The Establishment And Study Of Thermotolerance Model In The Liver Of Zebrafish (Brachydanio Rerio)

The objective of this study was to investigate the association among stress damage, Hsp70and apoptosis expression in the liver of zebrafish in various heat conditioning. Following experimental methods were taken into account:HE staining, transmission electron microscope,TUNEL staining, immunohistochemistry, ELISA.760zebrafish were divided into5groups randomly:group1(control, water temperature was28℃), group2(pre-conditioning exposure to30℃, thermo1), group3(pre-conditioning exposure to32℃, thermo2), group4(pre-conditioning exposure to32℃,thermo3) and group5(CTMax group), each group was consisted of150±2zebrafish. Group2-4were processed as following:zebrafish were stressed at3different mild temperature for4h and put into CTMax temperature (41.1℃) after recovery from acclimation temperature for2h. Group5was put into CTMax directly withour pre-conditioning. When the heat stress period was over, zebrafish were serially slaughtered quickly for sampling. Specimens for histopathological and immunohistochemical analysis were fixed in freshly prepared4%paraformaldehyde and2.5%glutaraldehyde in phosphate-buffered saline (pH7.4), and for quantifying the Hsp70protein and apoptosis levels were frozen in liquid nitrogen and stored at-70℃.The death curve of zebrafish showed that thermo groups had more steady mortality curve, which implied zebrafish gained thermotolerance after mild pre-heated stress that could resist the damage of second abrubt stress.After heat stress, the parenchyma cells of livers showed histopathological changes characterized by granular degeneration. Hsp70expressed mainly in the cytoplasm and nuclei of parenchyma cells. However, there were no obvious histological lesions in the control group. Homogeneous immunolabelling of Hsp70was observed in the hepatocytes and bile duct of liver; localization of apoptosis was observed in the hepatocytes, erythrocytes and bile duct of livers.We can learn from the ELISA results that Hsp70level increased in4thermo groups after heat conditioning and group2-3increased compared to control ones. The level of apoptosis in control group was pretty low but of which in group2-4were significantly higher than those of control ones (P<0.05)The dissimilarity of expression of Hsp70and apoptosis at different pre-conditioning temperature implied that Hsp70exerts its protective may effect by a temperature-specific mechanism.