| Objective:In order to provide experimental basis and reference for the further research about the pathogenesis of DEACMP,this experimental study explore the may pathogenesis of these two proteins by analysis those behavior,the expression and correlation of HIF-1? and HO-1 in brain hippocampus of rats.Methods:1. Sampling according to the random number table method and putting the 156 healthy male SD rats with SPF grade(body weight 280~320g) into three groups after screening: DEACMP group(group CO) and air control group(group AC)have 60 rats,the blank control group(group BC)have 36 rats.Then randomly put those rats into the six subgroups after exposure 1stday,3rdday,7thday,14 thday,21thday and 28 thday,CO group and AC group have 10 rats in each subgroups,BC group have 6 rats in each subgroups.2.CO group use a method of intraperitoneal inject CO by a modified multiple intervals way to establish animal model of DEACMP,AC group inject air with the same method,BC group do nothing.3.Preliminarily judge the model of DEACMP whether succeed by the changes in their memory and cognitive function before and after exposure,determination the concentration of Hb CO at the same time.Detecting the morphological changes of the rats' hippocampus cells by HE staining,the apoptosis of the rats' hippocampus CA1 area by TUNEL staining,the expression of iron ion in rats' hippocampus by prussian blue staining,the expression of HIF-1? and HO-1 in rats' hippocampus by envision method of immunohistochemistry and the expression of HO-1 in rats' hippocampus by semi-quantitative Western Blotting method.4.Analysing the experimental data with SPSS17.0 statistics software.Results:1.DEACMP animal model:The rats of CO group have found gradually that they become weakness, bradykinesia, less dynamic, transient paralysis, deep and shortness breathing after a few minutes with contamination, their lips mucosa become cherry red colour, some rats have found muscle rigidity, limbs twitch and even coma or death. As compared with the other two groups, the survival rats of CO group eat less, are more irritable and insensitive. The rats of AC group and BC group have no significant changes.2.The concentration changes of Hb CO in arterial blood: The concentration of Hb CO in rats continue to maintain more than 50% above 16 hours throughout the course of exposure.3.Morris water maze experiment: The comparative within or between groups about those three groups of rats in the average escape latency, total time through the platform quadrant and crossing platform times have no significant differences in statistically, but in the 14 thday,21thday and 28 thday after contamination, the CO group of rats have longer average escape latency, less time through the platform quadrant and less crossing platform times, the comparative within or between groups have significant differences in statistically(P<0.05).4.HE staining: In CO group of rats, those cells in hippocampus CA1 area have irregular arrangement and poorly level, the number of pyramidal cells become fewer, the intercellular become widened, the nuclear become fragmentation and condensation gradually and the cells become swelling, even degeneration and necrosis. Those cells in hippocampus CA1 area of AC group and BC group rats have no obvious abnormalities.5.Prussian blue staining: In the CO group of rats, the expression of iron ion was increased at the 1th day after contamination, reaching a peak at the 7th day, then gradually dereased. There wasn't obvious iron ion expressed in AC group and BC group.6.TUNELstaining:The apoptotic cells in the hippocampus area have growth trend, it reachs a peak at the 3rdday,then declining after that, the number is still higher than AC group and BC group at the 28 th day, the difference is statistically significant(P<0.05).There wasn't obviously apoptosis in AC group and BC group at each time point.7.The expression of HIF-1? and HO-1 protein:(1)The method of immunohistochemistry: The expression of HIF-1? and HO-1 protein in CO group have a trend of increased at first, reaching a peak at the 3rdday, and then gradually decreased from the 1stday to the 28 thday,but was still higher than AC group and BC group at the 28 thday,while the expression of HIF-1? and HO-1 protein in AC group and BC group had no significant change.(2)The method of Western Blotting: The expression of the HO-1 protein in hippocampus of CO group rats increased at the 1th day, and peaked at the 3rd day, then gradually decreased, and 28 th day was still higher than that at 1st day level.Conclusion:1.The modified multiple interval intraperitoneal injection of CO poisoning method operation is simple and convenient, the equipments required are common, the amount of CO will be small and easy to control, there isn't obviously waste and pollution, it can reach the condition of other model, just like the inhaled model, it is an ideal model which is closer to clinical practice.2. The Morris water maze experiment through the comparison of the changes of memory and cognitive function before and after exposure in rats, detecting the changes of Hb CO concentration in arterial blood of rats poisoned and contrasting the behavior change of poisoned rats to preliminary determination the DEACMP model is successful or whether.3. The expression of HIF-1 increased in hippocampus after acute CO poisoning, promoteing the expression of the downstream factor HO-1, it is likely to be one of the pathogenesis of DEACMP that both of them promote the occurrence of delayed apoptosis in hippocampal area. |
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