Associativity Study Between Actin Degradation Regularity And The Estimation Of Postmortem Interval

BackgroudActin is structural subunit of microfilaments and is the most abundant intracellular protein in a eukaryotic cell, which often constitutes as much as 50% of total cellular protein. Experiments had shown that actin in the liver tissue of rats could be detected at 8 days postmortem, but could not be examined after 10 days postmortem and within a certain postmortem interval ( <54h postmortem), the extent of anti-actin monoclonal antibody staining depletion in cardiac and skeletal muscle cells increases with the extension of postmortem interval. These observations indicate that the degradation pattern of the actin may be used as a parameter for PMI estimation. However, whether there are variability of actin postmortem degradation in different tissues? Whether the environmental factors( temperature and humidity), anthropic factors(modes of death) could affact the postmortem degradation of actin ? Whether there are correlationship between the morphologic changes of actin and the time since death? Whether the actin content in human tissues also decreases with the extension of the post-isolation time ? At present, we can't find reports in these filds. So, in this study, we try to find the answer for the above problems.Objective(1)To observe the postmortem degradation of actin in cardiac muscle, liver, spleen, lung, kidney, brain and skeletal muscle of rats,for the purpose of finding a new method of estimating the time since death.(2)To investigate the correlation between morphologic changes of actin in skeletal muscle of rats and the time since death.(3)To study the effects of ambient temperature on the actin postmortem degradation of rats.(4)To investigate the variability of actin postmortem degradation in skeletal muscle of rats at different ambient humidity.(5)The content changes of actin in the skeletal muscle of rats killed by different manners were investigated to elucidate evidence that can be used to determine the modes of death.(6)To study the relationship between the actin content in ex vivo skeletal muscle of rats and the time since isolation.(7)To observe the degradation of actin in ex vivo human pectoralis major within 168h after being excised. Methods(1) Twenty eight clear Sprague Dawley rats were killed by suffocating and put into an artificial climate incubator(set at 20℃)for 0, 24, 48, 72, 96, 120 and 168h, respectively. The actin contents in the above tissues were quantitated by western-blot assay and collected by Image Pro Plus 5.0 image analysis system, and the datas were then statistically analyzed with the SPSS 11.5 software.(2) The morphologic changes of actin filament in the skeletal muscle (adductor magnus of right hind leg) of twenty eight rats were observed by optical microscope, laser scanning confocal microscope (LSCM) and transmission electron microscope (TEM) at different postmortem intervals (0, 24, 48, 72, 96, 120 and 168h).(3) The differences of the content of actin in the adductor magnus of rats at different ambient temperature (10℃, 20℃and 30℃) and different postmortem intervals (0, 24, 48, 72, 96, 120 and 168h) were measured and analyzed with western-blot assay and image analysis technology.(4) The content changes of actin in the adductor magnus of rats at different ambient humidity (30%, 50% and 70%) were determined by using western-blot assay and image analysis technology, in the course of 168h after death.(5)Twelve SD rats were randomly allocated into 3 groups and killed by bleeding,suffocating,and electrocution,respectively.The contents of actin in the adductor magnus of rats killed by different manners at different postmortem intervals (0, 24, 48, 72, 96, 120 and 168h) were measured by western-blot.(6)The adductor magnus of four SD rats killed by suffocating were dissected and put into the artificial climate incubator set at 20℃. The actin content in the ex vitro specimens were quantitated with western blot assay and image analysis techniques at differernt time after being excised (0, 24, 48, 72, 96, 120 and 168h).(7) The actin content in the ex vitro human pectoralis major (n = 4) were quantitated with western-blot assay, in the course of 168h after being excised.Results(1)Actin content in all these tissues decreased gradually with the prolonged postmortem intervals(PMI). The degradation differences between the groups was statistically significant(P<0.05), with the degradation in the brain fastest,then the lung, the spleen,the liver, the kidney, the cardiac muscle and the skeletal muscle in order. There was a strong correlation between actin degradation and PMI, and the coefficient of determination (R2) exceeded 0.75 in all these tissues.(2)After death, the disappearance of the transverse striation and karyopycnosis, hypochromatosis of the skeletal muscle of rats can be observed by the optical microscope, and all that became more and more notable with the extension of PMI; the results of the TEM revealed that actin filament began to disintegrate with the lapse of PMI, and at last, the structure of sarcomere and actin filament disappeared; the results of LSCM showed that skeletal muscles revealed depletion of anti-actin antibody staining and the extent of staining depletion increased with extension of PMI, and nearly no actin positive product can be detected until 168h after death.(3)Within 168h postmortem, actin content in the adductor magnus of rats in different ambinet temperature groups all reduced gradually with the extension of the PMI, and significant differences can be observed between the different temperature groups at all the intervals (P<0.05). The interaction effect on the postmortem dagradation of actin between the ambinet temperature and PMI can be found via the statistical analysis. The multiple regression equation between actin content and ambient temperature, PMI were: Y=136689.8-465.5X1-1377.7X2,R2 =0.823(Y was the IOD value of the actin content, X1 was the PMI,which was the same as the below in the whole thesis; X2 was the ambient temperature).(4)The postmortem degradation rate of actin in the different humidity groups were differernt, and there were significant differences in actin content can be observed between the rats in different humidity groups at most of intervals. The multiple regression equation between actin content and ambient humidity, PMI and the R2 were: Y=136689.8-465.5X1-1377.7X2,R2 =0.823 (X2 was the ambient humidity).(5)There were significant differences in contents of actin in the adductor magnus between the hemorrhage shock group and suffocation group,electrocution group at most of the intervals (P<0.05). The multiple regression equation between actin content and modes of death, PMI were: Y=118112.8-438.6X1-4972.5X2,R2 =0.793 (X2 was the modes of death: 1- by bleeding,2-by suffocating,3-by electrocution).(6)Actin content in the ex vivo adductor magnus of rats reduced gradually with the extension of the PMI, and there was a proximate linear relationship between actin content and post-isolation time, and significant differences can be observed between ex vivo and in vitro groups(P<0.05). The multiple regression equation were:Y=82422.6-420.5X1+15884.3X2,R2 =0.865(X2 :the modes of preservation of the specimen: 1- in vitro, 2- ex vivo).(7)There were significant differences in actin content in human pectoralis major (P<0.05) after being excised, but no differences can be found in the reminers of the actin content in human pectoralis major and adductor magnus of rats at most of the intervals(F=1.22,P=0.334). The multiple regression equation between actin content and PMI and the R2 were: Y=108288.8-342.28X,R2=0.89(X was the time after isolation). Conclusion(1) Although with tissue disparity in degradation, the actin contents in cardiac muscle, liver, spleen, lung, kindey, brain and skeletal muscle of rats decreased gradually with prolonged PMI, which may potentially be a useful parameter for the late PMI estimation.(2)To observe the morphologic changes of the actin filament of skeletal muscle may be expected to be a useful method for the early PMI estimation.(3)The influence of temperature on the postmortem degradation of actin in skeletal muscle was evident, and the degradation rate tended to to be raised by higher temperature.(4)The postmortem degradation rate of actin in skeletal muscle were different between different humidity groups, and the rise of the ambient humidity tended to speed up the degradation rate.(5)The manners of death could influence the postmortem degradation rate of actin in the skeletal muscle of rats, the content changes of actin may provide a basis for determination of the manner of death.(6)There was a negative correlativity between actin content in the skeletal muscle of rats and the post-isolation time, and the postmortem degradation rate of actin was higher in vitro group than in ex vivo group.(7)The actin content in human pectoralis major decreased with the extension of the post-isolation time, which seems to be a useful biological indicator for the late PMI estimation.