Inhibition Of Drp1 After Traumatic Brain Injury Provides Brain Protection And Improves Behavioral Performance In Rats

BackgroudTraumatic brain injury(TBI)is a common cause of serious public health problems throughout the world,which has a high disability and mortality.Survivors of TBI experience various problems,including sensory and motor dysfunction as well as cognitive,emotional,and behavioral problems,which can occur either individually or in combination.Individuals who survive TBI may suffer lifelong disability,mostly due to cognitive disorders and behavioral and personality changes.TBI patients impose a tremendous burden on their families and society and increase demands on the healthcare system.Frontal lobe and temporal lobe contusion are widely existed in traumatic brain injury.The hippocampus plays a crucial role in memory and cognition,but it is highly vulnerable to brain trauma.TBI results in significant hippocampal atrophy in patients and a reduction in hippocampal volume,correlated with damaged verbal memory function.Hippocampal neurogenesis is strongly interrelated with hippocampus-dependent learning and memory,but the damage to the hippocampal neurogenesis is widespread post-injury.The brain is a highly aerobic,energy-demanding tissue and,as such,is dependent upon mitochondria to maintain cerebral function.Mitochondria not only provide cellular energy through ATP synthesis,but also play an important role in intracellular calcium buffering,reactive oxygen species(ROS)production,and apoptosis.Mitochondria continuously undergo fission and fusion,which helps to maintain mitochondrial circuitry and homeostasis.Recent studies have proven that there is an imbalance between increased energy demand to repair cell damage and decreased energy production caused by mitochondrial dysfunction during the pathogenic process of TBI.Excessive fission can lead to reduced mitochondrial respiration and ATP production,increased ROS generation,and release of apoptogenic factors,changes similar to those seen in the second injury after TBI.Mitochondrial membrane fusion is known to be mediated by mitofusin-1 and mitofusin-2(Mfn1 and Mfn2,respectively)and optic atrophy 1(OPA1).Mitochondrial fission is mediated by mitochondrial fission 1(Fis1)and dynamin-related protein 1(Drp1).Drpl is a large protein in the dynamin GTPase family that is critical for regulating mitochondrial division,size and shape.Drpl assembles from the cytosol onto mitochondria at focal sites of mitochondrial fission.Drpl is highly expressed in the nervous system.Several recent studies reported abnormal Drpl expression in the postmortem brains from APP cell lines,AD mouse models and AD patients.Moreover,recent studies have proven that inhibition of mitochondrial fission plays a protective role against neurodegeneration in the cerebellum and against neurotoxicity.Mitochondrial division inhibitor-1(Mdivi-1)is a selective inhibitor of Drp1.It has been shown to selectively target Drp1 in mammalian cells by binding an allosteric site and suppressing the ability of Drp1 to catalyze GTP hydrolysis as well as its self-assembly into ring-like structures around the mitochondria.Inhibition of Drpl by Mdivi-1 remarkably reduced the infarct volume and neurological deficits in MCAO mice.Mdivi-1 also protected primary neurons against glutamate excitotoxicity and attenuated ischemic brain damage in a mouse model.However,Mdivi-1 has rarely been investigated as a post-insult treatment for TBI.In our study,the potential therapeutic effects of Mdivi-1 were tested in a model of moderate traumatic brain injury.ObjectiveFinding out the protective effects of Mdivi-1 on TBI-induced neurological and psychological dysfunction.Results1.Drpl but not Phosphorylation of Drpl was Enhanced in the Hippocampus after TBIIn our studies,rats were subjected to TBI,and ipsilateral hippocampus tissue extracts prepared from animals at 3 h,6 h,12 h and 24 h post-injury were subjected to western blot analyses.The results indicated that the total Drpl levels in the ipsilateral hippocampus in the first 24 h after TBI began to increase at 6 h,peaked at 12 h,and then slightly decreased afterward.Phosphorylation of Drpl is an important posttranslational modification that affects protein activity,but the levels of Ser637,Ser616,Ser40 and Ser44 phosphorylation of Drpl in the hippocampus after TBI did not significantly change.2.Neuronal Apoptosis in the Cortical Border Zone and Hippocampal Dentate Gyrus Caused by TBI Was Rescued by Mdivi-1The border zone contains a large number of injured neurons,which are the focus of treatment after TBI.The hippocampus is vulnerable to TBI,and selective death of neurons in the hippocampal DG has been reported after TBI.We immunostained brain tissue against the apoptosis marker activated caspase-3 three days after TBI.The numbers of activated caspase-3-positive cells in the border zone and DG were significantly reduced in the Mdivi-1-treated TBI groups compared with the saline-treated TBI groups.3.Mdivi-1 Rescued Impaired Hippocampal Neurogenesis at 4 Weeks Following TBINeurogenesis includes the proliferation of neural precursor cells and the survival of newborn neurons.To identify neural precursor cell proliferation,we administered a single BrdU injection 2 h before TBI,we observed that the rats in the TBI group exhibited significantly decreased BrdU+ cells in the subgranular zone of the hippocampus DG compared with sham group,but the number of BrdU+ cells was increased after 4 weeks Mdivi-1 treatment.Next,we measured the survival of newborn neurons in the hippocampal DG by administering BrdU once daily for 4 days after injury.BrdU+ NeuN+ cells were significantly more numerous in the sham groups than in the TBI groups,and the TBI group with Mdivi-1 treatment had far more BrdU+ NeuN+ cells than the saline-treated TBI group.4.Inhibition of Drpl Attenuated TBI-induced Brain DamageWe treated rats with Mdivi-1(20 mg/kg body weight)for 3 days and measured the cortical lesion volume,compared with the TBI group treated with saline,the TBI group treated with Mdivi-1 showed a remarkably reduced cortical lesion volume.We observed that Mdivi-1 treatment significantly reduced the Evans Blue leakage and brain edema induced by TBI.Compared with the sham groups,the TBI groups exhibited significantly decreased SOD activity in the border zone.However,Mdivi-1 significantly restored the activity of SOD compared with saline treatment.To examine the long-term sequelae of TBI in rats,we measured the ipsilateral and contralateral hippocampal volumes 4 weeks after sham-treated or TBI.TBI caused profound ipsilateral hippocampal atrophy,but Mdivi-1 rescued the trend after 4 weeks treatment.5.Inhibition of Drpl Rescued TBI-Induced Spatial Learning ImpairmentsWe conducted a Morris water maze test at 2 weeks after sham surgery or moderate fluid-percussion brain injury.TBI rats had significantly longer escape latencies and path lengths compared with sham rats during the whole acquisition phase.On the third and fourth days of task acquisition,TBI rats treated with Mdivi-1 displayed a significant decrease in escape latencies and path lengths to reach the hidden platform compared with TBI rats treated with saline.At 24 h after the last acquisition trial,the rats were tested for retention in a probe trial during which the platform was removed.TBI rats treated with saline spent significantly less time in the target quadrant and crossed the target platform fewer times compared with TBI rats treated with Mdivi-1.6.Mdivi-1 Attenuated TBI-Induced Anxiety-Like BehaviorThe open-field test was used to evaluate anxiety-like behavior and locomotor activity 14 days post-injury.TBI rats spent less time in the center zone than sham rats,but Mdivi-1 treatment significantly increased the time TBI rats spent in the center zone compared with TBI rats treated with saline.We found no difference in total distance moved in the arena between sham animals and TBI animals.ConclusionsOur results provided some new insights into the protective effects of Mdivi-1 on TBI-induced neurological and psychological dysfunction.In this study,we observed that TBI increased the expression of total Drpl,but the phosphorylated sites of Drpl(Ser637,Ser616,Ser40 or Ser44)involved in TBI pathogenesis showed no alterations.Mdivi-1 treatment suppressed neuronal apoptosis in the border zone surrounding the cortical lesion area and in the hippocampal DG 3 days after TBI.Mdivi-1 also rescued impaired hippocampal neurogenesis in DG at 4 weeks following TBI.We established that I.P.delivery of Mdivi-1 significantly reduced the traumatic cortical lesion volume,BBB permeability,brain edema and oxidative stress 3 days post-injury.We also observed attenuated hippocampal atrophy when Mdivi-1 was administered for 4 weeks.The improved Morris water maze performance in the treated animals indicates that Mdivi-1 treatment could improve cognitive and behavioral outcomes 2 weeks after TBI.Furthermore,post-insult treatment with Mdivi-1 could attenuate TBI-induced anxiety-like behavior but did not alter locomotor activity.