N,N-diethyl m-toluamide (DEET) and permethrin to adult rats, alone or in combination, causes diffuse neuronal cell death and cytoskeletal abnormalities in the cerebral cortex and the hippocampus,and Purkinje neuron loss in the cerebellum.Exper Neurol 2001,172:153-71.
[26] Abdel-Rahman A,Shetty AK,Abou-Donia MB.Disruption of the blood-brain barrier and neuronal cell death in cingulate cortex,dentate gyrus,thalamus,and hypothalamus in a rat model of Gulf-War syndrome.Neurobiol Dis 2002b,10:306-26.
[27] Abdel-Rahman A,Abou-Donia S,El-Masry E,Shetty AK,Abou-Donia MB.Stress and combined exposure to low doses of pyridostigmine bromide,DEET,and permethrin produce neurochemical and neuropathological alterations in cerebral cortex,hippocampus and cerebellum.J Toxicol Environ Health Part A 2004a,67:163-92.
[28] Abdel-Rahman A,Dechkovskaia AM,Goldstein LB,Bullman SH, KhanW,ElMasry EM,et al. Neurological deficits induced by malathion,DEET,and permethrin,alone or in combination in adult rats.J Toxicol Environ Health Part A 2004b,67:331-56.
[29] Paul Barenberg,Howard Strahlendorf,Jean Strahlendorf.Hypoxia induces an excitotoxic-type of dark cell degeneration in cerebellar Purkinje neurons[J].Neuroscience Research 40 (2001) 245-254.
[30] Obernier JA,Bouldin TW,Crews FT. Binge ethanol exposure in adult rats causes necrotic cell death[J].Alcohol Clin Exp Res.2002,26(4):547-557.
[31] K. Ishida, H. Shimizu, H. Hida,et al. Argyrophilic dark neurons represent various states of neuronal damage in brain insults: some come to die and others survive [J].Neuroscience125 (2004) 633-644.
[32] Gallyas F,Zoltay G,Horváth Z,et al. An immediate morphopathologic response of neurons to electroshock; a reliable model for producing "dark" neurons in experimental neuropathology[J].Neurobiology (Bp).1993,1(2):133-46.
[33] J. M. Powers, G. T. Mann, R. Jones,et al.A reassessment of the significance of dark neurons in serotonergic cell groups[J].Neuropharmacology,1979,18(4):383-389.
[34] Bernard S.Jortner.The return of the dark neuron. A histological artifact complicating contemporary neurotoxicologic evaluation[J].NeuroToxicology 27(2006)628-634.