Effects Of Oxidative Stress On Neuritin,Peripheral Nerve Function And Ultrastructure In Rats With Diabetes

Objective:1.To explore effects of oxidative stress on neuritin level of blood circulation and nerve tissue in diabetic rats.2.To investigate the possible relationship between neuritin level of blood circulation and nerve tissue and nerve functionMethods:1.The Sprague Dawley(SD)male rats were randomly divided into 10 normal control rats(NC)and 30 STZ induced diabetic rats,which were further divided into the diabetes control(DC)and the lipoic acid treated diabetes(LD,20mg?kg-1?d-1 or HD,40mg?kg-1?d-1,intraperitoneally).2.The left sciatic nerve was stimulated proximally at the sciatic notch and distally at the ankle via bipolar electrodes with supramaximal stimuli(8 V)at 20 Hz.The latencies of the compound muscle action potentials were recorded via bipolar electrodes from the first interosseous muscle of the hindpaw and measured from the stimulus artifact to the onset of the negative M-wave deflection.MNCV was calculated by subtracting the distal latency from the proximal latency,and the result was divided into the distance between the stimulating and recording electrode.3.The caudal NCV(CNCV)was measured at a fixed distance,with the stimulating cathode positioned 120 mm distally to the tail hairline and the active electrode located100 mm proximal to the cathode.Neural responses were induced by stimulation at a frequency of 5 Hz.Rectal temperature was maintained at 37?C.4.A short sciatic nerve was pre-fixed with glutaraldehyde solution,after fixation, embedding,conventional producers,and double staining with uranyl acetate and lead citrate,we observed the nerve with Tecnai transmission electron microscope.5.The tail tip blood of rats was determined with FAD blood glucose dehydrogenase method,the content of MDA was detected with TBA method,SOD activity was assessed with dehydrogenase method,and neuritin was measured using ELISA kit.6.The samples(80?g protein/lane)were separated by SDS-PAGE using 15%acrylamide gels.Nonspecific binding was blocked by incubation in Tris-buffered saline containing 5% BSA for 1 h at room temperature and then incubated overnight at 4°C with anti-actin,anti-neuritin(1:200,Santa Cruz).Blots were washed and then incubated in horseradish peroxidase–conjugated anti-goat Ig G for 2h at room temperature.Protein bands were visualized using an Enhanced Chemiluminescence kit and quantified using image Lab image analysis software.7.The paraffin blocks of sciatic nerves and DRGs were cut into 5 um sections.Sections were deparaffinized using standard procedures.Briefly,sections were treated with 0.01 M PBS for antigen retrieval,blocked with 1% bovine serum albumin(Sigma-Aldrich)in PBS containing 0.05% Tween-20(Promega),and consecutively incubated with primary and secondary antibodies diluted in blocking solution.Nuclei were stained with 49,6-diamidino-2-phenylindole(DAPI).Result:1.The blood glucose levels of diabetic rats was significantly higher than NC,but there was no significant difference between DC and LD.2.Compared with NC,SOD and neuritin levels were decreased,MDA levels were increased in diabetes group in 2,4,6 weeks,respectively(P<0.05).DC showed slower MNCV and CNCV(P<0.05),and severer axonal degeneration and demyelination of sciatic nerves in 4,6 weeks.In contrast,LD had higher levels of SOD,neuritin,MNCV and CNCV,and lower MDA levels(P<0.05).3.In comparision with DC,the expression of neuritin in sciatic nerves and dorsal root ganglions was higher in NC(P < 0.05);the diabetic rats of 2,4,6weeks showed a falling in neuritin expression(P < 0.05).Conclusion:1.There was severe oxidative stress and falling levels of neuritin in rats with diabetes,these changes existed before the functional and structural abnormalities of sciatic nerves.2.Lipoic acid could improve oxidative stress,and there was a dose-effect relationship between them.3.Oxidative stress directly induced the abnormalities of sciatic nerves or via affecting neuritin levels.