| Objectives: We established an animal experimental model of excessive drinking of alcohol in early stage by giving different concentration of alcohol to healthy mice. The morphological changes were observed by light and electron microscopy. The changes of enzyme activity of cytochrom C oxidase (Cox) in myocardial cell were observed by enzyme cytochemisty. The effect of alcohol on apoptosis and the expression of apoptosis-associated gene-bc1-2 and bax were analysed by immunohistochemistry method. And the mechanisms of heart toxication caused by alcohol were investigated.Methods: The sixty healthy male mice were randomly divided into four groups: the alcohol-treated group I, II, III, and the control group. The alcohol-treated groups were given intraperitoneal injection by 10%, 20%, 30%(v/v) ethanol respectively once a day for 30 days. The control group was giving saline solution injection for the same duration as alcohol-treated group. Then the hearts were removed and processed by following procedures. Some specimens were made into paraffin section, stained by hematoxylin-eosin(HE) staining method and morphological changes were observed by light microscope. Myocardial apoptosis was analysed quantitatively by the nuclear fast red-crystal violet staining method. The expression of apoptosis-associated genes--bcl-2 and bax were showed by immunohistochemical method and image analysis technology was employed to study the quantity changes. Some specimens were prepared into transmission electron microscope (TEM) sample and the effect of alcohol on myocardial ultrastructural alteration was observed by TEM. For enzyme cytochemistry, other specimens were sliced into 15μm and 40μm frozen sections by cryostat microtome. The 15μm sections were stained by Cox and the enzyme activity was observed by light microscope. The 40μm sections were incubated in Cox incubation solution, then prepared to TEM samples and the enzyme activity changes of Cox on mitochondria were observed by TEM.Results: (1) Morphological observation shows: There were no remarkable abnormities of myocardial cell observed by HE staining in all experimental groups afteralcohol treatment. But under TEM, we found that the myofilaments in alcohol-treated groups arranged irregularly, some vacuole and dissolved myoneme could be observed. The cristae of mitochondria ruptured, inosculated and even disappeared and some mitochondria became bulgy and nonuniformed size. The nuclears were irregular and the thickness of karyotheca was nonuniformity. The chromatic agglutination was keeping to the side and the plaque form under the karyotheca. Along with increasing of the alcohol concentration, the alterations of ultrastructure were more and more obvious. (2) Nuclear fast red-crystal violet staining and pycnosis cell nuclears counting shows: Some nuclears of the experimental groups were stained purple, which chromatin agglutinated and some were pycnosis. Counting under the high power microscope, we found that the number of abnormal nuclears increased in alcohol-treated group than control group (P < 0.01). And that along with increasing of the alcohol concentration, the number of pycnosis cell were increasing gradually. (3) Immunohistochemisty staining and image analysis shows: The expression of bc1-2 in all experimental groups was weakened in alcohol-treated groups compared with control group (P < 0.01) and the granules were stained weak and maldistribution. In contrast, the expression of bax in all experimental groups was reinforced (P<0.01) and the granules engrained and gross. (4) The results of Cox staining: The enzyme activity in all experimental groups was decreased compared with the control group (P < 0.01). And that the changes were positive correlated to alcohol concentration. By the TEM, we found that the reaction products of Cox located on the inner membrane and cristae of mitochondria, that the electron density in all experimental groups reduced, and that most of mitochondria were devoid of the reaction products.Conclusions: (1) The structural changes of card...
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