急慢性酒精中毒大鼠凝血功能变化与外伤性蛛网膜下腔出血发生及死亡机制研究
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摘要
1背景及目的
酗酒是严重的社会公害之一,过度饮酒造成的酒精中毒已成为独立的、不容忽视的致死因素。在法医学鉴定中,经常遇见饮酒后较轻微头颈部外伤引发的外伤性蛛网膜下腔出血(traumatic subarachnoid haemorrhage, TSAH),其发生率约占TSAH的87%,死亡率达50%以上。由于目前对酒精和外力因素在TSAH发生及死亡机制中相互关系和作用程度大小尚无统一认识,致使此类案件的鉴定往往争议较大,亦给法庭审判量刑带来困难。因此,有必要深入研究酒后TSAH的发生及死亡机制,明确各因素在伤亡后果中的参与度,为此类案件法医学鉴定及审判量刑工作提供必要的科学证据和理论参考。
本研究通过建立急慢性酒精中毒大鼠打击模型,检测不同时相点大鼠血乙醇浓度、凝血功能及抗凝活性变化系列指标,观察各指标变化的内在联系,从血液的酒精毒理学角度探讨急慢性酒精中毒大鼠TSAH发生机制。同时,检测血栓烷A-2及其受体表达并监测心功能变化,从脑-心联系角度探讨急慢性酒精中毒大鼠TSAH死亡机制。
2材料与方法
2.1动物分组与模型建立
SPF级成年雄性SD大鼠90只,体重300±50g。实验动物分组见下表。实验大鼠灌胃和打击处置后观察0.5h,乙醚麻醉,腹主动脉采血并放血处死,立即开颅观察并记录TSAH发生率及出血量情况,留取脑、心、肝等组织冻存及固定处理。
2.2实验方法
观察实验大鼠的行为学、体重、血压、心电变化及脑组织病理学改变;气相色谱法检测血乙醇浓度(BAC);全自动凝血分析仪检测凝血酶原时间(PT)、凝血酶时间(TT)、活化部分凝血活酶时间(APTT)及纤维蛋白原(FIB);全自动血气分析仪检测pH值、血氧饱和度(SaO2)、氧分压(PO2)、碳酸氢根(HCO3-)、剩余碱(BE)等7项血气分析指标;ELISA法检测血浆中组织因子(TF)、组织因子途径抑制剂(TFPI)、组织型纤溶酶原激活剂(tPA)、纤溶酶原激活物抑制剂-1 (PAI-1)、D二聚体(D-D)及血栓烷B-2 (TXB-2)浓度;免疫组化法及免疫印迹法进行定性、定位及定量检测脑及心组织的tPA、TXA2R表达情况。统计TSAH发生率及死亡率,Fisher法分级TSAH。所有实验数据均采用SPSS 17.0及Excel 2007进行统计学处理和分析。
3结果
3.1行为学表现及体重、血压变化
急性灌酒组大鼠酒后0.5h内兴奋性增强,此后逐渐出现昏睡、翻正反射消失等急性酒精中毒表现,4-6h后逐渐清醒。慢性灌酒组大鼠每次灌酒后均有急性酒精中毒表现,灌酒1周后体重明显下降,至第4周末下降达60g以上(p<0.01)。第3周始尾动脉压自16.60±0.76 kPa升高至17.48±0.90 kPa (p<0.05)。打击后,实验组大鼠均呈典型的脑震荡表现,AA/AW/CW及部分CA组大鼠3-5sec后渐至正常,55%(11/20) CA组大鼠出现角膜反射消失、四肢抽搐、口唇发绀、呼吸由深大转至浅慢,2-4min内迅速死亡。
3.2心电变化
脑震荡性打击后,各实验组均出现一过性心电基线漂移,心率从正常的400 bpm增至500-600 bpm,约5-6sec后渐至正常,而慢性灌酒组死亡大鼠在心电恢复正常后30-60sec内,心率骤减至100-200 bpm伴心律不齐,而后逐渐减慢,直至消失,心电呈直线改变,此过程历时约2-4min。
3.3脑组织病理学观察和TSAH发生率及死亡率
急性灌酒组伤后脑组织轻度淤血、肿胀,多数有少量蛛网膜下腔出血,局限于脑干腹侧面或小脑表面,脑神经元及胶质细胞轻度水变性。慢性灌酒组伤后脑组织中至重度淤血、肿胀,多数有较多量蛛网膜下腔出血,广泛分布于全脑,脑神经元及胶质细胞水变性,可见噬神经和卫星现象;小脑蒲肯野细胞排列疏密不均,部分溶解消失;脑干神经核团多见“暗神经元”,呈深暗红色、胞体皱缩、胞浆及胞核浓缩、均质化、核不清或消失、轴突不规则扭曲等表现。急慢性组脑实质内均未见明显挫伤及出血改变。
急慢性灌酒组TSAH发生率分别为26%及85% (p<0.01),死亡率分别为0及55% (p<0.01),且死亡大鼠均发生TSAH。
3.4血乙醇浓度变化
灌酒后2h大鼠BAC达峰值188.16±14.71 mg/dL,0.5h至4h始终处于人醉酒下限值80 mg/dL之上,6h后仍达60.08±13.80 mg/dL。
3.5凝血功能变化及与BAC、TSAH的相关性分析
急性灌酒组各时相点血浆中PT、TT、APTT均不同程度延长,2h最明显,分别达28.50±2.08、35.49±1.15、24.56±1.09 sec (p<0.01);FIB先升高(0.5h,p<0.05)后下降(1h,p<0.05)再缓慢回升趋于正常。慢性灌酒组各指标亦不同程度延长和增高,与相应对照组(Control/CW)比较差异显著(p<0.05),但与AA-2h组比较差异不明显(p>0.05)。急性灌酒组PT、TT及APTT与BAC存在较强的相关性(分别r=0.789,0.818,0.752),呈正相关线性分布;FIB与BAC二者相关性不明显(p>0.05)。急性灌酒组各时相点TSAH发生率与对应的BAC存在高度正相关(r=0.974),决定系数R2=0.949。
3.6血气分析变化
急性灌酒组动脉血pH值、SaO2、PO2、HCO3-、BE均不同程度降低,2h达最低点(分别为7.26±0.05、97.77±0.36、12.70±3.67、14.17±0.95、-12.57±2.90) (p<0.05),此后缓慢回升,部分指标至6h仍未恢复正常范围。慢性灌酒组亦有不同程度降低,与相应对照组比较均有显著差异(p<0.05),急慢性灌酒组间差异不显著(p>0.05)。
3.7血浆TF/TFPI、tPA/PAI-1、D-D及TXB-2浓度变化
急性灌酒组各时相点TF不同程度降低,2h达最低点385.32±104.13 pg/ml (p<0.01),TFPI不同程度升高,2h达峰值1611.65±211.59 pg/ml (p<0.01);TF/TFPI先降后升,2h达最小值0.2391。慢性灌酒组二者与相应对照组及AA-2h组比较差异显著(p<0.05)。
急性灌酒组各时相点tPA及PAI-1均不同程度升高,2h分别达峰值6422.18±119.76 pg/ml (p<0.01)及21246.67±1346.07 pg/ml (p<0.01),tPA/PAI-1先升后降,2h达最大0.3023。慢性灌酒组tPA及PAI-1与相应对照组及AA-2h组比较差异亦显著(p<0.05)。
急性灌酒组各时相点D-D不同程度升高,0.5h-6h与相应对照组比较差异显著(p<0.05),2h达峰值2207.40±322.12 pg/ml (p<0.01)。慢性灌酒组D-D与相应对照组及AA-2h组比较有显著差异(p<0.05)。
急性灌酒组各时相点TXB-2略升高,1h与相应对照组差异显著(p<0.05)。慢性灌酒组TXB-2明显升高,与相应对照组及急性灌酒组比较均有高度显著差异(p<0.01)。
3.8脑及心组织tPA/TXA2R表达变化
目的蛋白tPA在各组额叶、小脑、脑干延髓腹侧及背侧面的免疫印迹膜67kDa处均出现清晰条带。三个对照组条带较窄细、色淡,急慢性灌酒组条带较粗深。IHC光密度分析结果显示,急性灌酒组tPA表达均明显增加,与相应对照组差异高度显著(p<0.01),2h表达最强,IOD值分别为65975.29±5961.34、70203.96±5406.58、64696.20±6394.44及68072.76±4471.79。慢性灌酒组tPA表达亦增强,与相应对照组差异亦高度显著(p<0.01),与AA-2h组比较,除额叶外,其余部位tPA表达量均有显著差异(p<0.05)。
目的蛋白TXA2R在脑干及心组织的免疫印迹膜58kDa处均出现较清晰条带,但是脑(除慢性灌酒组)及心的各实验组条带均较窄细,其中慢性灌酒组条带粗深,其IOD值达6188.04±391.11,与相应对照组、急性灌酒组以及同组的心脏差异均高度显著(p<0.01)。
4结论
(1) BAC与凝血功能、TSAH三者间存在明显的剂量-效应关系:BAC越高,凝血时间越长,TSAH发生率就越高;酗酒时间越长,凝血功能破坏越严重,TSAH发生率及死亡率就越高。
(2)急慢性酒精中毒均可抑制TF/TFPI的凝血启动机制、增强tPA/PAI-1的纤溶激活作用,破坏二者动态平衡,降低凝血功能,长期酗酒更显著,成为酒后TSAH高发生率的重要机制之一。
(3)急慢性酒精中毒导致的机体持续低氧状态,可促进血管内皮tPA过度表达、纤溶活性增强,构成酒精抑制凝血功能的重要机制之一。
(4)本研究首次从脑-心联系角度,探讨酒后TSAH死亡可能机制。认为:长期酗酒血液TXA-2含量增高、脑组织TXA2R表达增强,二者在TSAH脑组织中快速大量结合,通过5-羟色胺能信号通路,过度抑制低位脑干中线区的生命中枢,过度兴奋迷走神经以抑制心脏活动,可能是慢性酒精中毒TSAH高死亡率的重要机制之一。
1 Background and Objective
Alcohol abuse is a serious public hazard in modern society, and alcoholism has become one of the independent and notable lethal factors. In medicolegal examinations, the cases frequently involved in traumatic subarachnoid hemorrhage (TSAH) were caused by a minor blow on the head after drinking. The morbility and mortality of TSAH are about 87% and more than 50% in total death cases of TSAH. Although alcohol’s contributory role in head injury has been well discussed by researchers for many years, no conclusions have been drawn as to its exact role in the cause of TSAH, as well as death. As a result, in those cases involved, controversy and trouble often occur in the forensic identification and criminal investigation. Therefore, it is necessary to investigate the occurrence and death mechanisms of TSAH after drinking, to identify their respective contributories, and to provide the necessary scientific evidences and theoretical references for the medicolegal examination and forensic investigation.
In this study, the animal models of the acute and chronic alcoholism rats were established firstly, and the blood alcohol concentration (BAC) and the series of indicators of coagulation and anticoagulation were detected in order to observe the internal relations of them. As viewed from alcohol toxicology, it is an attempt to study the possible occurrence mechanisms of TSAH in the acute and chronic alcoholism rats. In addition, the expression of thromboxane A-2 & receptor and the changes of ECG had also been detected to explore the possible death mechanisms of TSAH from the view of the brain-heart connection.
2 Materials and Methods
2.1 Groups of animals and Establishment of model
Ninety male Sprague-Dawley rats weighting 250-350g were used in this study, and they were divided into five groups, including Acute Alcoholism and Strike group (AA, n=50), Acute Water and Strike group (AW, n=5), Chronic Alcoholism and Strike group (CA, n=20), Chronic Water and Strike group (CW, n=5), and Control (n=10). a) Edible spirituous liquor (52% v/v, 15ml/kg) was given intragastrically to AA for one time, and the rats were averagely divided into 0.5h, 1h, 2h, 4h and 6h, which received the concussion strike at the different times. b) Rats of AW were given the equivalent volume of distilled water, and they received the strike after 2h. c) The same spirituous liquor was given to CA twice every day at an interval of 6 hour for 4 weeks. The dosage was 8 ml/kg in the first 2 weeks, and 12 ml/kg in the last 2 weeks. Rats of CA received the concussion strike after 2h at the end of 4 weeks. d) Rats of CW were given the equivalent volume of distilled water, and the other treatments were same to CA. e) Blood and tissues of Control were obtained after bleeding execution directly.
The rats were observed half an hour after receiving the intragastric administration and strike. Then the blood of the abdominal aorta was collected after etherization. The craniotomy was operated immediately after bleeding execution, and the site and volume of TSAH were recorded with a digital camera. The brains, hearts, livers and other tissues were preserved by freezing and fixed in 10% formalin solution.
2.2 Experimental Methods
The general conditions of experimental rats were observed, which include the behavior, body weight, blood pressure, ECG and cerebral pathology. Gas chromatography was used in the assay of blood alcohol concentration (BAC). The automated coagulation analyzer was applied to detect the prothrombin time (PT), thrombin time (TT), activated partial thromboplastin time (APTT) and fibrinogen (FIB). The automatic blood gas analyzer was applied to determine pH, oxygen saturation (SaO2), Pressure of oxygen (PO2), bicarbonate (HCO3-), base excess (BE) and other seven indicators of blood gas analysis. ELISA was used in the assay of the tissue factor (TF), tissue factor pathway inhibitor (TFPI), tissue plasminogen activator (tPA), plasminogen activator inhibitor -1 (PAI-1), D dimer (D-D) and thromboxane B-2 (TXB-2) concentration in plasma. Immunohistochemistry methods and Western blotting were used for the qualitative and quantitative measurement of tPA and TXA2R expression in brain and heart.
The morbility and mortality of TSAH were calculated, and the volumes of TSAH were graded according to Fisher methods. All experimental data were treated with SPSS 17.0 and Excel 2007 for statistical analysis.
3 Results
3.1 Changes of performances, body weight and blood pressure
Excitability of acute alcoholism rats increased within a short time after drinking, and the signs of acute alcoholic intoxication were showed gradually after 0.5h, such as lethargy, loss of righting reflex, etc. The consciousness of rats came back after 4-6h. Chronic alcoholism rats had the same signs of the acute alcoholic intoxication after drinking every time. The body weights decreased after 1st week, and to 4th week the body weight decreased about 60g or more (p<0.01). The arterial blood pressures increased from 16.60±0.76 kPa to 17.48±0.90 kPa (p<0.05) from 1st to 3rd week. After concussion strike, the rats showed the typical signs of the concussion, and the rats (AA/AW/CW/part of CA groups) recovered gradually in 3-5sec. However, 55% of the chronic alcoholism rats had absent corneal reflex, raising head, limbs twitch, lips cyanosis, and the breathing from deep to slow, and then died in 2-4 min rapidly.
3.2 Changes of ECG
After concussion strike, all experimental groups had transient baseline drift of ECG. The heart rates sped up from the normal 400 to 500-600 bpm, and ECG came back after about 5-6 sec. While the heart rates in chronic alcoholism rats (death) dropped drastically to 100-200 bpm with cardiac arrhythmia after 30-60 sec, and then they slowed down gradually until the disappearance. The process took about 2-4min.
3.3 Cerebral pathology and morbility & mortality of TSAH
Acute alcoholism rat brains after strike showed mild congestion, swelling and a small amount of subarachnoid hemorrhage, and TSAH confined to the ventral surface of the brain stem or cerebellar surface. Neurons and glial cells also showed mild degeneration. Chronic alcoholism rat brains after strike showed moderate to severe congestion, swelling, and most amount of subarachnoid hemorrhage, and TSAH more widely distributed in the whole brain. Neurons and glial cells of the cerebrum showed degeneration and satellite phenomena. Purkinje cell of cerebellum arranged asymmetrically, and part of them dissolved away. The "dark neurons" were observed in the brainstem, and they showed dark red, cell body shrinkage, condensation of cytoplasm and nucleus, homogenization, unclear or disappear nucleus, and the irregular distortions of axon, etc. In addition, neither injury nor bleeding was found in brain tissue of the acute and chronic groups.
The morbility of TSAH in the acute and chronic alcoholism groups were 26% and 85% (p<0.01). The mortality of the two groups were 0 and 55% (p<0.01), and TSAH were found in all of the death rats.
3.4 Changes of blood alcohol concentration
BAC of rats raised to the maximum (188.16±14.71mg/dL) 2h after intragastric administration, and BAC were always above the legal limit of intoxication (80mg/dL) from 0.5h to 4h, and reached 60.08±13.80 mg/dL after 6h.
3.5 Correlation analysis of blood coagulation, BAC and TSAH
Compared with Control and AW groups, the PT, TT, APTT of the acute alcoholism group increased in different degrees at each time point, and that of AA-2h are the most significant, 28.50±2.08, 35.49±1.15, 24.56±1.09 sec (p<0.01). Content of FIB increased firstly (0.5h, p<0.05) and then decreased (1h, p<0.05), and it recovered to normal slowly. These of the chronic alcoholism group also extended and increased, and it was significant difference (p<0.05) with the Control/CW groups, but there was no significant difference with AA-2h group (p>0.05). In addition, there was a strong correlation between BAC and PT, TT, APTT (r=0.789, 0.818, 0.752), and they showed the positive correlation and linear distribution. There was no obvious correlation between BAC and FIB. Morbility of TSAH and BAC in different phases of acute alcoholism group showed the highly positive correlation (r=0.974), and the coefficient of determination R2 was 0.949.
3.6 Changes of blood gas analysis
Arterial pH, SaO2, PO2, HCO3- and BE in the acute alcoholism rats all reduced to different extent, and they reached the lowest points on 2h (7.26±0.05, 97.77±0.36, 12.70±3.67, 14.17±0.95, -12.57±2.90) (p<0.05). And then they recovered slowly, and some indicators had not returned to normal range until 6h. These of the chronic alcoholism group also decreased in various degrees, and the data compared with Control/CW group were significantly different (p<0.05). However, there was no significant difference between the acute alcoholism group and the chronic one (p>0.05).
3.7 Changes of TF/TFPI, tPA/PAI-1, D-D, TXB-2 in plasma
TF of the acute alcoholism group were reduced to varying degrees, and they reached the lowest point of 385.32±104.13 pg/ml (p<0.01) at 2h. However, TFPI increased to varying degrees, and they reached the peak of 1611.65±211.59 pg/ml (p<0.01) at 2h. TF/TFPI decreased firstly and then rose, and it was the minimum of 0.2391 at 2h. TF and TFPI of the chronic alcoholism group were significantly different compared with Control, CW and AA-2h groups (p<0.05).
tPA and PAI-1 of the acute alcoholism group all increased to varying degrees, and they reached the highest point of 6422.18±119.76 pg/ml (p<0.01) and 21246.67±1346.07 pg/ml (p<0.01) at 2h. tPA/PAI-1 rose firstly and then decreased, and it was the maximum of 0.3023 at 2h. tPA and PAI-1 of the chronic alcoholism group were significantly different compared with Control, CW and AA-2h groups (p<0.05).
D-D of the acute alcoholism group all increased to varying degrees, which had significant differences with that of Control/AW groups from 0.5h to 6h (p<0.05), and reached the maximum of 2207.40±322.12 pg/ml at 2h (p<0.01). D-D of the chronic alcoholism group were significantly different from that of Control, CW and AA-2h groups (p<0.05).
TXB-2 of the acute alcoholism group increased slightly, which had significant difference compared with that of Control/AW at 1h (p<0.05). TXB-2 of the chronic alcoholism group increased significantly, which had significant differences with that of Control, CW and AA-2h groups (p<0.01).
3.8 Expression of tPA/TXA2R in brain and heart
Interest protein tPA of the frontal lobe, cerebellum, ventral and dorsal of brainstem showed the clear bands on 67kDa of Western blot membranes. Bands of Control/AW/CW groups were narrow, thin and pale, but those of acute and chronic alcoholism groups were thick and dark. Optical density analysis of IHC showed that expression of tPA in the acute alcoholism group increased significantly compared with Control/AW group (p<0.01), whose highest value occurred at 2h. tPA expression of chronic alcoholism group also increased, which had highly significant differences with Control/CW groups (p<0.01), and other parts of brain except for the frontal lobe were statistical significances compared with AA-2h group (p<0.05).
Interest protein TXA2R of brainstem and heart showed clear bands of 58kDa on the Western blotting membranes. Bands of Control/AW/CW/AA groups in brainstem and heart were narrow, thin and pale, but those of CA group were thick and dark in brainstem with IOD value of 6188.04±391.11. They had highly significant differences with Control/CW groups and the heart in the same group (p<0.01).
4 Conclusions
(1) Signifcant dose-response relationships between BAC, coagulation and TSAH showed that, as BAC increase, the clotting times extend, and the morbility and mortality of TSAH also increase; The longer alcohol abuse, the more serious destruction of blood coagulation, and the higher morbility and mortality for TSAH.
(2) Acute and chronic alcoholism could reduce the clotting start function (TF/TFPI) and increase the fibrinolytic activity (tPA/PAI-1). The normal dynamic balances of them are damaged, and blood coagulation decreases. The more obvious changes are in chronic alcoholism, which is considered as one of the important mechanisms for the high morbility of TSAH.
(3) Acute and chronic alcoholism could lead to the sustained hypoxia in vivo, which could promote the expression of tPA and increase the fibrinolytic activity; it may be one of the key mechanisms that alcohol inhibits the coagulation function.
(4) It is the first time to explore the death mechanisms of TSAH after drinking in terms of the brain-heart connection. It is believed that chronic alcoholism could cause increasing contents of TXA-2 in blood and expression of TXA2R in brain. After TSAH, they combine quickly in brain tissues, and excite vagal activity and inhibit cardiac function, which may be one of the important mechanisms for the chronic alcoholism death from TSAH.
酗酒是严重的社会公害之一,过度饮酒造成的酒精中毒已成为独立的、不容忽视的致死因素。在法医学鉴定中,经常遇见饮酒后较轻微头颈部外伤引发的外伤性蛛网膜下腔出血(traumatic subarachnoid haemorrhage, TSAH),其发生率约占TSAH的87%,死亡率达50%以上。由于目前对酒精和外力因素在TSAH发生及死亡机制中相互关系和作用程度大小尚无统一认识,致使此类案件的鉴定往往争议较大,亦给法庭审判量刑带来困难。因此,有必要深入研究酒后TSAH的发生及死亡机制,明确各因素在伤亡后果中的参与度,为此类案件法医学鉴定及审判量刑工作提供必要的科学证据和理论参考。
本研究通过建立急慢性酒精中毒大鼠打击模型,检测不同时相点大鼠血乙醇浓度、凝血功能及抗凝活性变化系列指标,观察各指标变化的内在联系,从血液的酒精毒理学角度探讨急慢性酒精中毒大鼠TSAH发生机制。同时,检测血栓烷A-2及其受体表达并监测心功能变化,从脑-心联系角度探讨急慢性酒精中毒大鼠TSAH死亡机制。
2材料与方法
2.1动物分组与模型建立
SPF级成年雄性SD大鼠90只,体重300±50g。实验动物分组见下表。实验大鼠灌胃和打击处置后观察0.5h,乙醚麻醉,腹主动脉采血并放血处死,立即开颅观察并记录TSAH发生率及出血量情况,留取脑、心、肝等组织冻存及固定处理。
2.2实验方法
观察实验大鼠的行为学、体重、血压、心电变化及脑组织病理学改变;气相色谱法检测血乙醇浓度(BAC);全自动凝血分析仪检测凝血酶原时间(PT)、凝血酶时间(TT)、活化部分凝血活酶时间(APTT)及纤维蛋白原(FIB);全自动血气分析仪检测pH值、血氧饱和度(SaO2)、氧分压(PO2)、碳酸氢根(HCO3-)、剩余碱(BE)等7项血气分析指标;ELISA法检测血浆中组织因子(TF)、组织因子途径抑制剂(TFPI)、组织型纤溶酶原激活剂(tPA)、纤溶酶原激活物抑制剂-1 (PAI-1)、D二聚体(D-D)及血栓烷B-2 (TXB-2)浓度;免疫组化法及免疫印迹法进行定性、定位及定量检测脑及心组织的tPA、TXA2R表达情况。统计TSAH发生率及死亡率,Fisher法分级TSAH。所有实验数据均采用SPSS 17.0及Excel 2007进行统计学处理和分析。
3结果
3.1行为学表现及体重、血压变化
急性灌酒组大鼠酒后0.5h内兴奋性增强,此后逐渐出现昏睡、翻正反射消失等急性酒精中毒表现,4-6h后逐渐清醒。慢性灌酒组大鼠每次灌酒后均有急性酒精中毒表现,灌酒1周后体重明显下降,至第4周末下降达60g以上(p<0.01)。第3周始尾动脉压自16.60±0.76 kPa升高至17.48±0.90 kPa (p<0.05)。打击后,实验组大鼠均呈典型的脑震荡表现,AA/AW/CW及部分CA组大鼠3-5sec后渐至正常,55%(11/20) CA组大鼠出现角膜反射消失、四肢抽搐、口唇发绀、呼吸由深大转至浅慢,2-4min内迅速死亡。
3.2心电变化
脑震荡性打击后,各实验组均出现一过性心电基线漂移,心率从正常的400 bpm增至500-600 bpm,约5-6sec后渐至正常,而慢性灌酒组死亡大鼠在心电恢复正常后30-60sec内,心率骤减至100-200 bpm伴心律不齐,而后逐渐减慢,直至消失,心电呈直线改变,此过程历时约2-4min。
3.3脑组织病理学观察和TSAH发生率及死亡率
急性灌酒组伤后脑组织轻度淤血、肿胀,多数有少量蛛网膜下腔出血,局限于脑干腹侧面或小脑表面,脑神经元及胶质细胞轻度水变性。慢性灌酒组伤后脑组织中至重度淤血、肿胀,多数有较多量蛛网膜下腔出血,广泛分布于全脑,脑神经元及胶质细胞水变性,可见噬神经和卫星现象;小脑蒲肯野细胞排列疏密不均,部分溶解消失;脑干神经核团多见“暗神经元”,呈深暗红色、胞体皱缩、胞浆及胞核浓缩、均质化、核不清或消失、轴突不规则扭曲等表现。急慢性组脑实质内均未见明显挫伤及出血改变。
急慢性灌酒组TSAH发生率分别为26%及85% (p<0.01),死亡率分别为0及55% (p<0.01),且死亡大鼠均发生TSAH。
3.4血乙醇浓度变化
灌酒后2h大鼠BAC达峰值188.16±14.71 mg/dL,0.5h至4h始终处于人醉酒下限值80 mg/dL之上,6h后仍达60.08±13.80 mg/dL。
3.5凝血功能变化及与BAC、TSAH的相关性分析
急性灌酒组各时相点血浆中PT、TT、APTT均不同程度延长,2h最明显,分别达28.50±2.08、35.49±1.15、24.56±1.09 sec (p<0.01);FIB先升高(0.5h,p<0.05)后下降(1h,p<0.05)再缓慢回升趋于正常。慢性灌酒组各指标亦不同程度延长和增高,与相应对照组(Control/CW)比较差异显著(p<0.05),但与AA-2h组比较差异不明显(p>0.05)。急性灌酒组PT、TT及APTT与BAC存在较强的相关性(分别r=0.789,0.818,0.752),呈正相关线性分布;FIB与BAC二者相关性不明显(p>0.05)。急性灌酒组各时相点TSAH发生率与对应的BAC存在高度正相关(r=0.974),决定系数R2=0.949。
3.6血气分析变化
急性灌酒组动脉血pH值、SaO2、PO2、HCO3-、BE均不同程度降低,2h达最低点(分别为7.26±0.05、97.77±0.36、12.70±3.67、14.17±0.95、-12.57±2.90) (p<0.05),此后缓慢回升,部分指标至6h仍未恢复正常范围。慢性灌酒组亦有不同程度降低,与相应对照组比较均有显著差异(p<0.05),急慢性灌酒组间差异不显著(p>0.05)。
3.7血浆TF/TFPI、tPA/PAI-1、D-D及TXB-2浓度变化
急性灌酒组各时相点TF不同程度降低,2h达最低点385.32±104.13 pg/ml (p<0.01),TFPI不同程度升高,2h达峰值1611.65±211.59 pg/ml (p<0.01);TF/TFPI先降后升,2h达最小值0.2391。慢性灌酒组二者与相应对照组及AA-2h组比较差异显著(p<0.05)。
急性灌酒组各时相点tPA及PAI-1均不同程度升高,2h分别达峰值6422.18±119.76 pg/ml (p<0.01)及21246.67±1346.07 pg/ml (p<0.01),tPA/PAI-1先升后降,2h达最大0.3023。慢性灌酒组tPA及PAI-1与相应对照组及AA-2h组比较差异亦显著(p<0.05)。
急性灌酒组各时相点D-D不同程度升高,0.5h-6h与相应对照组比较差异显著(p<0.05),2h达峰值2207.40±322.12 pg/ml (p<0.01)。慢性灌酒组D-D与相应对照组及AA-2h组比较有显著差异(p<0.05)。
急性灌酒组各时相点TXB-2略升高,1h与相应对照组差异显著(p<0.05)。慢性灌酒组TXB-2明显升高,与相应对照组及急性灌酒组比较均有高度显著差异(p<0.01)。
3.8脑及心组织tPA/TXA2R表达变化
目的蛋白tPA在各组额叶、小脑、脑干延髓腹侧及背侧面的免疫印迹膜67kDa处均出现清晰条带。三个对照组条带较窄细、色淡,急慢性灌酒组条带较粗深。IHC光密度分析结果显示,急性灌酒组tPA表达均明显增加,与相应对照组差异高度显著(p<0.01),2h表达最强,IOD值分别为65975.29±5961.34、70203.96±5406.58、64696.20±6394.44及68072.76±4471.79。慢性灌酒组tPA表达亦增强,与相应对照组差异亦高度显著(p<0.01),与AA-2h组比较,除额叶外,其余部位tPA表达量均有显著差异(p<0.05)。
目的蛋白TXA2R在脑干及心组织的免疫印迹膜58kDa处均出现较清晰条带,但是脑(除慢性灌酒组)及心的各实验组条带均较窄细,其中慢性灌酒组条带粗深,其IOD值达6188.04±391.11,与相应对照组、急性灌酒组以及同组的心脏差异均高度显著(p<0.01)。
4结论
(1) BAC与凝血功能、TSAH三者间存在明显的剂量-效应关系:BAC越高,凝血时间越长,TSAH发生率就越高;酗酒时间越长,凝血功能破坏越严重,TSAH发生率及死亡率就越高。
(2)急慢性酒精中毒均可抑制TF/TFPI的凝血启动机制、增强tPA/PAI-1的纤溶激活作用,破坏二者动态平衡,降低凝血功能,长期酗酒更显著,成为酒后TSAH高发生率的重要机制之一。
(3)急慢性酒精中毒导致的机体持续低氧状态,可促进血管内皮tPA过度表达、纤溶活性增强,构成酒精抑制凝血功能的重要机制之一。
(4)本研究首次从脑-心联系角度,探讨酒后TSAH死亡可能机制。认为:长期酗酒血液TXA-2含量增高、脑组织TXA2R表达增强,二者在TSAH脑组织中快速大量结合,通过5-羟色胺能信号通路,过度抑制低位脑干中线区的生命中枢,过度兴奋迷走神经以抑制心脏活动,可能是慢性酒精中毒TSAH高死亡率的重要机制之一。
1 Background and Objective
Alcohol abuse is a serious public hazard in modern society, and alcoholism has become one of the independent and notable lethal factors. In medicolegal examinations, the cases frequently involved in traumatic subarachnoid hemorrhage (TSAH) were caused by a minor blow on the head after drinking. The morbility and mortality of TSAH are about 87% and more than 50% in total death cases of TSAH. Although alcohol’s contributory role in head injury has been well discussed by researchers for many years, no conclusions have been drawn as to its exact role in the cause of TSAH, as well as death. As a result, in those cases involved, controversy and trouble often occur in the forensic identification and criminal investigation. Therefore, it is necessary to investigate the occurrence and death mechanisms of TSAH after drinking, to identify their respective contributories, and to provide the necessary scientific evidences and theoretical references for the medicolegal examination and forensic investigation.
In this study, the animal models of the acute and chronic alcoholism rats were established firstly, and the blood alcohol concentration (BAC) and the series of indicators of coagulation and anticoagulation were detected in order to observe the internal relations of them. As viewed from alcohol toxicology, it is an attempt to study the possible occurrence mechanisms of TSAH in the acute and chronic alcoholism rats. In addition, the expression of thromboxane A-2 & receptor and the changes of ECG had also been detected to explore the possible death mechanisms of TSAH from the view of the brain-heart connection.
2 Materials and Methods
2.1 Groups of animals and Establishment of model
Ninety male Sprague-Dawley rats weighting 250-350g were used in this study, and they were divided into five groups, including Acute Alcoholism and Strike group (AA, n=50), Acute Water and Strike group (AW, n=5), Chronic Alcoholism and Strike group (CA, n=20), Chronic Water and Strike group (CW, n=5), and Control (n=10). a) Edible spirituous liquor (52% v/v, 15ml/kg) was given intragastrically to AA for one time, and the rats were averagely divided into 0.5h, 1h, 2h, 4h and 6h, which received the concussion strike at the different times. b) Rats of AW were given the equivalent volume of distilled water, and they received the strike after 2h. c) The same spirituous liquor was given to CA twice every day at an interval of 6 hour for 4 weeks. The dosage was 8 ml/kg in the first 2 weeks, and 12 ml/kg in the last 2 weeks. Rats of CA received the concussion strike after 2h at the end of 4 weeks. d) Rats of CW were given the equivalent volume of distilled water, and the other treatments were same to CA. e) Blood and tissues of Control were obtained after bleeding execution directly.
The rats were observed half an hour after receiving the intragastric administration and strike. Then the blood of the abdominal aorta was collected after etherization. The craniotomy was operated immediately after bleeding execution, and the site and volume of TSAH were recorded with a digital camera. The brains, hearts, livers and other tissues were preserved by freezing and fixed in 10% formalin solution.
2.2 Experimental Methods
The general conditions of experimental rats were observed, which include the behavior, body weight, blood pressure, ECG and cerebral pathology. Gas chromatography was used in the assay of blood alcohol concentration (BAC). The automated coagulation analyzer was applied to detect the prothrombin time (PT), thrombin time (TT), activated partial thromboplastin time (APTT) and fibrinogen (FIB). The automatic blood gas analyzer was applied to determine pH, oxygen saturation (SaO2), Pressure of oxygen (PO2), bicarbonate (HCO3-), base excess (BE) and other seven indicators of blood gas analysis. ELISA was used in the assay of the tissue factor (TF), tissue factor pathway inhibitor (TFPI), tissue plasminogen activator (tPA), plasminogen activator inhibitor -1 (PAI-1), D dimer (D-D) and thromboxane B-2 (TXB-2) concentration in plasma. Immunohistochemistry methods and Western blotting were used for the qualitative and quantitative measurement of tPA and TXA2R expression in brain and heart.
The morbility and mortality of TSAH were calculated, and the volumes of TSAH were graded according to Fisher methods. All experimental data were treated with SPSS 17.0 and Excel 2007 for statistical analysis.
3 Results
3.1 Changes of performances, body weight and blood pressure
Excitability of acute alcoholism rats increased within a short time after drinking, and the signs of acute alcoholic intoxication were showed gradually after 0.5h, such as lethargy, loss of righting reflex, etc. The consciousness of rats came back after 4-6h. Chronic alcoholism rats had the same signs of the acute alcoholic intoxication after drinking every time. The body weights decreased after 1st week, and to 4th week the body weight decreased about 60g or more (p<0.01). The arterial blood pressures increased from 16.60±0.76 kPa to 17.48±0.90 kPa (p<0.05) from 1st to 3rd week. After concussion strike, the rats showed the typical signs of the concussion, and the rats (AA/AW/CW/part of CA groups) recovered gradually in 3-5sec. However, 55% of the chronic alcoholism rats had absent corneal reflex, raising head, limbs twitch, lips cyanosis, and the breathing from deep to slow, and then died in 2-4 min rapidly.
3.2 Changes of ECG
After concussion strike, all experimental groups had transient baseline drift of ECG. The heart rates sped up from the normal 400 to 500-600 bpm, and ECG came back after about 5-6 sec. While the heart rates in chronic alcoholism rats (death) dropped drastically to 100-200 bpm with cardiac arrhythmia after 30-60 sec, and then they slowed down gradually until the disappearance. The process took about 2-4min.
3.3 Cerebral pathology and morbility & mortality of TSAH
Acute alcoholism rat brains after strike showed mild congestion, swelling and a small amount of subarachnoid hemorrhage, and TSAH confined to the ventral surface of the brain stem or cerebellar surface. Neurons and glial cells also showed mild degeneration. Chronic alcoholism rat brains after strike showed moderate to severe congestion, swelling, and most amount of subarachnoid hemorrhage, and TSAH more widely distributed in the whole brain. Neurons and glial cells of the cerebrum showed degeneration and satellite phenomena. Purkinje cell of cerebellum arranged asymmetrically, and part of them dissolved away. The "dark neurons" were observed in the brainstem, and they showed dark red, cell body shrinkage, condensation of cytoplasm and nucleus, homogenization, unclear or disappear nucleus, and the irregular distortions of axon, etc. In addition, neither injury nor bleeding was found in brain tissue of the acute and chronic groups.
The morbility of TSAH in the acute and chronic alcoholism groups were 26% and 85% (p<0.01). The mortality of the two groups were 0 and 55% (p<0.01), and TSAH were found in all of the death rats.
3.4 Changes of blood alcohol concentration
BAC of rats raised to the maximum (188.16±14.71mg/dL) 2h after intragastric administration, and BAC were always above the legal limit of intoxication (80mg/dL) from 0.5h to 4h, and reached 60.08±13.80 mg/dL after 6h.
3.5 Correlation analysis of blood coagulation, BAC and TSAH
Compared with Control and AW groups, the PT, TT, APTT of the acute alcoholism group increased in different degrees at each time point, and that of AA-2h are the most significant, 28.50±2.08, 35.49±1.15, 24.56±1.09 sec (p<0.01). Content of FIB increased firstly (0.5h, p<0.05) and then decreased (1h, p<0.05), and it recovered to normal slowly. These of the chronic alcoholism group also extended and increased, and it was significant difference (p<0.05) with the Control/CW groups, but there was no significant difference with AA-2h group (p>0.05). In addition, there was a strong correlation between BAC and PT, TT, APTT (r=0.789, 0.818, 0.752), and they showed the positive correlation and linear distribution. There was no obvious correlation between BAC and FIB. Morbility of TSAH and BAC in different phases of acute alcoholism group showed the highly positive correlation (r=0.974), and the coefficient of determination R2 was 0.949.
3.6 Changes of blood gas analysis
Arterial pH, SaO2, PO2, HCO3- and BE in the acute alcoholism rats all reduced to different extent, and they reached the lowest points on 2h (7.26±0.05, 97.77±0.36, 12.70±3.67, 14.17±0.95, -12.57±2.90) (p<0.05). And then they recovered slowly, and some indicators had not returned to normal range until 6h. These of the chronic alcoholism group also decreased in various degrees, and the data compared with Control/CW group were significantly different (p<0.05). However, there was no significant difference between the acute alcoholism group and the chronic one (p>0.05).
3.7 Changes of TF/TFPI, tPA/PAI-1, D-D, TXB-2 in plasma
TF of the acute alcoholism group were reduced to varying degrees, and they reached the lowest point of 385.32±104.13 pg/ml (p<0.01) at 2h. However, TFPI increased to varying degrees, and they reached the peak of 1611.65±211.59 pg/ml (p<0.01) at 2h. TF/TFPI decreased firstly and then rose, and it was the minimum of 0.2391 at 2h. TF and TFPI of the chronic alcoholism group were significantly different compared with Control, CW and AA-2h groups (p<0.05).
tPA and PAI-1 of the acute alcoholism group all increased to varying degrees, and they reached the highest point of 6422.18±119.76 pg/ml (p<0.01) and 21246.67±1346.07 pg/ml (p<0.01) at 2h. tPA/PAI-1 rose firstly and then decreased, and it was the maximum of 0.3023 at 2h. tPA and PAI-1 of the chronic alcoholism group were significantly different compared with Control, CW and AA-2h groups (p<0.05).
D-D of the acute alcoholism group all increased to varying degrees, which had significant differences with that of Control/AW groups from 0.5h to 6h (p<0.05), and reached the maximum of 2207.40±322.12 pg/ml at 2h (p<0.01). D-D of the chronic alcoholism group were significantly different from that of Control, CW and AA-2h groups (p<0.05).
TXB-2 of the acute alcoholism group increased slightly, which had significant difference compared with that of Control/AW at 1h (p<0.05). TXB-2 of the chronic alcoholism group increased significantly, which had significant differences with that of Control, CW and AA-2h groups (p<0.01).
3.8 Expression of tPA/TXA2R in brain and heart
Interest protein tPA of the frontal lobe, cerebellum, ventral and dorsal of brainstem showed the clear bands on 67kDa of Western blot membranes. Bands of Control/AW/CW groups were narrow, thin and pale, but those of acute and chronic alcoholism groups were thick and dark. Optical density analysis of IHC showed that expression of tPA in the acute alcoholism group increased significantly compared with Control/AW group (p<0.01), whose highest value occurred at 2h. tPA expression of chronic alcoholism group also increased, which had highly significant differences with Control/CW groups (p<0.01), and other parts of brain except for the frontal lobe were statistical significances compared with AA-2h group (p<0.05).
Interest protein TXA2R of brainstem and heart showed clear bands of 58kDa on the Western blotting membranes. Bands of Control/AW/CW/AA groups in brainstem and heart were narrow, thin and pale, but those of CA group were thick and dark in brainstem with IOD value of 6188.04±391.11. They had highly significant differences with Control/CW groups and the heart in the same group (p<0.01).
4 Conclusions
(1) Signifcant dose-response relationships between BAC, coagulation and TSAH showed that, as BAC increase, the clotting times extend, and the morbility and mortality of TSAH also increase; The longer alcohol abuse, the more serious destruction of blood coagulation, and the higher morbility and mortality for TSAH.
(2) Acute and chronic alcoholism could reduce the clotting start function (TF/TFPI) and increase the fibrinolytic activity (tPA/PAI-1). The normal dynamic balances of them are damaged, and blood coagulation decreases. The more obvious changes are in chronic alcoholism, which is considered as one of the important mechanisms for the high morbility of TSAH.
(3) Acute and chronic alcoholism could lead to the sustained hypoxia in vivo, which could promote the expression of tPA and increase the fibrinolytic activity; it may be one of the key mechanisms that alcohol inhibits the coagulation function.
(4) It is the first time to explore the death mechanisms of TSAH after drinking in terms of the brain-heart connection. It is believed that chronic alcoholism could cause increasing contents of TXA-2 in blood and expression of TXA2R in brain. After TSAH, they combine quickly in brain tissues, and excite vagal activity and inhibit cardiac function, which may be one of the important mechanisms for the chronic alcoholism death from TSAH.
引文
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[4] Filter ER, Fernandes JR. Fatal traumatic subarachnoid hemorrhage due to assault-related tear of the basilar artery [J]. J Forensic Leg Med. 2009; 16(7):414-416.
[5] Gray JT, Puetz SM, Jackson SL, et al. Traumatic subarachnoid hemorrhage: a 10-year case study and review [J]. Forensic Sci Int. 1999; 105(1):13-23.
[6] Deck JHN, Jaghada V. Fatal subarachnoid haemorrhage due to traumatic rupture of the vertebral artery [J]. Arch Pathol Lab Med. 1986; 110(6):489-493.
[7] Simonsen J. Traumatic subarachnoid hemorrhage in alcohol intoxication [J]. J Forensic Sci. 1963; 8(1):97-116.
[8] Simonsen J. Fatal subarachnoid hemorrhage in relation to minor head injuries [J]. J Forensic Med. 1967; 14(4):146-155.
[9] Simonsen J. Fatal subarachnoid hemorrhages in relation to minor injuries in Denmark from 1967 to 1981[J]. Forensic Sci Int. 1984; 24(1):57-62.
[10] Harland WA, Pitts JF, Watson AA. Subarachnoid haemorrhage due to uper cervical trauma [J]. J Clin Pathol. 1983; 36(12):1335-1341.
[11] Plant JR. Laceration of vertebral artery: A historic boxing death [J]. Am J Forensic Med Pathol. 1993; 14(1):61-67.
[12] Barbour RL, Gebrewold A, Altura BM. Optical spectroscopy and cerebral vascular effects of alcohol in the intact brain: effects on tissue deoxyhemoglobin, blood content, and reduced cytochrome oxidase [J]. Alcohol Clin Exp Res. 1993; 17(6):1319-1324.
[13] Hillbom M, Juvela S, Karttunen V. Mechanisms of alcohol-related strokes [J]. NovartisFound Symp. 1998; 216:193-204.
[14] Bruno V. E, Oscar SM. Cardiac arrhythmias and sudden death in subarachnoid hemorrhage [J]. Stroke. 1975; 6(4):382-386.
[15]任智英译.蛛网膜下腔出血猝死原因分析[J].国外医学脑血管疾病分册. 1994;2(2):118-119.见:北原孝雄[日].脑神经外科[M]. 1993; 21(9):781-786.
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[17] Martin AS. The brain–heart connection [J]. Circulation. 2007; 116(1):77-84.
[18]于晓军,吴家文.酗酒后外伤性蛛网膜下腔出血及其死因分析[J].法律与医学杂志. 1995; 2(1):27-29.
[19]于晓军,吴家文,吴梅筠.外伤性蛛网膜下腔出血的研究现状[J].法医学杂志. 1998; 14(1):46-49.
[20]于晓军,樊瑜波,吴家文等.大鼠外伤性蛛网膜下腔出血模型的建立及其与酒精关系的研究[A].柳兆荣.生物力学新进展[M].第1版.成都:成都科技大学出版社, 1996, pp188-194.
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[1]徐广涛,于晓军,吕俊耀.饮酒促发外伤性蛛网膜下腔出血6例及死因分析[J].汕头大学医学院学报. 2006; 19 (3):174-175.
[2] Filter ER, Fernandes JR. Fatal traumatic subarachnoid hemorrhage due to assault-related tear of the basilar artery [J]. J Forensic Leg Med. 2009; 16(7):414-416.
[3] Gray JT, Puetz SM, Jackson SL, et al. Traumatic subarachnoid hemorrhage: a 10-year case study and review [J]. Forensic Sci Int. 1999; 105(1):13-23.
[4] Deck JHN, Jaghada V. Fatal subarachnoid haemorrhage due to traumatic rupture of the vertebral artery [J]. Arch Pathol Lab Med. 1986; 110(6):489-493.
[5] Bruno V. E, Oscar SM. Cardiac arrhythmias and sudden death in subarachnoid hemo- rrhage [J]. Stroke. 1975; 6(4):382-386.
[6]任智英译.蛛网膜下腔出血猝死原因分析[J].国外医学脑血管疾病分册. 1994;2(2):118-119.见:北原孝雄[日].脑神经外科. 1993; 21(9):781-786.
[7]裴叶.蛛网膜下腔出血导致循环呼吸停止的探讨[J].中国急救医学. 1996; 16(3):34-37.
[8] Martin AS. The brain–heart connection [J]. Circulation. 2007; 116(1):77-84.
[9]于晓军,吴家文.酗酒后外伤性蛛网膜下腔出血及其死因分析[J].法律与医学杂志. 1995; 2(1):27-29.
[10]于晓军,吴家文,吴梅筠.外伤性蛛网膜下腔出血的研究现状[J].法医学杂志. 1998; 14(1):46-49.
[11]于晓军,樊瑜波,吴家文等.大鼠外伤性蛛网膜下腔出血模型的建立及其与酒精关系的研究[A].柳兆荣.生物力学新进展[M].第1版.成都:成都科技大学出版社, 1996, pp188-194.
[12] Wang HP, Yu XJ, Xu GH, et al. Alcoholism and traumatic subara- chnoid hemorrhage: an experimental study on vascular morphology and biomechanics [J]. J Trauma. 2010. (doi: 10.1097/TA.0b013e3181cda3b9)
[13] Wacker MJ, Best SR, Kosloski LM, et al. Thromboxane A2-induced arrhythmias in the anesthetized rabbit [J]. Am J Physiol Heart Circ Physiol. 2006; 290(4):1353-1361.
[14] Provencio JJ. Subarachnoid hemorrhage: A model for heart-brain interactions [J]. Cleve Clin J Med. 2007; 74(1):S86-90.
[15] Yalcin M, Cavun S, Yilmaz MS, et al. Activation of the central cholinergic system mediates the reversal of hypotension by centrally administrated U-46619, a thromboxane A2 analog, in hemorrhaged rats [J]. Brain Res.2006; 1118(1):43-51.
[16] Raneem OS, Michael L. Effects of alcohol on hemostasis [J]. 2005; Am J Clin Pathol 2005; 123(Suppl 1):S1-10.
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[3]徐广涛,于晓军,吕俊耀.饮酒促发外伤性蛛网膜下腔出血6例及死因分析[J].汕头大学医学院学报. 2006; 19 (3):174-175.
[4] Filter ER, Fernandes JR. Fatal traumatic subarachnoid hemorrhage due to assault-related tear of the basilar artery [J]. J Forensic Leg Med. 2009; 16(7):414-416.
[5] Gray JT, Puetz SM, Jackson SL, et al. Traumatic subarachnoid hemorrhage: a 10-year case study and review [J]. Forensic Sci Int. 1999; 105(1):13-23.
[6] Deck JHN, Jaghada V. Fatal subarachnoid haemorrhage due to traumatic rupture of the vertebral artery [J]. Arch Pathol Lab Med. 1986; 110(6):489-493.
[7] Simonsen J. Traumatic subarachnoid hemorrhage in alcohol intoxication [J]. J Forensic Sci. 1963; 8(1):97-116.
[8] Simonsen J. Fatal subarachnoid hemorrhage in relation to minor head injuries [J]. J Forensic Med. 1967; 14(4):146-155.
[9] Simonsen J. Fatal subarachnoid hemorrhages in relation to minor injuries in Denmark from 1967 to 1981[J]. Forensic Sci Int. 1984; 24(1):57-62.
[10] Harland WA, Pitts JF, Watson AA. Subarachnoid haemorrhage due to uper cervical trauma [J]. J Clin Pathol. 1983; 36(12):1335-1341.
[11] Plant JR. Laceration of vertebral artery: A historic boxing death [J]. Am J Forensic Med Pathol. 1993; 14(1):61-67.
[12] Barbour RL, Gebrewold A, Altura BM. Optical spectroscopy and cerebral vascular effects of alcohol in the intact brain: effects on tissue deoxyhemoglobin, blood content, and reduced cytochrome oxidase [J]. Alcohol Clin Exp Res. 1993; 17(6):1319-1324.
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[14] Bruno V. E, Oscar SM. Cardiac arrhythmias and sudden death in subarachnoid hemorrhage [J]. Stroke. 1975; 6(4):382-386.
[15]任智英译.蛛网膜下腔出血猝死原因分析[J].国外医学脑血管疾病分册. 1994;2(2):118-119.见:北原孝雄[日].脑神经外科[M]. 1993; 21(9):781-786.
[16]裴叶.蛛网膜下腔出血导致循环呼吸停止的探讨[J].中国急救医学. 1996; 16(3):34-37.
[17] Martin AS. The brain–heart connection [J]. Circulation. 2007; 116(1):77-84.
[18]于晓军,吴家文.酗酒后外伤性蛛网膜下腔出血及其死因分析[J].法律与医学杂志. 1995; 2(1):27-29.
[19]于晓军,吴家文,吴梅筠.外伤性蛛网膜下腔出血的研究现状[J].法医学杂志. 1998; 14(1):46-49.
[20]于晓军,樊瑜波,吴家文等.大鼠外伤性蛛网膜下腔出血模型的建立及其与酒精关系的研究[A].柳兆荣.生物力学新进展[M].第1版.成都:成都科技大学出版社, 1996, pp188-194.
[21] Wang HP, Yu XJ, Xu GH, et al. Alcoholism and traumatic subarachnoid hemorrhage: an experimental study on vascular morphology and biomechanics [J]. J Trauma. 2010. (doi: 10.1097/TA.0b013e3181cda3b9)
[22]陆一,高贵山,于晓军等.食用酒对脑组织Ngb、Hif-1α和Na+,K+-ATPase活力影响及其死亡机制[J].毒理学杂志. 2009; 23(2):96-99.
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