The Study Of TRP Channels After Traumatic Axonal Injury In The Rat’s Optic Nerve

[Background]Traumatic brain injury（TBI）is characterized by kinds of posttraumatic injuries.Which is resulting from mechanical forces on head.Traumatic axonal injury（TAI）,which is believed to contribute significantly to morbidity and mortality of TBI patients and is common in closed head injury（CHI）,occurs when a mechanical force was imposed to the head that results in shearing force within brain tissue,inducing white matter injuried characterized by axonal injury and capillary breakage.TAI contributes to more than 35%of morbidity and mortality in TBI patients without space occupying lesions.TAI is also believed to contribute to high rate of morbidity and mortality in focal brain injury patients.The mechanism of TAI is believed that it was injuried by shear force due to the differential density beteween grey matter and white matter when the brain suffers from the motion of acceleration or deceleration.The mechanism is different from other types of brain trauma,in which the external force is direct cause on the brain tissue.There is no breakthrough in clinical diagnosis and treatment for TAI,besides,there are also rare applications in forensic science.The reason why the diagnosis of TAI remains challenging in clinical sciences and forensic science is that the physiopathologic mechanism of TAI is still unclear.Ca²⁺influx is very important to neurons,especially to apotosis.Calcium overload in the axoplasm is considered to be crucial for secondary axonal injury,besides,intracellular accumulation of free calcium in the axoplasm is considered to cause an increase in Blood-brain barrier（BBB）permeability.Although the intracellular accumulation of free calcium in the axoplasm is considered to be crucial for secondary axonal injury,the pathway of shearing force signal leading to calcium overload in the axoplasm is unclear.There are many theories explain the calcium overload in the axoplasm,but none of them can explain it completely.That means it need more researchs to take to solve the problem.Transient receptor potential channels（TRP channels）are nonspecific cationic channels,most of which are permeable for Ca²⁺,and some also for Na⁺and Mg²⁺.They are widely expressed in the CNS and peripheral cell types,and are involved in numerous fundamental cell functions.TRP channels belong to mechanosensitive channels（MS channels）,which can transform mechanical signals to electric signals and biochemical signals when it is activated by mechanical forces stimuli.TRP channels may play the bridge roles in the relationship between shearing force and calcium overload in the axoplasm.Our preliminary work revealed that the expression of Trpc1and Trpv3 genes in rat injury model brain stem were significantly increased at 72 h post-injury in comprison with control group for neurobiological ion channels and transport proteins.Based on the above theoretical basis,we presume TRP channels（including TRPC1and TRPV3）play important roles in neurons injury,especially in the pathogenesis of TAI.Therefore,we use the optic nerve stretch injury model in rat to mimic TAI,and the pathological changes in optic nerve tissue was observed.Meantime,detecting the expression of subtypes of TRP channels,and to study the rules of TRPC1 and TRPV3in the optic nerve after stretch injury may provide an important foundation for clinical treatment and forensic diagnosis.[Objectives]1.We use the optic nerve stretch model in rat to mimic TAI.The pathological changes and the expression of TRP channels protein in optic nerve tissue at different time points was observed.2.TRPC1 and TRPV3 channels nonspecific blockers,including SKF-96365 and 2-APB,are used to explore the pathogenesis and mechanism of TRP channels in TAI by.The changes of axons and capillary in rat model are observed.[Materials and Methods]1.Adult healthy male SD rats with similar genetic background were randomly divided into the control group,TAI group and intervention groups（including TAI+SKF-96365 group and TAI+2-APB group）.The rats in TAI group and intervention groups were sacrificed at 12 h,24 h,48 h,and 72 h respectively.Each group includes 7 rats.2.TAI optic nerve stretch injury model was made in TAI group and intervention groups referring to Gennarelli method.In the control group,rats underwent the surgical procedure without stretch injury.The intervention groups were divided into TAI+SKF-96365 group and TAI+2-APB group,respectively.3.The optic nerve was removed at different timepoint after perfusion with normal saline.The specimen was stored in 4%paraformaldehyde for HE stain,The specimen was embeded in OCT for immunofluorescent staining,and the other was stored in sterile cryotubes for western blot.4.The expression of NF-L and the number of axonal retraction balls（ARBs）were detected by HE stain or/and immunofluorescent staining,to estimate the severity of axonal injury.The changes in morphology and expression of TRPC1 and TRPV3 were observed by immunofluorescent staining.The concentration of TRPC1 and TRPV3were detected by Western blotting.[Results]1.Pathological changes in rat optic injury of TAI experimental group are consistent with TAI or DAI morphology,axons became circuitous and enlarged and retraction balls are observed.HE stained and immunofluorescence staining showed that the scope and number of injury axons were increased.No NF-L-stained axons were found in the sham-operated group.2.Immunofluorescence staining showed that the number of NF-L positive axons were decreased in TAI intervention groups（including SKF-96365 group and 2-APB group）at the same time interval compared with TAI group.Besides,DMSO control groups（including SKF-96365 control group and 2-APB control group）were similar to TAI group.3.The expression of TRPC1 and TRPV3 in TAI group were significantly increased,and TRPC1 reached the peak at 48 h post-injury.4.Immunofluorescence staining showed that the number of TRPC1 positive expression were decreased in TAI intervention groups（including SKF-96365 group and2-APB group）at the same time interval compared with TAI group.Furthermore,the expression of TRPC1 in 2-APB intervention group was less than in SKF-96365intervention group,but the expression of TRPV3 was opposite.5.The result of WB are similar to immunofluorescence staining.[Conclusion]1.The expression of TRPC1 and TRPV3 channels proteins were upregulated after TAI.2.TRPC1 and TRPV3 channels may participate in the traumatic axonal injury.TRP channels blockers SKF-96365 and 2-APB alleviate the TAI damage.