The Role Of KCC2in Traumatic Brain Injury In Rats And The Neuroprotective Mechanisms Of Melatonin

Background and PurposeTraumatic brain injury (TBI) is a form of brain injury that leading to significant morbidity and mortality in the worldwide. The toxicity of excitatory amino acids is considered as an important factor of initial brain damage, which affects the prognosis of TBI patients. The neuronal-specific K+-Cl-co-transporter2(KCC2), a key molecule in maintaining neuronal Cl" homeostasis, plays an important role in modulating GABA-mediated inhibition in mature neurons. However, the expression pattern and pathophysiological role of KCC2in TBI-induced cortical neuronal injury remains unknown. On the other side, melatonin (Mel), which is primarily synthesized and secreted by the pineal gland during darkness in a normal diurnal cycle, exerts multiple neuroprotective effects, including anti-oxidant and anti-inflammatory properties. In the TBI models, Mel can alleviate head trauma-induced cell death. But its effect and underlying molecular mechanisms on neuronal cell death following TBI are less elucidated.This study aimed to explore whether KCC2downregulation is involved in the pathogenesis of secondary injury of TBI in a controlled cortical impact injury (CCI) model. In addition, we aimed to testify Mel could provide neuroprotective effect for TBI and investigate whether those neuroprotection is associated with its anti-apoptotic effect of neuron via activation of brain derived neurotrophic factor (BDNF)/extracellular regulated protein kinases (ERK)/KCC2signaling pathway.MethodsTwo separate experiments were conducted (Experiments1and2).Experiment1:The dynamic expression and distribution of KCC2in the injured cortex after TBI were investigated. After controlled cortical impact (CCI) was performed, the rats were randomly divided into7groups (0,3,6,12,24,72, and168h). The ipsilateral cerebral cortex (injured) was collected, and the temporal expression of KCC2protein was detected by Western Blot; According to different time after TBI (0,3,6,12,24,72, and168h), the ipsilateral cerebral cortex (injured) was collected, and the temporal expression of KCC2mRNA was analyzed by fluorescence quantitative PCR; Immunofluorescence technique are used to examine the distribution and localization of KCC2.Experiment2:The study aims to test the anti-apoptotic effect of Mel of cortical neuron following TBI. Additionally, this study explores whether activation of BDNF/ERK/KCC2signaling pathway is associated with Mel-mediated anti-apoptotic effects after TBI. The rats were randomly divided into sham+vehicle, TBI+vehicle, and SAH+melatonin (Mel) groups. The animals were intraperitoneally treated with Mel (10mg/kg) or vehicle at5min,1h,2h,3h,4h after injury. After24h of TBI, F JB staining was performed for evaluating the degenerated neuron density in injured cerebral cortex, and expression of cortical BDNF、ERK、p-ERK、KCC2and cleaved caspase-3in all groups were assayed. Neuronal apoptosis was examined by double immunofluorescence staining using TUNEL and NeuN; And the amount of brain water content and modified neurological severity score were recorded after24h of TBI.Results(1) Western Blot showed that KCC2protein levels decreased in ipsilateral cerebral cortex following TBI, which reached a statistically significant difference from12h group to168h group when compared with Oh group; Fluorescence quantitative PCR showed that KCC2mRNA levels decreased in ipsilateral cerebral cortex following TBI, which reached a statistically significant difference from6h group to168h group when compared with Oh group; Immunofluorescence double-staining demonstrated that KCC2was co-located with NeuN but not GFAP, indicated KCC2was expressed in neurons. In addition, KCC2has a distribution in the plasma membrane of neurons.(2) Compared with the sham+vehicle group, the TBI+vehicle group animals have significantly decreased expressions of BDNF、p-ERK、KCC2at24h after TBI. In addition, TBI+vehicle group animals showed increased degenerated neuron, neuron apoptosis, brain edema and neurological function deficit. Treatment with melatonin attenuated the downregulation of BDNF、p-ERK、KCC2, but inhibited the upregulation of cleaved caspase-3, and reduced neuronal apoptosis, brain edema, and improved neurologic outcomes.ConclusionOur work demonstrated that both protein and mRNA levels in the ipsilateral cerebral cortex are decreased following TBI, suggesting its pathogenic role in secondary brain injury after TBI. In addition, KCC2downregulation may involve in cortical neuronal apoptosis after TBI, while Mel treatment could restore KCC2expression, attenuate neuronal apoptosis, reduce brain edema and alleviate neurological deficit after TBI through activation of BDNF/ERK pathway.