The Study Of The Accumulation Of Brain Injury Leads To Severe Neuropathological And Neurobehavioral Changes After Repetitive Mild Traumatic Brain Injury

Objective: Traumatic brain injury(TBI)is a major public health problem with long-term neurobehavioral sequela.The epidemiological evidence suggests that TBI is one of the risk factors for the development of neurodegenerative diseases.Both the single TBI and repetitive TBI can lead to the development of neruodegeneration.The mechanism of accumulation of brain injury paly an role in neurodegeneration remains unclear.Therefore,in this study,we established a single and repetitive brain injury model in SD rats through a controled cortical impact injury instrument.The TBI model was used to study the accumulation of brain injury effects on the neurobehavioral and neuropathological changes after repetitive brain injury.Methods:Male,Sprague Dawley(SD)rats(aged 8 weeks,each weighing about 200g)were hit by controlled cortical injury.Rats were allowed to adapt to the vivarium for at least 1 week before experimental procedures.For this study,animals were randomly assigned to four experimental groups: single m TBI controls(s-ctl),single m TBI(s TBI),repetitive m TBI controls(r-ctl)and repetitive mild TBI(r TBI).Timing for all testing was from the point of last impact(i.e.,the amount of time out from the single impact for the single TBI rats and the time out from the fourth hit for the repetitive TBI rats).These rats were tested Modified Neurological Severity Score(m NSS)at 1 day,3 days,7 days,14 days and 30 days after m TBI.Then used Morris water maze to detect the spatial learning and memory after 30 days of brain injury.In addition,rats were used only once for an individual behavioral task at every time point.Neuronal immunofluorescence was used to detect neuronal loss at different time points after injury.Microglia and astrocyte immunofluorescence were used to detect the inflammatory response at different time points after injury,and the expression of inflammatory factors at the corresponding time points was detected by ELISA.Western blot was used to detect the expression of tau protein and APP protein at different time points after injury.Results:Neurobehavioral test results showed that repeated mild brain injury resulted in a more severe neurobehavioral outcome than single mild brain injury.The results of m NSS showed that the neurological score of r TBI group was higher than that of s TBI group at different time points after injury,which indicated that neurological damage caused by repeated mild brain injury was more severe(F3,40 = 649.57,P <0.0001).Morris water maze results showed that the escape latency was significantly longer than that of the s TBI group from day 25 to day 29 after repeated brain injur(F4,112 = 393.19,P < 0.001).The percentage of target quadrant after 30 days of brain injury showed that the ratio of r TBI group was significantly lower than that of s TBI group,the difference was statistically significant(F3,12 = 32.38,P < 0.001).The results of immunofluorescence detection showed that neuronal deletion occurred in the cerebral cortex and hippocampus after brain injury,and r TBI group was more severe than the s TBI neuron deletion.The activation of microglia and astrocytes reached the peak at 7 days after brain injury,and the number of microglia and astrocytes activated in r TBI group was higher than that of s TBI group at the same time.And more activated glial cells were detected in the r TBI group 30 days after brain injury.The result of ELISA was consistent with the results of immunofluorescence in the brain tissue after brain injury.The proinflammatory cytokines(TNF-?,IL-6,IL-10)reached the peak at 7 days after injury.And the proinflammatory cytokines(TNF-? and IL-6)expressed in r TBI group were higher than those in s TBI group at different time points after brain injury.The result of western blot assay showed that APP protein increased after brain injury,and the expression of APP in r TBI was higher than that the s TBI group,while tau protein and P-tau protein were not statistically different between the two groups after brain injury.Conclusion: Our study showed that the accumulation of brain injury could lead to more severe neuropathological and neurobehavioral changes after traumatic brain injury.The activation of chronic microglia and astrocyte could lead to the dysregulation of inflammatory cytokines which might play a role in the neurodegeneration after brain injury.The mechanism of brain injury and how the accumulation of TBI leads to increase the expression of neurodegeneration protein remain unclear.To investigate the mechanism of how the accumulation of TBI aggravates neurodegeneration is the key point to intervene the process of neurodegenerative disease after brain injury.