Study On The Occurrence And Death Mechanisms Of Traumatic Subarachnoid Haemorrhage And The Coagulation Changes In The Acute And Chronic Alcoholism Rats

1 Background and ObjectiveAlcohol 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 Methods2.1 Groups of animals and Establishment of modelNinety 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 MethodsThe 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 Results3.1 Changes of performances, body weight and blood pressureExcitability 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 ECGAfter 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 TSAHAcute 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 concentrationBAC 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 TSAHCompared 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 analysisArterial 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 plasmaTF 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 heartInterest 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.