Experimental Study On The Rat Model Of Diffuse Brain Injury And The Expression Of Apoptosis-regulatory Proteins In Traumatic Rat Brain

Objective: To develop rat model of graded diffuse brain injury from the modified Marmarou's mothod and to investigate the temporal and spatial characteristics of bcl-2, bax and caspase-3 gene alterations at protein level in injured rat brain and the molecular mechanism of neuronal apoptosis following diffuse traumatic brain injury.Methods: A modified Marmarou's impact device was used to establish the animal model of diffuse brain injury with varied degree by changing the impact conditions,including 80cm x 450g, 120cm x 450g and 180cm x 400g, that was applied on the rat head; The animal model was evaluated by observing the alterations of nervous system signs, vital signs, sensorimotor function and brain tissue morphology in rats following the different impact. Immunohistochemistry was performed to examine Bcl-2, Bax and Caspase-3 protein expression in different brain regions of rat at different times after the diffuse brain injury with moderate severity (120cm×450g); Flow Cytometer (FCM) was used to detect cell apoptosis quantitatively in brain hippocampus of rat after the moderate diffuse brain injury.Results:(1) Compared to sham injured rats, the significant alterations of nervous system signs, vital signs and sensorimotor function were observed in the rats that were subjected to the different impact; Histological examination revealed pathologic changes of diffuse brain injury in all impacted rats; Moreover, all above changes became more significant with the impact energy increasing. (2) After moderate diffuse brain injury, the decreased Bcl-2 protein expression was observed at cerebral cortex in rats from 6 hours to 12 hours, reached minimus level at 24th hours after injury (P<0.05) and returned to controls level after 7 days; In contrast, Bax protein expression in injured rat brain cortex increased gradually during 6-24 hours after the diffuse brain injury, reached a peak at 48th hours (P0.05) and was still higher at 7th days than that in controls after injury ; Caspase-3 protein expression was not observed at cortex of rat brain until 12 hours after injury, increased at 24th hours, reached to the highest level after 72 hours (P<0.01) and still remained high level at 7th days after injury. At cerebral hippocampus in rats, Bcl-2 protein expression increased at 6th hours after moderate diffuse brain injury (P0.05), but decreased at 12 hours, reached to the lowest level at 48th hours (P0.05) and still was lower than that in controls at 7th days after injury; The changes of Bax and Caspase-3 protein expression at hippocampus were similar to that occurred at cortex of rat brain following moderate diffuse brain injury, whereas the expression of the two proteins varied more significantly at cerebral hippocampus in rats after injury; Furthermore, at brain hippocampus in rats Bcl-2/Bax ratio (mean value ratio) had significant negativecorrelation with Caspase-3 protein expression after moderate diffuse brain injury. At brain white matter and thalamus in rats, the variations of all the proteins expression after moderate diffuse brain injury were not evident. (3) The cell apoptosis at cerebral hippocampus in rats increased significantly from 12 hours to 48 hours (P<0.01) after the moderate diffuse brain injury, reached to a peak at 72 hours (P<0.01) and still remained higher level after 7 days after injury. Moreover, the alteration of the cell apoptosis at brain hippocampus in rats correlated with both the decreased Bcl-2/Bax ratio and the increased Caspase-3 expression after moderate diffuse brain injury.Conclusion: A reliable, practical and reproducible rat model of graded diffuse brain injury have been developed from the modified Marmarou's mothod, and the moderate impact (120cmx450g), which have produced low animal death rate and resulted in significant pathological alteration in injured rat brain, is suitable to investigate the pathophysiological changes following diffuse traumatic brain injury. (2) The variations of apoptosis -regulatory proteins expression in brain hippocampus appear to be mo...