| SECTION ONEEstablishment of model of fluid percussion brain injury with different damage extent and pathological observations in ratsObjective: To establish rat models of fluid percussion brain injury with different damage extent and observed their pathological changes.Method: 1 Animals groups and model establishing: 160 adult male SD r- ats of clean grading, which came from the Experimental Animal Center of Hebei Medical University, were randomly divided into three groups: control group (8 rats), sham-operation group (8rats), injury group (144 rats).①the injury group were randomly divided into 0.1mpa injury group, 0.2mpa injury group, 0.3mpa injury group according to hit force. According the sacrifice time of 1 h, 6h, 12h, 24h, 3d, 7d after injury, each of the injury group is divid ed into six sub-groups, which was composed of 8 rats.③the control group are normal rats and only accepted anesthesia.④the sham group only is operated, not accepted the hydraulic shock. The rats of③④were sacrificed after operating, and those of other groups at corresponding time points, to prepere for brain sample.2 Experimental observed indexes:①The impact stress testing: the pressure sensors is used to test and record the practice impact pressure of brain injury location, while the computer is used for analysis of data.②Neuroethology observation: the rat dura in bone window was blue and there are a brief apnea and limb paralysis on one side in rats which are the standardization of TBI model-making successing. Neurological behavioral score cited from litera tur- es was employed to evaluate the neurological function in all groups.③Brain water content: the brain water content was measured by the way of dry and weight.④Pathological observation: light microscopy was used to observe in the pathological changes of all groups, and comparing the evolution of trends. The above results of the data were expressed with mean±standard deviation (x±s), which was analyzed by single factor analysis of variance andχ2 test a nd spss13.0 statistical software , p<0.05 was considered to be statistically significant.Results: 1 Test results of the impact pressure: Under the same impact pressures, there was no significant difference between measurable pressures in sub-groups. The measured pressures between the different impact forces had statistically significance.2 Neurobehavioral changes: The dura of bone window in the injury rats bec ame blue. As the impact pressure increased, there occured severe respiratory dysfunction: 0.1mpa injury group rats suffered atransient hypopnea after hydraulic shock. Respiratory apnea time in 0.2mpa blast injury group rats was 10.88±2.69s and that of 0.3mpa blast injury group rats was 20.60±3.02s. The neurological function score of injury groups was less than that of the normal control group. The neurological function score in 0.1mpa injury group recovered rapidly, had been no significant difference at 24h; Neurological sco rein 0.2mpa injury group was significantly reduced at various time points aft- er injury, gradually recovered at 3d to 7d, still showed a significant difference compared with the control group. The neurological function score in 0.3mpa injury group significantly reduced at different time points, recovered slowly, and had statistically significance to compare with the 0.1mpa injury group and 0.2mpa injury group.3 Cerebral water content: Brain water content of 0.1 mpa injury group began to increase at 1h, became worse at 6h, reached the peak at 24h(81.12±0.03%), then declined, and returned to normal level at 7d. Brain water content of 0.2 mpa injury group significantly increased at 1h, increased to 79.48±1.54% at 6h, and continued to increase at 24h, began to decline at 3d, showed signi fi- cant compared with the control group. Brain water content of 0.3mpa injury group increased most obviously: rose up to 79.16±0.02% at 1h, 6h reached to 81.82±0.06% at 12h, continuined to increase until 3d, gradually became nor mal at 7d, and there are still statistically significant compared with normal control group.4 Morphological changes: As the impact pressure increased, more severe brain injury occurred with range-increasing of brain injury, epidural and subdural hemorrhage also gradually expanded. with the impact pressure increasing, cerebral edema also appear earlier, ballooning degeneration of glial cells and neurons nuclear pyknosis, necrosis, and reduction of the cell number occured ahead of time. Extent and scope of the lesion expended, accompanying with inflammatory cell infiltration and exudation of red blood cells. Conclusion: slight, moderate and severe injury model of brain damage can be reproduced, with characteristics of stable quality and similarity of the human brain injury and pathological changes.SECTION TWO Effect of high-dose methylprednisolone on AQP4 expression aft- er moderate fluid percussion brain injury in ratsObjective: High-dose Methylprednisolone were used to treat the mo dera te fluid percussion brain injury model. AQP4 protein and its mRNA were examined by IHC, Western Blot and RT-PCR, to explore effect of high-dose Methylprednisolone on AQP4 of the brain edema and to render the theoretical evidence for clinic use.Method: 1 Animals groups and model establishing: 160 adult male SD rats of clean grading, which came from the Experimental Animal Center of Hebei Medical University, were randomly divided into three groups: normal control group (4 rats), sham operation group (4 rats), injury (48 rats) and inter vention group (48 rats). the injury groups and intervention groups according to1 h, 6h, 12h, 24h, 3d, 7d six time points after brain injury were also divided into six subgroups, each of which comprised 8 rats. The intervention group w- as given instantly MP by intravenous injection after operation: the first dose was designed as 30mg/kg, laterly as 5.4mg/kg, administered once every 6h, until the 3d, the dose was changed to 10mg/kg at 4~7d. Sham-operated rats were anesthetized and surgically prepared in the exact same manner, but did not receive brain injury. normal control group did not accept any treatment. Animals of normal control and sham operation group was killed by decap- itation after injury, those of intervention group was disposeded with same way at 1h, 6h, 12h, 24h, 3d and 7d after injury.2 Experimental observed indexes: Immunohistochemical, Western Blot and RT-PCT was used to analysis of AQP4 protein and mRNA. The brain water content (BWC) was measured by Wet-Dry Method.3 Statistical analysis: Above data were expressed with mean±standard de viation( x±s)and analyzed by SPSS13.0 statistical software. the samples in g- roup were compared using the method of single factor analysis of variance and the samples between groups were compared using the method of indepen dent samples t test.Results: 1 Pathological changes: Compared with the control group, the glial cells of intervention groups was ballooning and expanded extravascular space was reduced significantly.2 Brain water content: Compared with the control group, brain water content in the injury group at 1h began to increase, gradually increased at 6h and 12h, reached the maximum at 24h (82.74±1.11%, p <0.05), began to decline at 3d, gradually nearly attend to normal level at 7d after brain injury. Compared with the injury group, MP can significantly reduced brain water content of the intervention group at 6h, 12h, 24h, 3d (77.71±1.15%, 78.18±1.36%, 80.11±0.84%, 79.95±0.85%) , while at the other two time points such as 1h and 7d, brain water content were less obvious (p> 0.05).3 AQP4 protein expression: Compared with the control group, the expression of AQP4 protein at 1h began to increase, increased gradually at 6h and 12h, (immunohistochemistry 0.126±0.018, Western-blot 1.168±0.165, p <0.05), reached its maximum at 24h, began to decline at 3d, decreased gradually to be near normal at 7d. Compared with the injury group, AQP4 expression of the intervention group decreased at 6h,12h,24h,3d(immunohisto-chemistry 0.062±0.029,0.079±0.010,0.098±0.016,0.080±0.021; Western blot 0.511±0.131, 0.571±0.111, 0.832±0.123, 0.752±0.918). AQP4 protein expression of four time points such as 6h, 12h, 24h, 3d was significantly decreased and the difference was significant, while at the other two time points such as 1h and 7d, AQP4 protein expression did not significant reduced(p> 0.05).4 AQP4mRNA expression: Compared with the control group, AQP4mRNA expression began to increase at 1h, increased gradually at 6h and 12h, rise up to the maximum (0.885±0.110, p <0.05)at 24h, began to decline at 3d, be came to be near to normal level at 7d after brain injury. Compared with the injury group, AQP4mRNA expression in intervention group at 6h, 12h, 24h, 3d (0.519±0.106,0.598±0.112,0.665±0.121,0.636±0.103) significantly d ecreased, this difference was significant, while at the 1h and 7d, AQP4 mRNA expression did not reduced significantly ( p> 0.05).Conclusion: 1 AQP4 protein and mRNA increase in brain edema from fluid percussion brain injury in rats.2 High-dose MP can alleviate brain edema from fluid percussion brain injury in rats by down-regulation of AQP4 protein and its gene expression.
|
PDF Full Text Request