| Background: Study of SCD is one of the most important fields in forensic medicine,and the postmortem diagnosis of EMI is a focus as well as difficult problem in forensic pathology .It is unlikely to find special changes with either unaided eye or light microscope for myocardial infarction less than 6 hours. Without the use of special methods, it is impossible to get the histological diagnosis of EMI as a cause of sudden death. A series of pathophysiologic changes have been taken place in the ischemic myocardial before the appearance of characteristic morphology on the routine histology stains. Although early ischemic myocardial injury can be detected biochemically, histochemically,enzyme histochemically, fluorescence histochemically , electronmicroscopicly. These methods are limited in forensic practice,either because they are too complexed to be operated, or because they are too unspecific, instabile ,expensive , or easy to be influenced by autolysis and putrefaction of tissue . However immunohistochemical technology, as a swift, convenient and practical method can show the qualitative and quantitative changes of proteins in the ischemic myocardial cell, which make it possible to diagnose EMI postmortem. Many studies have verified that there are two ways of death of cardiac muscle cell,they are necrosis and apoptosis. Immunohistochemical stain of cTnI and caspase-3 is reliable and sensitive marker for detect the necrosis and apoptosis of cardiac muscle cell. They can be applied to detect the necrosis and apoptosis of ischemic myocardial cell. Whether an immunohistochemical marker can be used in forensic practice or not depends on not only its specificity and sensitivity, but also the influence of autolysis and putrefaction of tissue on antigen . At present, what needs studying further is postmortem stability of immunohistochemical markers used in forensic practice and the regularity of its expression alteration in different postmortem intervals.。Objective: The present study used an animal model of early myocardial ischemia by ligating the left anterior descending branch of coronary artery of rabbits. In order to explore its postmortem stability and the regularity of its expression alteration, we detected the expression of immunohistochemical markers cTnI and caspase-3 in myocardium at different postmortem interval . The applicability Value of the two immunohistochemical markers was evaluated for diagnosis of SCD in forensic practice.Methods:15 healthy adult rabbits, optional sex, weighing 2000g～ 2500g were selected and divided into experimental group (n=10) and control group at random(n=5).In the experimental group, an animal model of early myocardial ischemia was established by ligating the left anterior descending branch of coronary artery at the below 2.0mm of auricula sinistra cordis of rabbits, then the rabbits was sacrificed at 3h after the ligation and myocardium was taken from the ischemic region. In the control group, the rabbits were sacrificed directly without ligation, and myocardium was taken from the left ventrical. The myocardium was fixed after1h,2h,4h,8h,16h,1d,2d,3d,4d,5d,6d,7d,10d,14d and 21d of postmortem stored at 4℃,made as serial paraffin sections, then treated with HE staining and cTnI,caspase-3 immunohistochemical staining respectively. Observe the sections stained with HE of the two groups by light microscop, as well as the sections stained with immunohistochemical methods according to the criterier of IHC stains, then come out an overall estimation. At the same time, image analysis technique was applied to detect and compare the area and intensity of positive reaction of cTnI and caspase-3 in each sample of the two groups stored at 4℃for different time intervals after death.Results1 HE staining : Myocardial cell of control groups stored at 4℃within 2d after death showed clear transverse striation, cellular structure and no abnormal changes . During 3-7 days, Cardiac muscle cell of control groups was slightly blur and cellular cloudy swelling after death, and after 7d,further blur take place. There were some pathological changes of ischemic myocardium such as cellular cloudy swelling, hydropic degeneration, blur or disapearanse of transverse striation, fortified eosnophilia of endochylema, nucleus contract with deep staining. Within 7d after death ,the ischemic myocardium showed hydropic degeneration, blur or disapearanse of transverse striation, fortified eosnophilia of endochylema, nucleus contract with deep staining. Stored at 4℃for 7-21d, the two groups experienced similar changes in morphous of tissue and in ischemic myocardial cell were similar to control group's, but the waviness of muscle fiber remains to be seen .2 IHC staining2.1 Expression of cTnI: Expression of cTnI showed strong positive with homogeneous distribution in normal myocardial cytoplasm, no conspicuous depletion, negative in interstitium. It showed irregularity punctiform, focus, patch depletion regions with ununiform distribution in ischemic myocardial cytoplasm, and the positive reaction was weakened in partial regions. Compared with the serial paraffin section stained with HE, the endochylema of cadiocytes showed fortified eosnophilia, nucleus contract with deep staining.The results of image analysis indicate that the intensity of positive reaction trends weaker as well as smaller in the area of positive reaction along with prolongation of postmortem interval. The intensity of positive reaction in the two groups stored at 4℃was differential apparently within 7d(P<0.01); within 6d the area of positive reaction in the two groups was not differential apparently(P>0.05); within 7d the area of positive reaction in the two groups was differential apparently(P<0.01)。After 10d,the distinction of the response intensity and areal between two groups was not obviously(P>0.05)。2.2 Expression of caspase-3: Expression of caspase-3 showed punctiform weakly positive or negative in normal myocardial cytoplasm. It showed focal,patchy strong positive in ischemic myocardial cytoplasm. The results of image analysis indicate that the intensity of positive reaction of caspase-3 trends weaker as well as smaller in the area of the positive reaction along with prolongation of postmortem interval. The changes of intensity of positive reaction in the two groups stored at 4℃was not differential apparently within 3d(P>0.05); and within 5d the area of positive reaction in the two groups was differential apparently(P<0.01)。After 6d,the distinction of the response intensity and areal between two groups was not obviously(P>0.05)。Conclusion: The cTnI and caspase-3 showed good stability after death. The expression of cTnI in normal and ischemic in cardiac muscle tissue stored at 4℃are obviously different within 7d. Within 5d, the expression of caspase-3 in normal and ischemic in cardiac muscle tissue stored at 4℃are obviously difference. It can be conclude that immunohistochemical detection of cTnI can be used in corpse stored at 4℃for 7d or less than 7d after death, and immunohistochemical detection of caspase-3 can be used in corpse stored at 4℃for 5d or less than 5d after death..
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