Essential Role Of TRPC6 In Regulating Diffuse Axonal Injury Through Ca²⁺/CaMKⅡβ Pathway

SubjectDiffuse axonal injury（DAI）is a kind of serious traumatic brain injury（TBI）caused by shear force during head rotational and accelerated movement,which results in the rupture of axon and blood vessels.Up to now,there is no special diagnostic method and treatment.Most of patients suffer from DAI have bad prognosis with mortality over 40%.The traditional treatment includes Dehydration agents,free radical scavengers,craniotomy,complications prevention and treatment of neurotrophic drugs,etc.Chief among them is drug therapy.Therefore,it is essential to understand the mechanism of DAI in early pathophysiological process,and to find a better effective and safe therapy.Calcium overload is one of the recognized mechanisms for the occurrence and development of DAI.TRPC6 widely expresses in nervous system,which is one of non-selective cation channels,involves or not calcium regulation remains to be studied.This study aims to observe the expression changes of TRPC6 in mechanical force-induced neuronal injury,and elucidate its function in molecular mechanism of DAI,and to investigate whether TRPC6 inhibitors have neuroprotective effects during DAI to provide theoretical and experimental basis for the treatment of DAI.Methods1.Animal model for DAI:Male SD（Sprague-Dawley）rats,weighted 300±20g,were randomly divided into sham operation group（Sham）,SKF group（SKF）,DAI group（Force）and treatment group（Force+SKF）.DAI model was made by linear acceleration load according to Marmorou method.The rat in Sham group and the SKF group were separately intraperitoneal injected with 35 mg/kg saline and 5 mg/kg SKF.The treatment group were given 5 mg/kg SKF 2 hours before hitting.The nerve function of each group was scored daily.2.Cellular model for mechanical injury:Primary cortical neurons were randomly divided into control group（Control,Cnt）,GsMTx-4 group（GsMTx-4）,mechanical force group（Force）and GsMTx-4 treatment group（Force+GsMTx-4）.).The Force group neurons were cultured with normal neuronal culture for the six day before applying mechanical force with specific force device.In the Force+GsMTx-4 group,the culture medium containing GsMTx-4（1 m M）was replaced on the sixth day,and the mechanical force was performed 6 hours later.The neuron in each group was observed daily and photographed.3.TRPC6 was detected by Western Blot and IF both in animal model and the primary neuron.4.The cytoplasmic Ca²⁺concentration was detected by laser confocal microscopy in the ninth day of neuronal cell culture,for observing the changes of cytoplasmic Ca²⁺concentration after the neuron was injured by mechanical force.5.The expression levels of CaMKⅡβand p-CaMKⅡβprotein in neuron for cellular model were detected by Western blot after pretreatment with TRPC6.6.Morphological changes were observed by immunofluorescent staining ofα-Tubulin for the cellular model.7.24 hours after the success of animal model,brain tissue was taken during rat was anesthetized by chloral hydrate for Nissel’s staining.Neuron loss in cerebral cortex and hippocampus region was observed.Result1.The expression of TRPC6 protein detected with Western blot in the Force group（both animal and cell）was higher than that in the Sham/Control group.2.Immunofluorescent detection showed TRPC6 protein was mainly expressed in the cytomembrane and cytoplasm,and the colocalization area of TRPC6 andα-Tubulin increased in the Force group.3.The intracellular cytoplasmic calcium concentration increased after the mechanical force was applied on the neuron,which can be inhibited by TRPC6,showed injury of neuron was related with TRPC6 channel activation.4.The levels of CaMKⅡβand p-CaMKⅡβin primary cortical neurons detected with western blot revealed that Ca²⁺/CaMKⅡβchannel was activated when DAI happens.TRPC6 channel inhibitors significantly down-regulated the protein expression of CaMKⅡβand p-CaMKⅡβ.5.Morphological changes in Control group,Force group,GsMTx-4 group and Force+GsMTx-4 were observed by neuron tracer,and the number of neurite was compared.The neuron curled,thickened and even ruptured under light microscope30min after mechanical force was applied on it.With the time,the unbroken axons gradually return to normal.Immunofluorescence staining withα-Tubulin showed the axonal rupture in the Force group was more than that in the GsMTx-4 treatment group,and number of neurite was significantly reduced.The number of neurite was more in GsMTx-4 treatment group than that in the Force group.6.Nissl staining showed the neurons in the cortex in the Force group were irregularly distributed,with the cytoplasm shrinks and nuclear pyknosis.The loss of neuron in the Force+SKF group was significantly decreased.There was no significant difference in neuronal survival between the Sham group and the SKF group.7.The m NSS neurological function score revealed that SKF can reduce the m NSS score and improve the neurological function at an early stage compared with the Force group.Neurological function of rats in the Sham group and the SKF group was normal.ConclusionThe expression of TRPC6 increases in rat’s neuron when DAI happens.TRPC6 plays a role in the regulation of calcium ions which involve in the development of DAI.TRPC6 takes part in occurrence and development of DAI by activating Ca²⁺/CaMKⅡβ.