Study On Relation Between The Expression Of S-100β& MBP And Traumatic Subarachnoid Haemorrhage In Brain Of The Acute & Chronic Alcoholism Rats

Background and objectiveTraumatic subarachnoid haemorrhage(TSAH) is an independent brain damage, and it differs from pathological subarachnoid haemorrhage and haemorrhage caused by cerebral contusion. TSAH combine with traumatic epidural hemorrhage and subdural hemorrhage compose the three cavity haemorrhage.It is reported TSAH is related with alcoholism by foreigners. There isn't systematic study in domestic and foreign reports about the mechanism of occurrence and death. To explore the mechanism of occurrence and death, can offer the evidence for analysis the cause of death and judicial sentencing for forensic.In the prophase, our group has been established acute and chronic alcoholism TSAH model in rats successfully.It indicate that the morbility of TSAH after alcohol intragastric administration and concussion strike: chronic group82.4%,acute group 28.6%, mortality rate: chronic group58.8﹪, acute group 0%,indicate the morbility and the mortality of chronic alcohol intragastric administration group is far more than acute alcohol intragastric administration group;We have confirm that the vessel wall toxic substances MMP-9,vessel wall morphology and biomechanical, the substance Ngb,Hif-1α,EPO,Na+,K+-ATPase related to oxygen and energy metabolism of brain, the substance tPA,Cyt-C,Caspase-9,Caspase-3,Bcl-2,Bax related to cell apoptosis participate in the mechanism of occurrence and death of TSAH.The research base on the former work,make further efforts to explorer the relationship between the high mortality of chronic alcoholism and glial cells.we select the improtant indicator that has a close connection with the structural functional of central nervous fiber S-100βand MBP of glial cells,to explore the expression of S-100βand MBP in alcoholism rat'brain and the correlation with mechanism of TSAH.Materials and Methods1 Animals model and groupsSixty-one male SD rats,each weighted 300±50g,breeding in several cages,devided into different groups randomly. 1.1 Actue alcoholism modelTwenty-eight rats were devided into four groups:alcohol i.g execute group, alcohol i.g strike group, water i.g execute group, water i.g strike group. With 7 rats in each group. Alcohol i.g group with white wine ( the hongxing winery of beijing er guo tou,52%v/v)i.g once 15ml/kg, water i.g with the same doze water.The strike group were given concussion strike after water or alcohol i.g.1.2 Alcoholism modelThirty-three rats were devided into four group: chronic alcohol i.g execute group, chronic alcohol i.g post-12h strike group, chronic alcohol i.g strike survival group, chronic alcohol i.g strike death group.With 7 rats in the former two groups and 19 rats in chronic alcohol i.g strike group.(10 survival and 9 death).All group rats alcohl i.g , twice a day with 8h interval, 8ml/kg for 2 weeks, 12ml/kg for the last 2 weeks, The strike group were given concussion strike after the last alcohol i.g. post-2h(or post-12h).2 MethodsAll strike groups were given concussion strike and lay up for 4h, and then giving etherization, thoracotomy and sucked blood about 5ml in the left ventricular. If the rats died after strike,they were given thoracotomy and sucked blood in the left ventricular about 5ml immediately, left ventricular-artery intubate, dripping 4% neutral paraformaldehyd to fix brain tissue,then were operated decapitation to get the brain tissue and placed in 4% neutral paraformaldehyde fixed, sectioning, paraffin embeddig, cut sections and HE staining. Fast green myelin staining was used to oberserve histomorphology of brain tissue and myelin tissue,immunohistochemistry of S-100βand MBP, The pathological changes was quantified by Image-Pro Plus 6.0 image analytical system, blood alcohol concentration was measureed.All datas were analyzed by SPSS 15.0, T-test and Independent-samples One-way ANONA .Results1 Alcoholism rats'behavior and blood alcohol concentrationThe acute alcohol i.g group rats had more movement and increased excitability post-0.5h alcohol i.g. But after that, the rats showed typical alcoholic intoxication symptom such as decreased movement, clumsiness, ataxia, lethargy, slow breath and delayed reaction. The post-2h BAC of alcohol i.g group rats was 180.16±16.58 mg/dL, the post-2h BP was 14.13±0.34Kpa which was lower than pro-0.5h BP of 17.02±0.88 Kpa (P<0.01).The movement and BP of water i.g group rats had not change (P>0.05).For chronic alcoholism group rats, with the increase of time, the rats showed typical chronic alcoholism symptom, such as weight decline, dystrophia, the less eating, side of hemiplegia. Their weight decreased from 307.2±28.9g (pro-i.g) to 251.8±19.6g (after 4w alcoholism) (P<0.01); Their BP increased from 16.60±0.76Kpa (pro-i.g) to 18.16±0.82 Kpa (after 4w alcoholism) (P<0.01). The rats showed alcoholic intoxication symptom such as side-lying, lethargy and can not turn post-0.5h alcohol i.g. The post-2h BAC of chronic alcohol i.g group rats was 208.50±32.35mg/dL which was higher than acute group (P<0.01).2 The morbility and mortality of TSAH, pathology observtionThe morbility of the acute alcohol i.g strike group was 28.6%,with no rats mortality; There is no morbility and mortality happen in the water i.g strike group; The morbility and mortality of the chronic alcoholism strike group is 84.2% and 52.6%. The morbility and mortality of the chronic alcoholism post-12h strike group is 71.4% and 28.6%,the mortality is lower than chronic alcoholism strike group.Acute water i.g group's brains had limited congestion; water i.g strike group's brains were smooth and fixed-like pale,alcohol i.g execute group's brains suffuse congested in the frontal and cerebellum, acute alochol i.g strike group's brain surface and brain stem had thin SAH , chronic alochol i.g strike group's brains had sufuse and thick SAH,especially in the brain stem front side.For the acute alcohol i.g.group, under microscope, the brain nerve cell swelt mildly, the space expanded slightly aroud the cell; brain stem and cerebellum SAH occurred in rats of the acute strike group. For the chronic alcohol i.g.group, under microscope,the brain neurons arrayed disorderly, decreased in number, had condensed nuclei and more neuronophagia phenomenon; purkinje cells arranged in loose, their shape was irregular triangular-like, synapse was not clear, some nuclei condensed and dissolved away partly. Medulla oblongata of brain stem was found to have more dark cells, which had homogenized cytoplasm, condensed dark stained nucleus neurons named dark cells,brain stem,frontal and cerebellum with thick SAH. of chronic alcohol i.g strike group.Fast green myelin stain(stained myelin and nucleus show dark green and red),water i.g execute group's brain stem,corpus callosum and cerebellum nerve fiber liner array, dense arrangement;acute alcohol i.g strike group myelin array loosen slightly, space expanded, stain light slightly;chronic alcohol i.g strike group myelin loosen, space expanded obvious and stain inhomogeneous,it is obviously that the nerve fiber unregular thickening, twist and fracture,space around expanded obvious.3 The expression of S-100βand MBP3.1 The number of positive cells of S-100βThe number of positive cells in acute strike group was more than that in execute group(P<0.05).There is no different in strike group of the number of positive cells, execute group as well(P>0.05).The number of positive cells in chronic alcohol i.g post-12h strike survival group and chronic alcohol i.g strike survival group is more than that in execute group and death group(P<0.05); There is no different in the two former group,the two latter group as well (P>0.05).The number of positive cells in chronic alcohol i.g post-12h strike survival group and chronic alcohol i.g strike survival group is more than that in acute group.(P<0.05); The number of positive cells in chronic alcohol i.g execute group and death group is more than that in acute execute group(P<0.05),have no different with strike group (P>0.05).3.2 The IOD of S-100βThe IOD in acute strike group was higher than that in execute group(P<0.05). There is no different in strike group of IOD,the execute group as well(P>0.05).The IOD of chronic alcohol i.g execute group is the higher than that in other chronic alcohol i.g group(P<0.05); chronic alcohol i.g strike death group is the lower than other chronic alcohol i.g group(P<0.05). chronic alcohol i.g post-12h strike survival group and chronic alcohol i.g strike survival group have no different (P>0.05).Except chronic alcohol i.g strike death group, The IOD of other chronic alcohol i.g group is higher than that in acut group; The IOD of chronic alcohol i.g strike death group have no different with that in acute strike group(P>0.05),but higher than that in execute group(P<0.05).3.3 The IOD of MBPThe brain stem and corpus callosum IOD in acute strike group is lower than that in execute group(P<0.05). There is no different in strike group of IOD, execute group as well(P>0.05). The cerebellum IOD in all acute group have no different (P>0.05).The IOD of chronic alcohol i.g execute group is the highest, higher than that in other chronic strike group(P<0.05).The corpus callosum and cerebellum IOD of chronic strike group have no different (P>0.05),brain stem IOD of chronic strike death group higher than that in chronic strike survival group(P<0.05),there is no different in survival group (P>0.05).Expect chronic alcohol i.g execute group's IOD have no different with acute strike group (P>0.05), all chronic group IOD is higher than that in acute group(P<0.05).Conclusions1. Alcoholic intoxication and chronic alcoholism can have synergistic role with brain concussion strike in causing TSAH, chronic alcohol abuse is an important factor of the mortality in TSAH.2. The expression of S-100βwas higher and MBP was lower in glial cells of chronic alcoholism brain which might be substance basis of TSAH death mechanism and might take part in the occurrence mechanism of alcoholic cerebral atrophy and alcoholic encephalopathy in chronic abuse.