The Role Of Microtubule Protectant Epothilone D In The Process Of Nerve Injury And Repair

Objective:TON is the main cause of vision loss in traumatic injury.It mainly manifests as edema of nerve tissue and microcirculatory disturbance.However,there is no uniform clinical diagnosis and treatment standard for TON treatment.If the treatment is not timely,serious blindness can be caused.The axon changes in TON are mainly the process of the further apoptosis of neurons caused by the disintegration of microtubule microwires.In recent years,a large number of literatures have reported that the microtubule-associated protein,Tau protein,has stable and promoting microtubule polymerization.This function is mainly regulated by the phosphorylation level of Tau.Tau phosphorylation has long been considered as one of the pathogenesis of neurodegenerative diseases such as AD and PD.The literature shows that the use of microtubule stabilizer epothilone in Tau transgenic mice can improve cognitive impairment and reduce Tau lesions in mice.In this paper,the microtubule stabilizer epothilone was first applied to the optic nerve to establish the traumatic optic neuropathy model in rats.It was studied whether the administration of epothilone after TON would result in the repair and regeneration of optic nerve axons and retinal ganglion cells.Observe whether there will be improvement of the optic nerve conduction pathway and changes in Tau protein phosphorylation.Provide reference for the treatment of TON and other neurodegenerative diseases.Methods:Animal model of traumatic optic neuropathy was established by clamping the optic nerve with 50 g force.24 adult female Sprague-Dawley rats were randomly divided into experimental group(n=12)and control group(n=12).In the experimental group,epothilone was intraperitoneally injected with 1 mg/kg of epothilone,and the left eye's optic nerve clamped the injury model.The right eye separated the optic nerve without clamping.The control group received the same volume of solvent DMSO intraperitoneally,and the left optic nerve clamped the injury model.The right eye separates the optic nerve and cannot hold it.The experimental group and the control group were obtained at 3 D and 7 D at the time points after the injury,and there were 6 rats at each time point.Western blot experiment(n=3)observed the changes of Tau protein expression and phosphorylation of retina and optic nerve at the corresponding time points after administration of epothilone and DMSO respectively;observed by immunofluorescence histochemical staining(n=3)Changes of optic nerve axon regeneration and changes of retinal ganglion cells(RGCs)after optic nerve injury in rats and at different time points;changes in optic nerve microtubules were observed by immunofluorescence staining(n=3)Axon regeneration was co-expressed.At the same time,10 other rats in the experimental group were simultaneously established with the FVEP detection model.Changes in FVEP were detected at 1D and 28D time points,respectively.Sixty adult female Sprague-Dawley Sprague-Dawley rats were randomly divided into two groups,each group(n=30),and 30 rats in group one were treated with 50 g force-shaving optic nerve on the same day to establish a rat model of optic nerve clamping injury.In 1D,3D,7D,14D,28D,6 samples were taken at each time point.Western blot experiment(n=6)was used to observe the changes of Tau protein expression and phosphorylation in the retina and optic nerve after injury.In the second group of 30 SD rats,a gripping model was established by the same method as in the appeal.The ID,3D,7D,14D,and 28D were performed on the Roland electrophysiological apparatus at the Ophthalmology Clinic of the Second Affiliated Hospital of Kunming Medical University for FVEP detection.The SD rats were observed.The optic nerve conduction pathway changes at different time points after optic nerve injury.Results:Western blot showed that the content of Tau protein in the optic nerve of TON continued to decrease and then returned to normal.The change of Tau protein 396/404 phosphorylation in the posterior optic nerve of TON was consistent with the change of Tau protein content;the optic nerve Tau protein 396 at 14 D after TON.The degree of phosphorylation at the/404 site increased;after TON,the Tau protein content decreased rapidly and then increased;after TON,the change of Tau protein 404 phosphorylation was similar to that of Tau protein,but the 396 site was different;The role of retinal Tau protein 396/404 sites is different in space-time.Tau protein content of optic nerve continued to decrease after epothilone treatment of TON;Tau phosphorylation of optic nerve increased after epothilone treatment of TON;Tau protein content of retina increased after epothilone treatment of TON;retinal Tau of epothilone after TON treatment The phosphorylation of-396 phosphorous increased continuously,and the phosphorylation of 404 increased transiently and then decreased.Immunofluorescence histochemical staining showed that there was a significant difference in the proportion of RGCs between the 3D group and the solvent control group(P<0.05),and there was a statistically significant difference in the ratio of RGC between the 7D group and the solvent group(P<0.05).P<0.05).The nucleus increased near the pinch point in the optic nerve of the TON injury eye,and the increased nuclei spread to the distal end of the clamping site;the nerve-specific ?-3 Tublin suggested that the same hole appeared away from the clamping site,and the residual ?-3 Tublin Irregular form,showing a kink;TON after dosing in the optic nerve of the eye,although the same phenomenon of nuclear increase,but only limited to the clamping site,does not spread to the distal end of the clamping site,nerve-specific P-3 Tublin staining was not found in the distal part of the clamping site,and the intact ?-3 Tublin morphology was similar to the normal eye;in the solvent control group,it was difficult to detect the expression of GAP43 in the optic nerve;in the treatment group,A large amount of GAP43 expression was detected in the clamped lesions.In the solvent control group,the expression of GAP43 decreased and it was difficult to co-localize with ?-3 Tublin.The expression of GAP43 and ?-3 Tublin increased in the treated group,and the expression of GAP43 increased.There are two kinds of GAP43 expression patterns in the axon regeneration of the optic nerve:nuclear localization and expression along with ?-3 Tublin fibrillation;and nuclear localization of GAP43 for astrocyte expression through optic nerve astrocytes and GAP43 Co-staining revealed that GAP43 was co-expressed with GFAP-labeled astrocytes,confirming that part of GAP43 originated from astrocytes.Roland electrophysiological showed that typical FEVP waveforms appeared in the sham eye,indicating that exposure of the optic nerve alone does not affect the FVEP's various indicators;the surgical eye's FVEP showed a clear waveform change,which was manifested as N1 wave delay.The amplitude of P1-N1 wave was reduced.Compared with sham-operated eyes,the FVEP N1 peak in the surgical eye had significant delay at each measurement time point(P<0.01).The average delay time was 14.34 ms;the surgical eye was sham-operated The amplitude difference of FVEP P1-N1 in the eyes was significantly smaller at each measurement time point(P<0.01),and the average increase was reduced by-40.30 ?s;during the development of TON injury,the surgical eye was compared with the FVEP P1-of the sham eye.N1 amplitude reduction did not significantly improve or aggravate at different time points,while N1 delayed degrees of mild natural remission occurred during 3D-7D(P<0.05).After TON administration of epothilone D,there was an improvement in the optic nerve conduction pathway in FVEP.Conclusion:1.After Animal model of traumatic optic neuropathy,the visual electrical signal detected by the FVEP can be slowed down and the quality of the electrical signal is reduced.After the optic nerve is clamped,the visual electrical signal transmission pathway may be hindered.2.Rat TON model can cause the decrease of free Tau protein content and increase of phosphorylation level in optic nerve;RGC cell body in the retina undergoes sustained free Tau depletion process after injury and Tau is not detected in the intracellular body.Phosphorylation.3.After TON given epothilone D,the optic nerve pathway was improved;4.After TON,epothilone D inhibited the apoptosis of retinal ganglion cells;After TON,epothilone D was given,both optic nerve and retina appeared.Axonal regeneration;GAP43 in the optic nerve indicating regeneration near the nucleus and co-localization with GFAP,GAP43 protein providing axonal growth may be derived from astrocytes 5.TON after administration of microtubule stabilizer epothilone D,the level of free Tau protein in the optic nerve decreased,and the free Tau protein level in the retina increased;but the phosphorylation levels of Tau-396/404 in both sites were enhanced to different degrees.