The Mechanism Of L-lactate Preconditioning Improves Neurological Deficits In Rats With Traumatic Brain Injury By Potentiating GPR81 Signaling Pathway

Objective:The model of traumatic brain injury(TBI)in SD rats was established to observe the effects of L-lactate preconditioning on TBI and the expression of GPR81,MCT2,and plasticity-related protein in cortex and hippocampus of rats after TBI.In order to investigate whether L-lactate preconditioning had the neuroprotective effect on TBI rats and further to explore the possible mechanism of lactate preconditioning on neuroprotection.Methods:72 adult male SD rats(280~300 g)were randomly divided into three groups: sham injury group(sham group),traumatic brain injury group(TBI group)and L-lactate preconditioning group(TBI+Lac group).Each group was divided into four sub-groups,including 12 h,24h,48 h,and 72 h group(n = 6/sub-group).The rats in the L-lactate preconditioning group were injected intraperitoneally 30 minutes before brain injury.The m NSS of each group was measured at 12 h,24h,48 h,and 72 h after operation respectively,and the time point of the lowest score was selected.At this time point,SD rats in each group were decapitated and than their brain tissues were taken.Western blot and immunofluorescence staining were used to detect the localization and quantification of GPR81,MCT2,PSD95,GAP43,and BDNF proteins in brain tissue.In addition,q RT-PCR was used to detect the expression of GPR81 m RNA and MCT2 m RNA,and HE staining was used to detect the pathological changes of cortex.Results:1.The results of modified neurological severity score L-lactate preconditioning decreased the m NSS of rats at all time points after TBI and played a neuroprotective effect.The results were as follows: compared with the sham group,the m NSS began to rise at 12 hour time point in the TBI+Lac group,reached the peak at 24 hour time point and then decreased slowly,but it was still higher than that in the sham group.However,compared with the TBI group,the score began to decrease at 12 hour time point and reached the trough at 24 hour time point in the TBI+Lac group,but it was still lower than that in the TBI group.At 24 hour time point,L-lactate preconditioning had the best neuroprotective effect.The results showed that the neurological functions of rats were reduced after TBI,while that can be improved by L-lactate preconditioning at 12 h,24h,48 h,and 72 h in TBI rats,especially,at 24 hour time point.2.The results of Western blot(1)The expression of GPR81 was increased in the ipsilateral cortex and hippocampus of rats at 24 hour time point in TBI group compared with the sham group.Interestingly,L-lactate preconditioning led to further enhancement of GPR81 expression in the ipsilateral cortex and hippocampus of rats in TBI+Lac group compared with the TBI group.The expression of MCT2 was decreased in the ipsilateral cortex and hippocampus of rats at 24 hour time point in TBI group compared with the sham group,but the expression of MCT2 was increased significantly in the ipsilateral cortex and hippocampus of rats in TBI+Lac group compared with the TBI group.(2)The expression of PSD95,GAP43,BDNF,and Trk B were decreased in the ipsilateral cortex and hippocampus of rats at 24 hour time point in TBI group compared with the sham group,but the expression of PSD95,GAP43,BDNF,and Trk B were increased significantly in the ipsilateral cortex and hippocampus of rats in TBI+Lac group compared with the TBI group.3.The results of q RT-PCR The m RNA expression of GPR81 was increased in the ipsilateral cortex and hippocampus of rats at 24 hour time point in TBI group compared with the sham group. Furthermore,L-lactate preconditioning led to further enhancement of GPR81 expression in the ipsilateral cortex and hippocampus of rats in TBI+Lac group compared with the TBI group.The m RNA expression of MCT2 was decreased in the ipsilateral cortex and hippocampus of rats at 24 hour time point in TBI group compared with the sham group.Furthermore,L-lactate preconditioning increased the expression of MCT2 in the ipsilateral cortex and hippocampus of rats in TBI+Lac group compared with the TBI group.4.The results of HE staining The morphology of the cells was normal and the structure was intact,and there was no necrotic area in sham group.Compared with the sham group,there were obvious necrotic areas in the injured cortex,obvious edema of brain cells,balloon change,enlarged intercellular space and fewer neurons at 24 hour time point in the TBI group;However,compared with the TBI group,the area of cortical injury,the cell edema and the number of pyknotic and missing neurons decreased significantly in the TBI+Lac group,and the number of survival cells increased significantly.5.The results of immunofluorescence staining(1)TBI significantly increased the immunoreactivity of GPR81 immunoreactive positve cell in the ipsilateral cortex and hippocampus CA1 and CA2 of rats at 24 hour time point compared with the sham group.Interestingly,L-lactate preconditioning futher increased the immunoreactivity of GPR81 immunoreactive positve cell in the ipsilateral cortex and hippocampus CA1 and CA2 of rats.TBI significantly decreased the immunoreactivity of MCT2 immunoreactive positve cell in the ipsilateral cortex and hippocampus CA1 and CA2 of rats at 24 hour time point compared with the sham group.Importantly,L-lactate preconditioning significantly increased the immunoreactivity of MCT2 immunoreactive positve cell in the ipsilateral cortex and hippocampus CA1 and CA2 of rats.Furthermore,GPR81 and MCT2 were expressed in the neurons of the cerebral cortex,hippocampal CA1 and CA2 regions of rats,which were co-located widely with the neuronal marker Neu N.(2)TBI significantly decreased the immunoreactivity of PSD95,GAP43,and BDNF immunoreactive positve cells in the ipsilateral cortex and hippocampus CA1 and CA2 of rats at 24 hour time point compared with the sham group.Importantly,L-lactate preconditioning significantly increased the immunoreactivity of PSD95,GAP43,and BDNF immunoreactive positve cells in the ipsilateral cortex and hippocampus CA1 and CA2 of rats.Conclusions:1.After TBI,SD rats showed neurological deficit behavior,which was the most serious at 24 hour time point.2.After TBI,the expression of GPR81 was increased in the ipsilateral cortex and hippocampus of SD rats.Meanwhile the expression of MCT2 and plasticity-related proteins(PSD95,GAP43,and BDNF)were decreased significantly.3.L-lactate preconditioning reduced the neurological deficit behavior of SD rats with TBI and played a neuroprotective effect,which was the best at 24 hour time point.4.L-lactate preconditioning led to further enhancement of GPR81 expression in the ipsilateral cortex and hippocampus of SD rats with TBI,while reversed the low expression of MCT2 and plasticity-related proteins(PSD95,GAP43,and BDNF),furthermore,improved synaptic plasticity and played a neuroprotective role.