| Acute myocardial ischaemia is one of the commonest reasons for sudden death due to heart disease. It is often seen in forensic medicine. Often the morphological changes characterizing myocardial ischaemia cannot be seen by examination with unaided eyes or by observation with microscopy as most cases of acute myocardial ischaemia result in death within several hours, even within several minutes. Thus, how to identify early stage myocardial ischaemia by means of morphological change presents major challenge for forensic pathological research. Cardiac troponin T(cTnT) located on the thin myofilament, is one of the subunits of Troponin(Tn). At present, many studies have demonstrated that when myocardial infarction occurs, the concentration of cTnT in patients' blood rises significantly and this can reflect the severity of myocardial infraction and its prognosis. The immunohistochemical study on cTnT in early myocardial ischaemia has not yet been reported. Desmin, the structural5protein in the intermediates filament of myocardium, is a component of cytoskeleton protein. Often, desmin may break away from its position with myocardial cell injury due to early myocardial ischaemia. This has been reported internationally as well as national , but with some difference of opinion.. Connexin 43(CX43) is the junction protein in the myocardial cell gap-junction, found mainly in the myocardial intercalated disc. CX43 is associated with information transmission between myocardial cells. At present, it has been proven by observation and study using the immunohistofluorescence method and confocal laser scanning microscopy that abnormal changes occur in heart diseases related to myocardial ischaemia. But immunohistochemical study on CX43 in early myocardial ischaemia.has not yet been reported.The aim of this research is to use immunohistochemical methods to detect cTnT, Desmin and CX43 in early rat myocardial ischaemia of an experimental model, and to record their distribution. By doing so, we hope to provide an important index and theoretical foundation for thepathological diagnosis of early myocardial ischaemia.Materials & Methods1. Group selection and development of rat model.Sixty SD rats were chosen(provided by Zhejiang Scientific Institute of Medicine), both male and female, aged 4+ months, weighing 300?5g, divided at random into 6 groups: control group, the 15 min hemostasis group, the 30 min hemostasis group, the Ih hemostasis group, the 2h min hemostasis group, the 3h hemostasis group. Each group included 10 rats, the hearts of rats in the control group were excised after anaesthesia . The heartsof the rat in the rest of the groups were taken out at different time respectively after ligation of the left coronary artery according to a previously documented technique.2. Specimen Fixation and sectionsThe heart was immediately fixed in 10% formaldehyde solution and then removed from solution after 24 hours. Then a 2mm thick slice of tissue was excised from the mid position of the heart. After routine paraffin embedment, continuous Sum section were made by the Leitz 1512 microtome.3. H&E and Immunohistochemical stainingRoutine staining with H&E stain was performed. The primary antibodies included anti-cTnT antibody , anti-Desmin antibody and anti-CX43 antibody. All procedures were carried out according to Evision kit instructions, then DAB display and slide were examined by routine microscopy.4. Image AnalysisThe immunohistochemical sections of each group were examined using the Olympus-BH2optical microscope. Five visual fields were chosen at random of the induced hemostasis sites of the left ventricular wall and ventricular septum. These picture were loaded into the computer through a video camera, and images were analyzed with HPIAS-1000(High Resolution Pathological Image Analysis System). The size of the unstained area and the average gray density was measured.5. Statistical AnalysisThe data were expressed with meanD(standard deviation) and7analyzed... |
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