Effects And Mechanisms Of Delayed Chronic Acidic Postconditioning On Neurological Functional Recovery After Traumatic Brain Injury

Objective:Traumatic brain injury（TBI）is a sudden disease with high fatal and disable rate.Because of TBI-induced primary injury and secondary injury induced by tissue ischemia,hypoxia and edema,permanent neurological dysfunction is the mainly complication of TBI.However,no safe and curative methods are available except rehabilitation training.Our previous studies found that postconditioning with CO₂ inhalation within minutes after the onset of reperfusion can protect against cerebral ischemia/reperfusion injury,and firstly proposed a concept of rapid acidic postconditioning as a new intervention for cerebral ischemia.On this basis,we aim to investigate the effects and mechanisms of delayed chronic acidic postconditioning on neurological functional recovery after cryogenic TBI.Methods:Adult male ICR mice were subjected to croyogenic TBI by exposing the right motor and sensory cortex to liquid nitrogen.In order to reduce the pH value in brain,mice received daily acidic postconditioning by inhaling 10%or 20%CO₂ for three types of time-course（three cycles of 5 min inhalation/5 min break;three cycles of 5 min inhalation/10 min break;three cycles of 10 min inhalation/10 min break）at different time-window（2,3,7 d post-injury）,the daily acidic postconditioning continued until the animals were sacrificed.DPCPX（1 mg/kg,i.p.）,a selective antagonist of A₁ receptor,was administrated at 30 min before daily acidic postconditioning.SCH58261（1 mg/kg,i.p.）,a selective antagonist of A_2AA receptor,was administrated at 60 min before daily acidic postconditioning.Beam walking test was used to evaluate motor function at different time-point after cTBI.Toluidine blue staining was used to measure the infarct volume.Detected the expression of axonal regeneration associated protein（growth associated protein 43,GAP-43）and synapse reorganization associated protein（synaptophysin,SYP）by immunofluorescence staining,area and thickness of glial scar and the total number of Iba1⁺cells were measured.Results:Delayed chronic acidic postconditioning dose-dependently and time-dependently enhanced the motor functional recovery after cTBI.In the condition of daily treatment with three cycles of 10 min CO₂ inhalation/10 min break（initiated at 2 d post-injury）,both 10%and 20%CO₂ reduced the percentage of footslips at 14 d post-injury,and the effect of 20%CO₂ was the most robust（P<0.01）;in the condition of 20%CO₂,all three types of time-course decreased percentage of footslips at 14 d post-injury,and three cycles of 10min CO₂ inhalation/10 min break had the most strong effect（P<0.01）;in the condition of20%CO₂ and three cycles of 10 min inhalation/10 min break,daily acidic postconditioning which initiated at 2 d or 3 d post-injury significantly improved the motor function at 14 d（P<0.01）and 21 d（P<0.05）post-injury,but daily acidic postconditioning within 2-7 d or7-21 d post-injury showed a weak effect on alleviating motor dysfunction without significance.Correspondingly,acidic postconditioning also reduced infarct volume at 14 d or 21 d post-injury.Immunofluorescence staining results showed that the expression of GAP-43（P<0.01）and synaptophysin（P<0.05）in peri-infarct cortex were down-regulated by cTBI at 14 d post-injury.And daily acidic postconditioning（three cycles of 10 min20%CO₂ inhalation/10 min break）within 3-21 d post-injury enhanced the expression of GAP-43（P<0.01）and synaptophysin（P<0.05）at 14 d post-injury.In addition,delayed chronic acidic postconditioning also attenuated glial scar area（P<0.05）but had no effect on glial scar thickness and total number of Iba1⁺cells.Furthermore,DPCPX（a selective antagonist of A₁ receptor）and SCH58261(a selective antagonist of A_2AA receptor)inhibited the effect of delayed chronic acidic postconditioning on motor function,infarct volume,and the expression of GAP-43 and synaptophysin at 14 d post-injury.Conclusion:Delayed chronic acidic postconditioning,which initiates at sub-acute stage of TBI,promoted neurological functional recovery and reduced infarct volume after injury.And it can enhance axonal regeneration and synapse reorganization by activation of A₁ and A_2AA receptors,which is a critical mechanism in acidic postconditioning-induced functional recovery.This study provides a new therapy for rehabilitation after TBI.