The Role Of Integrin-focal Adhesion Complex And TRPC Channels On Neuron Injury By Stretch Forces

Background and Objective Diffuse axonal injury（DAI）is a kind of diffuse and closed brain injury that occurs after traumatic brain injury,which is mainly manifested as axonal injury.Due to the lack of early diagnostic criteria and the absence of specific treatments in DAI,most patients are usually in severe condition with a high rate of mortality and a poor prognosis.Moreover,DAI is easy to misdiagnosed in forensic practices because of its morphological characteristics.Therefore,exploration of the pathophysiology mechanism of DAI is of great significance for clinical and forensic practices.At present,it is widely accepted that rotational acceleration and/or angular acceleration was produced after head injury,which makes the brain tissue suffer from shear forces,causing a series of biochemical changes and eventually the destruction of the cytoskeleton,the damage of axons and the loss of neurons.However,axonal injury is a delayed response and not directly caused by external forces,which is resulted from complex biochemical changes and the transduction between the mechanical signal and the biochemical signal.Studies have shown that there are two major pathways of mechanical signal transduction.One is the mechanical-sensitive receptor pathway,in which integrins have attracted great attention,and the other is the mechanosensitive channel pathway.Integrins are a cluster of mechanical-sensitive receptors and belong to the family of adhesion factors,which are present in all tissues and could transmit mechanical signals from the extracellular matrix（ECM）into the cell.Transient Receptor Potential Canonical（TRPC）is one of mechanosensitive channel and is related to mechanical transduction.It is widely expressed in the nervous system,which could affect the concentration of Ca²⁺ in neurons.However,the role of integrin and TRPC in DAI remains unclear.The aims of this study were:（1）to clarify the expression of integrins in neuron and to elucidate the role of integrin in the mechanism of cytoskeletal injury after DAI;（2）to clarify the role of the TRPC channel in neuronal injury caused by mechanical force.Methods 1.Part 1（1）Establish DAI in vitro model by stretching the primary cortical neurons: SD mouse cortical neurons were cultured,and the neurons were in good state on the seventh day,and it was randomly divided into control group,injury group and RGD peptide（integrin antagonist）intervention group.The control group was untreated,and the RGD peptide intervention group was cultured with RGD peptide before force loading.Then,the stretching force is administered in the injury group and RGD peptide intervention group.At last,the neuronal morphology was observed.（2）Immunofluorescence was used to observe the expression of integrin β1 and FAK in neurons.（3）Western blot was used to detect the expression of integrin and FAK.（4）Immunofluorescence was used to observe the cytoskeleton after DAI.2.Part 2（1）The methods to establish the DAI model were same as that in part1.It was randomly divided into control group,injury group and SKF（TRPC channel blocker）intervention group.The control group was untreated,and the SKF intervention group was cultured with SKF intervention before force loading.Then,the stretching force is administered in the injury group and SKF intervention group.Finally,concentration of Ca²⁺ was detected.（2）Immunofluorescence was used to detect the co-localization of NF-L and Ca MKII.（3）Flow cytometry was used to detect neuronal apoptosis after injury.Results 1.Part1（1）The parameters of the DAI model by force loading were set as follows: the deformation distance was unified to 10 mm,and the deformation time was 1.5s.It is found that the neuronal axon is distorted and even seemed as "S" shape,and the formation of the axonal retraction ball can be seen,accompanied by neuronal death.The number of axons is reduced,and the morphology is unclear.（2）Integrin β1 and FAK were expressed in the neurons abundantly,which can be used for subsequent experiments.（3）Compared with the control group,the expression of FAK in the injury group was upregulated（p<0.05）after force loading while the expression of integrin β1 was not changed.（4）Compared with the control group,the NF-L fluorescence intensity of neurons in the injury group was enhanced,while the NF-L fluorescence intensity in the RGD intervention group was weaker than that in the injury group;The fluorescence intensity of α-tubulin in injury group was weaker than that in the control group,but the α-tubulin fluorescence intensity in the RGD intervention group was enhanced compared with that in the injury group.2.Part 2（1）An increase of calcium concentrations（p<0.05）was detected after force loading,but intracellular calcium concentrations in neurons with SKF intervention was decreased.（2）After injury,the NF-L fluorescence intensity in the injury group was enhanced compared with that in the control group,while the NF-L fluorescence intensity in the SKF intervention group was weaker than that in the injury group.After injury,the Mander’s overlap coefficient between NF-L and Ca MKII continued to decrease,and the Mander’s overlap coefficient after SKF intervention was significantly higher than that in the injury group.（3）In terms of apoptotic cells,compared with the control group,the proportion of apoptosis after SKF intervention decreased overall,especially in the post-injury 12 hours,but there was no significant effect on living cells.Conclusions 1.Part 1: The expression of Integrin β1 does not change significantly,but the expression of FAK is upregulated,which is the main downstream molecules of integrin,thereby mediating the destruction of cytoskeleton after diffuse axonal injury.2.Part 2: TRPC channel are activated after DAI and ca²⁺/CaMKII mediates the destruction of cytoskeletons and neuron apoptosis.