| Objective: In this study, we use Morris watermaze experiment, histopathology, immunohistochemical detectionmethod, Tunel apoptosis detection techniques in rats and pathologicalchanges through the establishment of DEACMP rat model, to explorethe HIF-1α and VEGF in the corresponding table of changes in animalmodels, to determine its role in the pathogenesis of DEACMP, to providenew ideas and methods for further research on DEACMP, and to provideexperimental basis for clinical treatment. Methods:78healthy adult maleSD rats (SPF) were randomly divided into three groups: controlgroup (BC group),18; air control group (AC group),30; delayedencephalopathy after acute carbon monoxide poisoning group (CO group),30. The CO group was established by intraperitoneal injection of delayedencephalopathy rat model of acute carbon monoxide poisoning, AC groupwere treated with intraperitoneal injection of injected air. CO group andAC group were divided into6sub groups according to1d,3d,7d,14d,21d,28d six time points after poisoning, each sub group5rats. Therats in the model of the Morris water maze test memory, orientation andcognitive function. The rats of7d,14d,21d,28d time point subgroups were sacrificed in the brain before, also using Morris water maze to testthe changes of cognitive function. Each ratbrain tissuesections were used HE to observe the pathologic changes in hippocampusof rats staining, immunohistochemical staining to detect thehippocampus HIF-1α, VEGF protein. The apoptosis of pyramidal cells inhippocampus will be detected by TUNEL apoptosis detection method. Weuse SPSS17.0software for statistical analysis of the results. Results:1.The establishment of DEACMP rat model:(1) Toxic reaction appeared inthe gas of intraperitoneal injection of CO rats after CO. After exposureto5~15min has manifested as irritability, hyperactivity, mildactive behavior; after20min exposure rats started antifeedant lessdynamic, jitter, shortness of breath, increased respiratory rate, lip nasalmucosa and lower abdomen, acral skin showed obvious cherry red, andmove slowly, abnormal gait. Some rats appear manic, hit thecage, convulsions, opisthotonus, then paralysis, coma.Given theventilation, warm, about36.7%rats died in the period of24h, the survivalrats from exposure observation to fully awake cost about8hours, after28days, rats had slow reaction, reduced food intake, reduced activitiesof performance, the other rats daily activities had no obvious abnormal.(2) AC group rats were injected with air, no obvious abnormalbehavior, no death.2. Morris water maze test results:(1)Navigationexperiment: three groups had no significant difference in the average escape latency in the exposure before or after poisoning7d(P>0.05); afterpoisoning14d,21d,28d, the average escape latency of CO group waslonger than BC, AC group, there was statistically significantdifference (P<0.05). No significant difference between BC group and ACgroup (P>0.05).(2) spatial probe test:1) The IV quadrant (platformquadrant swim time/total time): before exposure, exposure after14d,the IV quadrant swimming time/total time of the three groups was nosignificant difference (P>0.05); after poisoning21d,7d,28d, CO groupwas different with the AC, BC group, there was statistically significantdifference (P<0.05); BC group, AC group at each time point, there was nostatistical difference (P>0.05); CO group decreased with time change.2)The times of crossing platform: each time point, the three group after theplatform number was no statistical difference (P>0.05); in each group atdifferent time points after platform times, no statistical difference(P>0.05);28d CO group had lower than the other groups; the COgroup changes over time, through the platform number decreasing.3. Pathological change of cerebral tissue morphology: HE stainingshowed,(1) at each time point in group CO in the brain of ratswith extensive pathological changes in hippocampal CA1region, theemergence of a large number of different degrees of degeneration andnecrosis of neurons. Mainly for the neuronal soma swelling,shrinkage, cell number decreased slightly, cell deformation spindle or triangle, cell layer to thin, enlarged intercellular space, nuclearpyknosis, indistinct nucleoli, nuclear week of halo, cytoplasm andcell nucleus is unclear, the cell structure disorder, infiltrationin hyperplasia around necrosis neurons showed different degreesof glial cells (After exposure to the21d subunit in rat brain slices canalso see box shadow left necrosis neurons were absorbed, can alsobe seen around the glial cells).(2) neurons in BC and the size of eachtime point in group AC in rat cerebral cortex and hippocampus wasnormal, nuclear membrane intact, clear nucleolus, nucleus was lightblue, color uniformity, the intact organelle. Among them, the CA1regionof the hippocampus area has two to three layers of pyramidal cells, neatlyarranged in dense, full nucleus, clear boundary, Nissl body rich, dark,clear nucleolus.4. Immune group of HIF-1α and VEGF protein stainingexpression:(1) the expression of HIF-1α: HIF-1α in BC group and ACgroup was significantly higher than expression, CO expression in the1dafter the exposure, the3d reached the peak, then decreased gradually, and28d still had a higher expression of HIF-1α content, expression of COgroup at each time point were higher than those in BC group and ACgroup, there were significant differences (P<0.01).(2)The expression ofVEGF: VEGF in BC group and AC group expressed less; group CO withthe exception of the14d, the rest of the time points on the expression ofVEGF was higher than that in BC and AC group, there was statistically significant difference (P<0.05); group CO VEGF expressionincreased rapidly from exposure after1D, reached the peak at aftergradually reduced in the3d, to the21d again when forming peakssignificantly increased, and then decreased, and the28d VEGFe x p r e s s i o n l e v e l o f n e a r l y f i r s t d a y s, s h o w e d i n c r e a s e d,decreased, increased and reduced dynamic change trend.5.TUNELapoptosis detection results: Group CO after exposure for the1d detectedin the hippocampus CA1area with a small amount of nerve cellapoptosis, began to increase at3d, reached the peak at7~14days (seventhdays for peak), the28d is still a small amount of apoptosis, apoptosisindex was significantly increased in BC group and AC group, there wasstatistically significant difference(P<0.01). Conclusions:1. Theadvantages of modified multiple interval of intraperitoneal injection ofCO rat model of DEACMP: low requirement of equipment, convenientoperation, less interference, the dosage of CO, less accurate, and thesimilar pathophysiological changes as human DEACMP process. So it isa perfect kind of animal model for the study of DEACMP.2. Morriswater maze experiment by navigation training and space explorationtest in order to clear the changes of learning and memory abilityand spatial memory ability of rats, to cognitive behavioral objectiveevaluation function of rats, Is initially determined DEACMP rat modelis successfully established a more reliable basis.3. The expression of HIF-1α after acute carbon monoxide poisoning increased expression maypromote VEGF, start the apoptosis signal transduction pathways, thuspromoting apoptosis and involving in the pathogenesis of DEACMP. |
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