急慢性酒精中毒大鼠脑组织Ngb、HIF-1α、Epo表达及神经元凋亡与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,外伤性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。国内外的调查研究发现,TSAH多数是发生在大量饮酒后,经轻微暴力作用于头面部发生,发生率高达87%。本课题组既往研究,建立了大鼠急慢性灌酒后TSAH模型:灌酒后大鼠TSAH发生率为急性组28.6﹪、慢性组82.4﹪,死亡率为急性组0﹪、慢性组58.8﹪,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,初步探讨了饮酒促发TSAH的发生机制。同时,发现了慢性灌酒组的脑组织暗神经元明显多于急性灌酒组,但是慢性灌酒大鼠TSAH高死亡率及其死亡机制是否与暗神经元之间存在关系,则尚不清楚。而这正是关于TSAH案例法医学鉴定、死因分析,及其司法审判量刑中极其重要而迫切需要解决的问题之一。
本研究旨在前期所建立的大鼠灌酒后TSAH模型基础上,观察脑组织脑红蛋白(Ngb)、缺氧诱导因子1α(HIF-1α)、促红细胞生成素(Epo)等脑组织氧代谢指标的表达,及凋亡因子Bcl-2、Bax,脑组织细胞凋亡、暗神经元数量的变化,探讨急慢性酒精中毒对脑组织形态、代谢和功能的影响,及TSAH高死亡率的相关机制,以期为此类案件的法医学鉴定和临床防治提供科学理论依据。
材料与方法
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。
急性灌酒模型:40只大鼠随机分:急性灌酒组,一次性胃内灌酒(北京红星酒厂二锅头,52%v/v)15ml/ kg;急性灌水组,灌同等量的自来水;再分别于灌酒或灌水后2h,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。
慢性灌酒模型:40只大鼠随机分:慢性灌酒组和灌水组,给予灌酒(北京红星酒厂二锅头,52%v/v)或灌水四周,每天9:00和15:00间隔6小时灌两次,前2周每次灌8ml/kg、后2周每次灌12ml/kg。最后一次灌酒和灌水后2h,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。
未打击组于灌胃后2h,打击组于打击后0.5h,开胸暴露心脏,经左心室-主动脉插管,直接灌注4%中性多聚甲醛液处死兼固定,剪开右心耳放血。约0.5h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5um,分别HE染色观察脑组织形态变化,Ngb,HIF-1α,Epo,Bcl-2和Bax免疫组织化学染色,及TUNEL标记细胞凋亡,观察其表达情况。病理真彩色图像分析系统4.0进行体视量化病理学观测,实验数据用SPSS15.0统计软件(Independent-samples T-test and One-way ANONA)及Excel进行分析处理。
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;2h后呼吸频率63.5±9.4次/分和血压106.1±4.6mmHg均较灌酒前85.1±9.3次/分和133.8±5.5mmHg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±22.2g较灌酒前的389.7±31.7g明显下降(P<0.05);灌水组大鼠第4周体重为405.1±20.8g较灌水前的390.1±24.7g增长明显(P<0.05);大鼠灌酒后第四周基础血压为152.3±13.5mmHg较灌酒前的135.0±11.2上升(P<0.05),最后一次灌酒后血压为140.3±10.4较灌酒前的152.3±13.5 mmHg低(P<0.05),打击后升高为151.2±10.6mmHg(P<0.05)。灌水打击组打击后血压为148.1±13.5mmHg较打击前137.1±12.6mmHg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。
急性灌水组大鼠脑表面光泽,表面散在细小血管纹理。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。急性单纯灌酒组脑表面光泽,大片淤血,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。
慢性灌酒组大鼠脑表面光泽,大片淤血,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,神经元数量减少,胶质细胞增生。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。
3 Ngb表达观察
急慢性灌水及灌水打击对照组大鼠脑神经元内均有少量Ngb的表达。急慢性灌酒组和急慢性灌酒打击组大鼠脑组织各区Ngb阳性细胞数,IOD值分别高于急慢性灌水组和急慢性灌水打击组(P<0.01)。急性灌酒打击组Ngb阳性细胞数23.0±3.48多于急性灌酒组19.7±2.37 (P <0.01)。慢性灌酒打击组的Ngb阳性细胞数20.0±2.67及IOD值4070.3±226.12与慢性灌酒组阳性细胞数18.0±2.83、IOD值3922.5±319.62有显著的统计学意义(均为P <0.01)。慢性灌酒组的Ngb阳性细胞数18.0±2.83低于急性灌酒组19.7±2.37 (P <0.01),而慢性灌酒打击组脑组织各区NgbIOD值4070.3±226.12与急性灌酒打击组6380.6±234.21有统计学意义(海马、小脑区P <0.01,脑干区P <0.05)。
4 HIF-1α表达观察
急慢性灌水及灌水打击对照组大鼠脑神经元内有散在少量细胞HIF-1α轻度表达。急慢性灌酒组和急慢性灌酒打击组大鼠脑组织各区HIF-1α阳性细胞数,IOD值分别高于急慢性灌水组和急慢性灌水打击组(P<0.01)。急性灌酒打击组HIF-1α阳性细胞数24.2±4.82多于急性灌酒组21.2±3.25 (P<0.01)。慢性灌酒打击组的HIF-1αIOD值3862.6±289.24也高于慢性灌酒组3502.7±312.91(P<0.01)。慢性灌酒组的脑组织各区HIF-1α阳性细胞数18.5±2.45,IOD值3502.7±312.91低于急性灌酒组21.2±3.25、3878.6±212.31 (均为P<0.01),而慢性灌酒打击组脑组织HIF-1α阳性细胞数21.1±2.46也比急性灌酒打击组24.2±4.82要少(P<0.01)。
5 EPO表达观察
急慢性灌水及灌水打击对照组大鼠脑神经元内有散在少量细胞Epo表达。急慢性灌酒组和急慢性灌酒打击组脑组织各区Epo阳性细胞数,IOD值分别高于急慢性灌水组和急慢性灌水打击组(P<0.01)。急性灌酒打击组Epo阳性细胞数21.9±3.55多于急性灌酒组20.0±3.00(P<0.01)。慢性灌酒打击组的EpoIOD值3821.6±336.21也比慢性灌酒组3654.1±304.74要高(P<0.01)。慢性灌酒组的脑组织各区Epo IOD值3654.1±304.74均低于急性灌酒组3908.1±312.81(P<0.01) ,而慢性灌酒打击组脑组织各区EpoIOD值3821.6±336.21也均低于急性灌酒打击组4192.6±351.62 (P <0.01)。
6 Bcl-2,Bax的表达观察及Bcl-2/Bax的比值
急慢性灌酒组和急慢性灌酒打击组大鼠脑组织各区Bcl-2和Bax的阳性细胞数分别高于急慢性灌水组和急慢性灌水打击组(P<0.01)。急性灌酒打击组Bcl-2 IOD值2952.9±358.08多于急性灌酒组2782.7±381.22(P<0.01)。慢性灌酒组的脑组织各区Bcl-2IOD值2638.3±346.43均低于急性灌酒组2782.7±381.22(P<0.01),而慢性灌酒打击组脑组织各区Bcl-2 IOD值2720.7±352.76均比急性灌酒打击组2952.9±358.08要低(P<0.01)。
急性灌酒打击组脑组织各区Bax阳性细胞数11.2±2.78多于急性灌酒组10.4±2.44(P<0.05)。慢性灌酒组的脑组织各区BaxIOD值2806.3±282.35高于急性灌酒组2618.7±223.61。(P<0.01)
急性灌水及灌水打击组各脑区Bcl-2/Bax的平均比值分别为1.090、1.083,慢性灌水及灌水打击组分为1.053、1.081,急性灌酒及灌酒打击组为1.144、1.188,以上各组比值均大于1,而慢性灌酒及灌酒打击组各脑区Bcl-2/Bax的比值为0.946、0.912,比值均小于1。
7 TUNEL标记细胞凋亡结果
慢性灌酒组和慢性灌酒打击组可见较多的黄棕色阳性细胞,慢性灌酒组和慢性灌酒打击组大鼠脑组织各区细胞凋亡阳性细胞数4.4±0.52、4.9±0.57均分别高于慢性灌水组1.8±0.42和慢性灌水打击组1.9±0.57 (P<0.01),同时也高于急性灌酒组1.8±0.42及急性灌酒打击组1.6±0.52 (P<0.01)。慢性灌酒打击组的脑组织各区阳性细胞数4.9±0.57也高于慢性灌酒组4.4±0.52 (P<0.05)。
结论
1、急慢性酒精中毒可促发TSAH,慢性酒精中毒与TSAH高发生率及死亡率之间存在显著相关性。提示酗酒后TSAH及其死亡案件的死因分析时,应充分考虑酒精的急慢性毒理学效应,其在TSAH死亡案件中与头颈部外伤应属于协同死因。
2、慢性酒精中毒可通过抑制脑组织Ngb、HIF-1α、Epo等氧代谢通路相关因子表达,Bcl-2/Bax比值明显降低、神经细胞凋亡及暗神经元数量明显增加,可能参与酒精性脑萎缩、脑功能障碍的发生机制。
3、证实了急慢性酒精中毒可诱发脑组织暗神经元出现,特别是明显酒精中毒的暗神经元可能构成酗酒后TSAH及其死亡的重要的神经病理学基础,参与死亡机制的过程。
Background and objective
Traumatic subarachnoid haemorrhage(TSAH) is an independent brain damage,and it differes from pathological SAH and SAH caused by cerebral contusion,TSAH makes up the three cavity haemorrhage with traumatic haemorrhage of cerebral dura mater and cerebral dura mater hypostegal cavity. It is reported that TSAH is often occur after alcoholism with trivial strike to the face,the incidence rate was high as 87%. Our group has established TSAH model after drinking,and the result is that the incidence rate of TSAH: the acute group is 28.6%,the chronic group is 82.4%; the mortality rate: the acute group is 0%,the chronic group is 58.8%.According three aspects of Ngb、Hif-1α、Na+ , K+-ATPase aerobic and energy metabolism,tPA、MMP-9 neuron toxicity,blood vessel morphous vitodynamics,explored the occurrence mechanism of TSAH caused by alcohol drinking,and discovered the dark neuron,but we still didn’t know the relation between the high mortality rate in the chronic fuddled group and the dark neuron. So it is still a tough problem in analysis of cause of death and judicial trial.
We established TSAH model in alcoholism rats,observe the expression of Ngb and HIF-1α,Epo,Bcl-2 and Bax,apoptosis cells and the dark neuron was detected in the brain tissue. With the aerobic metabolism and basic pathological change of the brain,to explore the effects of morphous,metabolism and function of neurons after excessive drinking,and the possible mechanisms or relationship between excessive drinking and the happening of fatal TSAH,providing science theory for the TSAH medicolegal expertise and clinical prevention and cure.
Materials and Methods
Animals and groups: 80 male SD rats,each weighted 300-400g,breeding in several cages,devided into different groups randomly.
Acute fuddled model:40 rats devided randomly: acute fuddled groups,The edible alcohol of Erguotou of Peking(52%v/v)was injected into rats’stomach through esophagus 15ml/ kg per rat;Used same amount of tap water in the acute sham group.2 hours after injecting alcohol or water,giving an concussion hit to the rats,to establish to acute fuddled group and the acute sham and hit group,10 rats in each groups.
Chronic fuddled model:40 rats devided randomly: chronic fuddled group,chronic sham group. The edible alcohol of Erguotou of Peking(52%v/v)was injected into rats’stomach through esophagus,injected at 9:00 and 15:00 every day with 8 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. 2 hours after the last time injecting,giving an concussion hit to the rats,to establish to chronic fuddled group and the chronic sham and hit group,10 rats in each groups.
2 hours after injecting in the non-hitting groups,and 0.5hours after hitting groups,rats were dissected, the heart of the rat were exposed,aortic cannula through the left ventricle. the rats would be fixed by perfuse 4% neutral paraformaldehyde directly,cutting the right auricula to bloodletting.0.5 hour later,got the rats’brain,washed it,and putted it into the 4% neutral paraformaldehyde to fixing about six hours,then dehydration, immersing paraffin,embedding,tissue slice with 5um thick,to observe the Morphologic change by HE staining,Ngb,HIF-1α,Epo,Bcl-2 and Bax immunohistochemistry and TUNEL marking apoptosis cells,viewing its expression.To observe the pathological changes by patho-true-color image analytical system quantizationly,datas would be analyzed by SPSS 15.0 and Excel software.
Results
1. BAC and animal’s behavior
The BAC is 114.67±67.37 mg/dL after 2h of gavaging wine.The rats appeared 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 disappearing,the respiration torpidity,2h later,the breathing frequency and blood pressure decreased(P<0.01),but the control group had no change; 0.5h after being beaten,the blood pressure of acute fuddled group and the control group set up(P<0.05),but the breathing frequency had no change. In the chronic group,the hair becomes rough and dim,the spirit becomes frustrated,rats were weightless and decreased food-intake,the basic blood pressure set up , body weight decreased after 2 weeks(P<0.05) , the body weight of control group increased(P<0.05) after 4 weeks; 4 weeks later,the blood pressure of chronic fuddled group rised(P<0.05),after the last gavaging wine,the blood pressure drop out(P<0.05),and rised after being beaten(P<0.05),the control group also rised after being beaten(P<0.05),but the basic blood pressure of control group was not change; the breathing frequency of fuddled group and control group was not change,just decreased after gavaging wine(P<0.05).
2. Incidence rate and death rate of TSAH and SAH,Histomorphology
The incidence rate and death rate of TSAH are 20% and 0 in acute fuddled and hit group, but 50% and 40% in chronic fuddled and hit group respectively.There were no TSAH in the acute and chronic sham and hit groups.The incidence rate of spontaneous SAH are 10% in acute fuddled group and 0 in chronic group.
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. 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.The neuron and gliacyte were midrange hydropic degeneration,the gap of cell was enlarged,nissl's body was disappeared in the chronic fuddle beaten group,and dark neurons could be seen,it was prunosus,cell body shrinkage, clarifixation , sometimes the nucelus was disappeared , axis cylinder irregular twist , neuronophagia could be seen,the quantity of neuron was decreased,the gliacyte hyperplasy. Thick layer blood could be seen in the surface brain of TSAH in the chronic fuddled beaten group.
3 The expression of Ngb
There were some Ngb expression in the brain neuron of the acute and chronic sham groups,and the acute and chronic sham and hit groups.Ngb positive cells and IOD value in the acute and chronic fuddled groups,acute and chronic fuddled hit groups,higher than the acute and chronic sham groups,acute and chronic sham hit groups(P<0.01). Posotive cells of Ngb in the acute fuddled and hit group 23.0±3.48 exceeded the acute fuddled group 19.7±2.37 (P<0.01). There was noticeable statistical significance between the chronic fuddled hit group posotive cells of Ngb 20.0±2.67 and the chronic fuddled group 18.0±2.83 (P <0.01). Posotive cells of Ngb in the chronic fuddled group 18.0±2.83lower than the acute fuddled group 19.7±2.37 (P<0.01). there was noticeable statistical significance between the chronic fuddled hit group IOD 4070.3±226.12 and the acute fuddled hit group 6380.6±234.21 (hippocamp,cerebellum P<0.01,brain stemP <0.05)
4 The expression of HIF-1α
There were some HIF-1αexpression in the brain neuron of the acute and chronic sham groups,and the acute and chronic sham and hit groups. HIF-1αpositive cells and IOD value in the acute and chronic fuddled group,acute and chronic fuddled hit group higher than the acute and chronic sham groups,acute and chronic sham hit groups(P<0.01). Positive cells of HIF-1αin the acute fuddled and hit group 24.2±4.82 exceeded the acute fuddled group21.2±3.25 (P<0.01).The HIF-1αIOD value in the chronic fuddled hit group 3862.6±289.24higher than the chronic fuddled group3502.7±312.91 (P<0.01). HIF-1αpositive cells 18.5±2.45and IOD value 3502.7±312.91in the chronic fuddled group lower than the acute fuddled group 21.2±3.25、3878.6±212.31 (P <0.01). HIF-1αpositive cells and IOD value in the chronic fuddled hit group 21.1±2.46 also lower than the acute fuddled hit group 24.2±4.82 (P <0.01).
5 The expression of Epo
There were some Epo expression in the brain neuron of the acute and chronic sham groups,and the acute and chronic sham and hit groups. Epo positive cells and IOD value in the acute and chronic fuddled group,acute and chronic fuddled hit group higher than the acute and chronic sham groups,acute and chronic sham hit groups(P<0.01). Positive cells of Epo in the acute fuddled hit group 21.9±3.55 exceeded the acute fuddled group 20.0±3.00 (P <0.01). The Epo IOD value in the chronic fuddled hit group 3821.6±336.21 higher than the chronic fuddled group 3654.1±304.74 (P <0.01).Epo IOD in the chronic fuddled group 3654.1±304.74 lower than the acute fuddled group 3908.1±312.81 (brain stemP <0.05,othersP < 0.01). Epo IOD in the chronic fuddled hit group 3821.6±336.21 also lower than the acute fuddled hit group 4192.6±351.62 (P < 0.01).
6 The expression Bcl-2 and Bax,ratio of Bcl-2/Bax
Bcl-2 positive cells and IOD value in the acute and chronic fuddled group,acute and chronic fuddled hit group higher than the acute and chronic sham groups,acute and chronic sham hit groups(P<0.01). Bcl-2 IOD in the acute fuddled hit group 2952.9±358.08 exceeded the acute fuddled group 2782.7±381.22 (P<0.01).Bcl-2 IOD in the chronic fuddled group 2638.3±346.43 lower than the acute fuddled group 2782.7±381.22 (P <0.01),the chronic fuddled group 2720.7±352.76 were also lower than the acute fuddled hit group 2952.9±358.08 (P <0.01)
Positive cells of Bax in the acute fuddled hit group 11.2±2.78 exceeded the acute fuddled group 10.4±2.44 (P <0.05). Bax IOD in the chronic fuddled group 2806.3±282.35 higher than the acute fuddled group 2618.7±223.61 (P<0.01).
The average ratio of Bcl-2/Bax in the acute sham group,and acute sham hit group were 1.090、1.083,the chronic sham group,and chronic sham hit group were1.053、1.081,the acute fuddled group,and acute fuddled hit group were1.144、1.188,above ratio were all much than one, the chronic fuddled group,and chronic fuddled hit group were0.946、0.912,less than one.
7 TUNEL marked apoptosis cells results
There were many yellowish-brown color positive cells in the chronic fuddled group and chronic fuddled hit group, apoptosis cells in the chronic fuddled group and chronic fuddled hit group exceeded the chronic sham group and the chronic sham hit group (P<0.01),and also higher than the chronic fuddled group and the chronic fuddled hit group(P<0.01). Apoptosis cells in the chronic fuddled hit group also higher than the chronic fuddled group(frontal lobe,hippocamp P<0.01,cerebellum and brain stem P<0.05).
Conclusions
1.Acute and chronic alcoholism would tend to occur TSAH,there was a significant dependablity between chronic alcoholism and TSAH high incidence and mortality rate.When we analyze the TSAH cases,we should thoroughly consider the toxicological effect of acute and chronic alcoholism,and with head and neck injury as coordination death cause.
2.The effect of chronic alcoholism could inhibit the Ngb、HIF-1α、Epo such aerobic metabolism factors,lowed down the ratio of Bcl-2/Bax,increased the numbers of nueron apoptosis and dark neuron,which may be participate the mechanism of alcohol encephalanalosis and disordered brain function.
3.It was proved that acute and chronic alcoholism could induce the occur of dark neuron, especially the dark neuron in the alcoholism may be constitute the important neuro-pathology foundation,participates the process of death mechanism.
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,外伤性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。国内外的调查研究发现,TSAH多数是发生在大量饮酒后,经轻微暴力作用于头面部发生,发生率高达87%。本课题组既往研究,建立了大鼠急慢性灌酒后TSAH模型:灌酒后大鼠TSAH发生率为急性组28.6﹪、慢性组82.4﹪,死亡率为急性组0﹪、慢性组58.8﹪,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,初步探讨了饮酒促发TSAH的发生机制。同时,发现了慢性灌酒组的脑组织暗神经元明显多于急性灌酒组,但是慢性灌酒大鼠TSAH高死亡率及其死亡机制是否与暗神经元之间存在关系,则尚不清楚。而这正是关于TSAH案例法医学鉴定、死因分析,及其司法审判量刑中极其重要而迫切需要解决的问题之一。
本研究旨在前期所建立的大鼠灌酒后TSAH模型基础上,观察脑组织脑红蛋白(Ngb)、缺氧诱导因子1α(HIF-1α)、促红细胞生成素(Epo)等脑组织氧代谢指标的表达,及凋亡因子Bcl-2、Bax,脑组织细胞凋亡、暗神经元数量的变化,探讨急慢性酒精中毒对脑组织形态、代谢和功能的影响,及TSAH高死亡率的相关机制,以期为此类案件的法医学鉴定和临床防治提供科学理论依据。
材料与方法
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。
急性灌酒模型:40只大鼠随机分:急性灌酒组,一次性胃内灌酒(北京红星酒厂二锅头,52%v/v)15ml/ kg;急性灌水组,灌同等量的自来水;再分别于灌酒或灌水后2h,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。
慢性灌酒模型:40只大鼠随机分:慢性灌酒组和灌水组,给予灌酒(北京红星酒厂二锅头,52%v/v)或灌水四周,每天9:00和15:00间隔6小时灌两次,前2周每次灌8ml/kg、后2周每次灌12ml/kg。最后一次灌酒和灌水后2h,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。
未打击组于灌胃后2h,打击组于打击后0.5h,开胸暴露心脏,经左心室-主动脉插管,直接灌注4%中性多聚甲醛液处死兼固定,剪开右心耳放血。约0.5h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5um,分别HE染色观察脑组织形态变化,Ngb,HIF-1α,Epo,Bcl-2和Bax免疫组织化学染色,及TUNEL标记细胞凋亡,观察其表达情况。病理真彩色图像分析系统4.0进行体视量化病理学观测,实验数据用SPSS15.0统计软件(Independent-samples T-test and One-way ANONA)及Excel进行分析处理。
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;2h后呼吸频率63.5±9.4次/分和血压106.1±4.6mmHg均较灌酒前85.1±9.3次/分和133.8±5.5mmHg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±22.2g较灌酒前的389.7±31.7g明显下降(P<0.05);灌水组大鼠第4周体重为405.1±20.8g较灌水前的390.1±24.7g增长明显(P<0.05);大鼠灌酒后第四周基础血压为152.3±13.5mmHg较灌酒前的135.0±11.2上升(P<0.05),最后一次灌酒后血压为140.3±10.4较灌酒前的152.3±13.5 mmHg低(P<0.05),打击后升高为151.2±10.6mmHg(P<0.05)。灌水打击组打击后血压为148.1±13.5mmHg较打击前137.1±12.6mmHg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。
急性灌水组大鼠脑表面光泽,表面散在细小血管纹理。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。急性单纯灌酒组脑表面光泽,大片淤血,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。
慢性灌酒组大鼠脑表面光泽,大片淤血,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,神经元数量减少,胶质细胞增生。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。
3 Ngb表达观察
急慢性灌水及灌水打击对照组大鼠脑神经元内均有少量Ngb的表达。急慢性灌酒组和急慢性灌酒打击组大鼠脑组织各区Ngb阳性细胞数,IOD值分别高于急慢性灌水组和急慢性灌水打击组(P<0.01)。急性灌酒打击组Ngb阳性细胞数23.0±3.48多于急性灌酒组19.7±2.37 (P <0.01)。慢性灌酒打击组的Ngb阳性细胞数20.0±2.67及IOD值4070.3±226.12与慢性灌酒组阳性细胞数18.0±2.83、IOD值3922.5±319.62有显著的统计学意义(均为P <0.01)。慢性灌酒组的Ngb阳性细胞数18.0±2.83低于急性灌酒组19.7±2.37 (P <0.01),而慢性灌酒打击组脑组织各区NgbIOD值4070.3±226.12与急性灌酒打击组6380.6±234.21有统计学意义(海马、小脑区P <0.01,脑干区P <0.05)。
4 HIF-1α表达观察
急慢性灌水及灌水打击对照组大鼠脑神经元内有散在少量细胞HIF-1α轻度表达。急慢性灌酒组和急慢性灌酒打击组大鼠脑组织各区HIF-1α阳性细胞数,IOD值分别高于急慢性灌水组和急慢性灌水打击组(P<0.01)。急性灌酒打击组HIF-1α阳性细胞数24.2±4.82多于急性灌酒组21.2±3.25 (P<0.01)。慢性灌酒打击组的HIF-1αIOD值3862.6±289.24也高于慢性灌酒组3502.7±312.91(P<0.01)。慢性灌酒组的脑组织各区HIF-1α阳性细胞数18.5±2.45,IOD值3502.7±312.91低于急性灌酒组21.2±3.25、3878.6±212.31 (均为P<0.01),而慢性灌酒打击组脑组织HIF-1α阳性细胞数21.1±2.46也比急性灌酒打击组24.2±4.82要少(P<0.01)。
5 EPO表达观察
急慢性灌水及灌水打击对照组大鼠脑神经元内有散在少量细胞Epo表达。急慢性灌酒组和急慢性灌酒打击组脑组织各区Epo阳性细胞数,IOD值分别高于急慢性灌水组和急慢性灌水打击组(P<0.01)。急性灌酒打击组Epo阳性细胞数21.9±3.55多于急性灌酒组20.0±3.00(P<0.01)。慢性灌酒打击组的EpoIOD值3821.6±336.21也比慢性灌酒组3654.1±304.74要高(P<0.01)。慢性灌酒组的脑组织各区Epo IOD值3654.1±304.74均低于急性灌酒组3908.1±312.81(P<0.01) ,而慢性灌酒打击组脑组织各区EpoIOD值3821.6±336.21也均低于急性灌酒打击组4192.6±351.62 (P <0.01)。
6 Bcl-2,Bax的表达观察及Bcl-2/Bax的比值
急慢性灌酒组和急慢性灌酒打击组大鼠脑组织各区Bcl-2和Bax的阳性细胞数分别高于急慢性灌水组和急慢性灌水打击组(P<0.01)。急性灌酒打击组Bcl-2 IOD值2952.9±358.08多于急性灌酒组2782.7±381.22(P<0.01)。慢性灌酒组的脑组织各区Bcl-2IOD值2638.3±346.43均低于急性灌酒组2782.7±381.22(P<0.01),而慢性灌酒打击组脑组织各区Bcl-2 IOD值2720.7±352.76均比急性灌酒打击组2952.9±358.08要低(P<0.01)。
急性灌酒打击组脑组织各区Bax阳性细胞数11.2±2.78多于急性灌酒组10.4±2.44(P<0.05)。慢性灌酒组的脑组织各区BaxIOD值2806.3±282.35高于急性灌酒组2618.7±223.61。(P<0.01)
急性灌水及灌水打击组各脑区Bcl-2/Bax的平均比值分别为1.090、1.083,慢性灌水及灌水打击组分为1.053、1.081,急性灌酒及灌酒打击组为1.144、1.188,以上各组比值均大于1,而慢性灌酒及灌酒打击组各脑区Bcl-2/Bax的比值为0.946、0.912,比值均小于1。
7 TUNEL标记细胞凋亡结果
慢性灌酒组和慢性灌酒打击组可见较多的黄棕色阳性细胞,慢性灌酒组和慢性灌酒打击组大鼠脑组织各区细胞凋亡阳性细胞数4.4±0.52、4.9±0.57均分别高于慢性灌水组1.8±0.42和慢性灌水打击组1.9±0.57 (P<0.01),同时也高于急性灌酒组1.8±0.42及急性灌酒打击组1.6±0.52 (P<0.01)。慢性灌酒打击组的脑组织各区阳性细胞数4.9±0.57也高于慢性灌酒组4.4±0.52 (P<0.05)。
结论
1、急慢性酒精中毒可促发TSAH,慢性酒精中毒与TSAH高发生率及死亡率之间存在显著相关性。提示酗酒后TSAH及其死亡案件的死因分析时,应充分考虑酒精的急慢性毒理学效应,其在TSAH死亡案件中与头颈部外伤应属于协同死因。
2、慢性酒精中毒可通过抑制脑组织Ngb、HIF-1α、Epo等氧代谢通路相关因子表达,Bcl-2/Bax比值明显降低、神经细胞凋亡及暗神经元数量明显增加,可能参与酒精性脑萎缩、脑功能障碍的发生机制。
3、证实了急慢性酒精中毒可诱发脑组织暗神经元出现,特别是明显酒精中毒的暗神经元可能构成酗酒后TSAH及其死亡的重要的神经病理学基础,参与死亡机制的过程。
Background and objective
Traumatic subarachnoid haemorrhage(TSAH) is an independent brain damage,and it differes from pathological SAH and SAH caused by cerebral contusion,TSAH makes up the three cavity haemorrhage with traumatic haemorrhage of cerebral dura mater and cerebral dura mater hypostegal cavity. It is reported that TSAH is often occur after alcoholism with trivial strike to the face,the incidence rate was high as 87%. Our group has established TSAH model after drinking,and the result is that the incidence rate of TSAH: the acute group is 28.6%,the chronic group is 82.4%; the mortality rate: the acute group is 0%,the chronic group is 58.8%.According three aspects of Ngb、Hif-1α、Na+ , K+-ATPase aerobic and energy metabolism,tPA、MMP-9 neuron toxicity,blood vessel morphous vitodynamics,explored the occurrence mechanism of TSAH caused by alcohol drinking,and discovered the dark neuron,but we still didn’t know the relation between the high mortality rate in the chronic fuddled group and the dark neuron. So it is still a tough problem in analysis of cause of death and judicial trial.
We established TSAH model in alcoholism rats,observe the expression of Ngb and HIF-1α,Epo,Bcl-2 and Bax,apoptosis cells and the dark neuron was detected in the brain tissue. With the aerobic metabolism and basic pathological change of the brain,to explore the effects of morphous,metabolism and function of neurons after excessive drinking,and the possible mechanisms or relationship between excessive drinking and the happening of fatal TSAH,providing science theory for the TSAH medicolegal expertise and clinical prevention and cure.
Materials and Methods
Animals and groups: 80 male SD rats,each weighted 300-400g,breeding in several cages,devided into different groups randomly.
Acute fuddled model:40 rats devided randomly: acute fuddled groups,The edible alcohol of Erguotou of Peking(52%v/v)was injected into rats’stomach through esophagus 15ml/ kg per rat;Used same amount of tap water in the acute sham group.2 hours after injecting alcohol or water,giving an concussion hit to the rats,to establish to acute fuddled group and the acute sham and hit group,10 rats in each groups.
Chronic fuddled model:40 rats devided randomly: chronic fuddled group,chronic sham group. The edible alcohol of Erguotou of Peking(52%v/v)was injected into rats’stomach through esophagus,injected at 9:00 and 15:00 every day with 8 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. 2 hours after the last time injecting,giving an concussion hit to the rats,to establish to chronic fuddled group and the chronic sham and hit group,10 rats in each groups.
2 hours after injecting in the non-hitting groups,and 0.5hours after hitting groups,rats were dissected, the heart of the rat were exposed,aortic cannula through the left ventricle. the rats would be fixed by perfuse 4% neutral paraformaldehyde directly,cutting the right auricula to bloodletting.0.5 hour later,got the rats’brain,washed it,and putted it into the 4% neutral paraformaldehyde to fixing about six hours,then dehydration, immersing paraffin,embedding,tissue slice with 5um thick,to observe the Morphologic change by HE staining,Ngb,HIF-1α,Epo,Bcl-2 and Bax immunohistochemistry and TUNEL marking apoptosis cells,viewing its expression.To observe the pathological changes by patho-true-color image analytical system quantizationly,datas would be analyzed by SPSS 15.0 and Excel software.
Results
1. BAC and animal’s behavior
The BAC is 114.67±67.37 mg/dL after 2h of gavaging wine.The rats appeared 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 disappearing,the respiration torpidity,2h later,the breathing frequency and blood pressure decreased(P<0.01),but the control group had no change; 0.5h after being beaten,the blood pressure of acute fuddled group and the control group set up(P<0.05),but the breathing frequency had no change. In the chronic group,the hair becomes rough and dim,the spirit becomes frustrated,rats were weightless and decreased food-intake,the basic blood pressure set up , body weight decreased after 2 weeks(P<0.05) , the body weight of control group increased(P<0.05) after 4 weeks; 4 weeks later,the blood pressure of chronic fuddled group rised(P<0.05),after the last gavaging wine,the blood pressure drop out(P<0.05),and rised after being beaten(P<0.05),the control group also rised after being beaten(P<0.05),but the basic blood pressure of control group was not change; the breathing frequency of fuddled group and control group was not change,just decreased after gavaging wine(P<0.05).
2. Incidence rate and death rate of TSAH and SAH,Histomorphology
The incidence rate and death rate of TSAH are 20% and 0 in acute fuddled and hit group, but 50% and 40% in chronic fuddled and hit group respectively.There were no TSAH in the acute and chronic sham and hit groups.The incidence rate of spontaneous SAH are 10% in acute fuddled group and 0 in chronic group.
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. 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.The neuron and gliacyte were midrange hydropic degeneration,the gap of cell was enlarged,nissl's body was disappeared in the chronic fuddle beaten group,and dark neurons could be seen,it was prunosus,cell body shrinkage, clarifixation , sometimes the nucelus was disappeared , axis cylinder irregular twist , neuronophagia could be seen,the quantity of neuron was decreased,the gliacyte hyperplasy. Thick layer blood could be seen in the surface brain of TSAH in the chronic fuddled beaten group.
3 The expression of Ngb
There were some Ngb expression in the brain neuron of the acute and chronic sham groups,and the acute and chronic sham and hit groups.Ngb positive cells and IOD value in the acute and chronic fuddled groups,acute and chronic fuddled hit groups,higher than the acute and chronic sham groups,acute and chronic sham hit groups(P<0.01). Posotive cells of Ngb in the acute fuddled and hit group 23.0±3.48 exceeded the acute fuddled group 19.7±2.37 (P<0.01). There was noticeable statistical significance between the chronic fuddled hit group posotive cells of Ngb 20.0±2.67 and the chronic fuddled group 18.0±2.83 (P <0.01). Posotive cells of Ngb in the chronic fuddled group 18.0±2.83lower than the acute fuddled group 19.7±2.37 (P<0.01). there was noticeable statistical significance between the chronic fuddled hit group IOD 4070.3±226.12 and the acute fuddled hit group 6380.6±234.21 (hippocamp,cerebellum P<0.01,brain stemP <0.05)
4 The expression of HIF-1α
There were some HIF-1αexpression in the brain neuron of the acute and chronic sham groups,and the acute and chronic sham and hit groups. HIF-1αpositive cells and IOD value in the acute and chronic fuddled group,acute and chronic fuddled hit group higher than the acute and chronic sham groups,acute and chronic sham hit groups(P<0.01). Positive cells of HIF-1αin the acute fuddled and hit group 24.2±4.82 exceeded the acute fuddled group21.2±3.25 (P<0.01).The HIF-1αIOD value in the chronic fuddled hit group 3862.6±289.24higher than the chronic fuddled group3502.7±312.91 (P<0.01). HIF-1αpositive cells 18.5±2.45and IOD value 3502.7±312.91in the chronic fuddled group lower than the acute fuddled group 21.2±3.25、3878.6±212.31 (P <0.01). HIF-1αpositive cells and IOD value in the chronic fuddled hit group 21.1±2.46 also lower than the acute fuddled hit group 24.2±4.82 (P <0.01).
5 The expression of Epo
There were some Epo expression in the brain neuron of the acute and chronic sham groups,and the acute and chronic sham and hit groups. Epo positive cells and IOD value in the acute and chronic fuddled group,acute and chronic fuddled hit group higher than the acute and chronic sham groups,acute and chronic sham hit groups(P<0.01). Positive cells of Epo in the acute fuddled hit group 21.9±3.55 exceeded the acute fuddled group 20.0±3.00 (P <0.01). The Epo IOD value in the chronic fuddled hit group 3821.6±336.21 higher than the chronic fuddled group 3654.1±304.74 (P <0.01).Epo IOD in the chronic fuddled group 3654.1±304.74 lower than the acute fuddled group 3908.1±312.81 (brain stemP <0.05,othersP < 0.01). Epo IOD in the chronic fuddled hit group 3821.6±336.21 also lower than the acute fuddled hit group 4192.6±351.62 (P < 0.01).
6 The expression Bcl-2 and Bax,ratio of Bcl-2/Bax
Bcl-2 positive cells and IOD value in the acute and chronic fuddled group,acute and chronic fuddled hit group higher than the acute and chronic sham groups,acute and chronic sham hit groups(P<0.01). Bcl-2 IOD in the acute fuddled hit group 2952.9±358.08 exceeded the acute fuddled group 2782.7±381.22 (P<0.01).Bcl-2 IOD in the chronic fuddled group 2638.3±346.43 lower than the acute fuddled group 2782.7±381.22 (P <0.01),the chronic fuddled group 2720.7±352.76 were also lower than the acute fuddled hit group 2952.9±358.08 (P <0.01)
Positive cells of Bax in the acute fuddled hit group 11.2±2.78 exceeded the acute fuddled group 10.4±2.44 (P <0.05). Bax IOD in the chronic fuddled group 2806.3±282.35 higher than the acute fuddled group 2618.7±223.61 (P<0.01).
The average ratio of Bcl-2/Bax in the acute sham group,and acute sham hit group were 1.090、1.083,the chronic sham group,and chronic sham hit group were1.053、1.081,the acute fuddled group,and acute fuddled hit group were1.144、1.188,above ratio were all much than one, the chronic fuddled group,and chronic fuddled hit group were0.946、0.912,less than one.
7 TUNEL marked apoptosis cells results
There were many yellowish-brown color positive cells in the chronic fuddled group and chronic fuddled hit group, apoptosis cells in the chronic fuddled group and chronic fuddled hit group exceeded the chronic sham group and the chronic sham hit group (P<0.01),and also higher than the chronic fuddled group and the chronic fuddled hit group(P<0.01). Apoptosis cells in the chronic fuddled hit group also higher than the chronic fuddled group(frontal lobe,hippocamp P<0.01,cerebellum and brain stem P<0.05).
Conclusions
1.Acute and chronic alcoholism would tend to occur TSAH,there was a significant dependablity between chronic alcoholism and TSAH high incidence and mortality rate.When we analyze the TSAH cases,we should thoroughly consider the toxicological effect of acute and chronic alcoholism,and with head and neck injury as coordination death cause.
2.The effect of chronic alcoholism could inhibit the Ngb、HIF-1α、Epo such aerobic metabolism factors,lowed down the ratio of Bcl-2/Bax,increased the numbers of nueron apoptosis and dark neuron,which may be participate the mechanism of alcohol encephalanalosis and disordered brain function.
3.It was proved that acute and chronic alcoholism could induce the occur of dark neuron, especially the dark neuron in the alcoholism may be constitute the important neuro-pathology foundation,participates the process of death mechanism.
引文
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