| Liver, the important organ to produce heat, is involved in ther-moregulation. Effect of liver on thermoregulation is increasingly attached importance in recent years. However, researches on effect of iN-OS on pathophysiologic and physiologic features in liver are not reported. Therefore, based on the wide pathophysiologic and physiologic effects of iNOS, we evaluated the expression of iNOS in livers in endo-toxic fever caused by LPS.METHODSMethods: (1) measurement of body temperature : The animals were put into the special designed box in order to make them adapt to environments three days before experiment. Each animal was caged in a 20 - 241 environment with food and water available except during the period of experimentation. A thermistor probe was inserted approximately 8cm into the rectum and taped to tail in the experiment to measure body temperature. E coli endotoxin diluted with unpyrogenic saline was injected i. p. in dose of 1mg/kg. During body temperature measure, we will measure temperature three times and look their mean value as basal body temperature. The rectal temperature of all the animals was recorded at 1 h intervals until the end of experiment. In orderto avoid differences in circadian baseline, experiment were started at thirty past eight in the morning. (2) Imrnunohistochemistry: rats were deeply anesthetized and perfused transcardially with 0. 01M phosphate buffered saline (PBS,Ph7.4) for 10 minutes, followed by 500ml of phosphate buffered 4% paraformaldehyde (Ph7.4). The liver tissues were removed, stored in the same fixative for 6 hours, submerged at 4 癈 in PBS containing 15% sucrose. Tissues were embedded using OCT and five series of sections were cut at 10 [xm the next day. Tissue sections incubated in 3% hydrogen peroxide in PBS for 10 minutes at room temperature; After blocking nonspecific binding by 5% normal goat serum for 30 minutes, the polyclonal iNOS antibody (dilution, 1:100) was applied in PBS for 24 hours at 4T1; the slides were subsequently incubated goat anti -rabbit IgG (1: 100 in PBS diluent) for 30 minutes at 37 癈. The sections were incubated in DAB for 10 minutes. Dehydration was performed in a graded ethanol. The sections were mounted onto coverglasses using resin. Then the sections were visualized under bright microscope. We used the image quantitative a-nalysis technique to determine the mean grey value and integrated optical density and threshold erea % of iNOS. Temperature responses were assessed as changes from pre ?injection values ( ). Results are expressed as the mean s. e. mean . The statistic analysis used the one - way analysis of variance test.RESULTSResults show that LPS administration produced a rise in deep body temperature of rats. Fever was maximal about 6h after injection then subsequently decreased but was still seen to be greater than con-trol levels. Body temperature in LPS groups is much higher than that in the control group which elevated 1. 38T1 at 6h; LPS not only produced a fever but also increased in the activity of iNOS in liver. The activity of iNOS between groups of LPS and control had outstanding differences (P<0.001). Microscope observations: positive staining for iNOS was observed in inflammatory cells, macrophages, kuffer cells and hepatocytes, especially at the canalicular membrane. Microscopic image quantitative amalysis result: integrated optical density and threshold area % of iNOS in LPS groups are much higher than those in the control group ( P < 0. 01) and are most prominent at 6 hours after LPS injection. In contrast, the mean grey value of LPS groups is lower than that in the control groups ( P < 0. 01) and is minimal at 6 hours after LPS administration. Change in body temperature between the LPS groups and the control group has positive correlation-ship with the increasing of iNOS activity in liver.CONCLUSION1. LPS can induce the expression of iNOS in a wide variety of cells in endotocxin - treated rats and show the greatest staining in 6 hours after treatment.2. These positive hepatocy... |
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