| Objective: This experimental study aims to explore the changes of hippocampus in HIF-1αand Caspase-9 in the rat model called DEACMP. Providing experimental foundation for further research of DEACMP as well as the theory of clinical prevention and treatment of this disease.Methods: First, randomly put 156 healthy adult male SD rats into three groups, which have been screened in the water maze before. The three groups are group DEACMP( group CO,60 rats), group sham operation( group SO,60 rats) and the blank control group( group BC,36 rats). Group CO repeatedly uses the intraperitoneal injection at intervals to establish the rat model DEACMP; group SO uses the same method to inject the same amount of air, while group BC has no any treatment measures at all. Six time points after the exposure of rats to poison are selected, namely: 1st day, 3rd day, 7th day, 14 th day, 21 st day and 28 th day, and the rats are divided into six subgroups according to the six time points. Achievements can be made by exploring the average time and space of rats’ escaping the latency period at the following five time points, that is, the day before the Morris test,7thday, 14 thday, 21 thday, and 28 th day.And in this experimental study HE dyeing, Tunel,immunohistochemistry Western-blot are used to detect the pathological changes of hippocampus in rat models, apoptosis and protein expression of HIF-1αand Caspase-9. Results: 1.The establishment of the DEACMP model:(1)After being poisoned, rats in group CO showed significantly acute poisoning reactions, skin mucous membrane also appeared cherry-red. The rats started to be irritable, shortness of breath.Severely-poisoned rats first had convulsions, then became unconscious and finally died. Compared with the rats in the blank control group, rats that survived in group CO had less daily activities, slept more, reacted more slowly and ate less. 2. Experimental result of Morris water maze:(1) Place navigation test: The time difference rats used to escape latency on the day they were poisoned and on the 7th day showed statistic difference(P> 0.05). At other time points like on the 14 th day, 21 thday and 28 th day, the average time of escaping latency used by rats in group CO was statistically different when compared with the time used in group BC and SO, and the state on 7th day after being poisoned(P<0.05). The average time used in group CO prolonged, while comparison among the 14 th day, 21 thday and 28 th day showed no statistic difference(P>0.05). Comparison between group BC and Group SO at each time point also showed no statistic difference(P>0.05). Spatial probe test: Section IV quadrant exercise time / total time percentage: on the day before the rats poisoned and 7 days later, they showed no statistic difference(P>0.05). After being poisoned, Section IV quadrant exercise time in group CO and group SO and BC on 14 th day, 21 thday and 28 th day was short, meanwhile the total time percentage reduced, when compared with the state on 7th day of group SO and BC, the difference is statistically meaningful(P<0.05). In group CO, comparison among 14 th day, 21 thday and 28 th day showed no statistic difference(P>0.05). Comparison between group BC and Group SO at each time point also showed no statistic difference(P>0.05).(2)Trajectory: rats in group SO and group BC tended to have linear motion and find a platform in a short time, besides, the trajectory of the rats in two groups showed no significant changes before and after being poisoned. After being poisoned, trajectory of rats in group CO scattered, seemed to have circular type motion, and it took a long time for them to find the platform. 3. Pathological changes of HE dyeing in the brain tissue: cell morphology in the hippocampus of group BC and group SO at each time point was normal, 3-4 layers of cells were arranged in good order,and cell cytoplasm evenly appeared pale blue, Nucleolus and the boundary were clear, and cell membrane showed integrity. Pathological changes occurred widely in the brain tissue of rats in group CO, reduction in the number of cells, disorder of cells, intercellular space expansion, nuclear fragmentation, anachromasis and pyknotic. 4. Tunel apoptosis detection results: apoptotic cells in hippocampus of rats at each time point in group BC and SO is small, AI difference was not statistically significant(P> 0.05). AI in group CO at all time points after being poisoned was more than those in group BC and SO, there were significant differences(P <0.05) and 7 days after being poisoned, it reached the peak of apoptosis.5. Immunohistochemical staining and the expression of HIF-1αand protein Caspase-9:(1) The expression of HIF-1αand protein Caspase-9: HIF-1αexpression in group BC and SO at all time points was not frequent and had no significant difference in statistics(P> 0.05). After being poisoned, HIF-1αexpression levels at each time point in group CO were higher than those in group BC and group SO. On 1st day after exposure to poison, expression increased and on 3rd day it reached a peak to reach the peak, followed by a decreasing trend. The comparison at all time points among the groups in the first three days were significantly different in statistics(P <0.05)(2) Caspase-9 expression in group BC and group SO is not frequent; expression 1d increased expression 3d reached a peak and then gradually declined in group CO, to 28 d expression, it was still more frequent than those in group SO and BC, Section difference on 3thday, 7th day and 14 th day was statistically significant(P> 0.05). 6. Test results of Caspase-9 protein with Western Blot method: positive activation body expression of Caspase-9 displayed as black stripe on the film, sizes of stripe and shades of color were relevant to the number of expression. Activation body expression of Caspase-9 in group CO was much more than that in group SO and BC. Its tendency wass that when expression on 1st day in group CO increased, reached the peak on 3rd day, then declined, until 28 th day, its expression was still more than that in the other two groups. Conclusion: 1.Repeated use of the intraperitoneal injection at intervals to establish the rat model DEACMP is a relatively ideal method of researching DEACMP to establish an animal model.2. Morris water maze test can objectively evaluate cognitive abilities in rats, combined with the behavior changes of rats, the blood COHb as criteria can be used as a preliminary standard to judge whether DEACMP occurred to rats.3. The occurrence of delayed encephalopathy in acute carbon monoxide poisoning may be related to damage on hippocampal neurons delayed venereal disease 4.High expression level of HIF-1αin DEACMP may promote the expression of apoptosis-related factors Caspase-9, inducing apoptosis, playing a role in promoting apoptosis in DEACMP. |
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