Autophagy Promotes Peripheral Nerve Regeneration And Motor Recovery Following Sciatic Nerve Crush Injury In Rats

Background:Peripheral nerve injury(PNI)is one of a common trauma,although it can be recover of function through regeneration and reconstruction.After Peripheral nerve injury,degeneration of axons appear fracture,nerve conduction interruption,demyelinating diseases etc.Accompanied with the nerve edema,a series of pathological change was observed in the distal,including Wallerian degeneration.It brings great suffering and economic burden to patients.Rapamycin is a fungal compound approved by the food and Drug Administration Americanused as immunity inhibitor,it can enhance the function of autophagy by inhibiting mTORl cell signaling pathway protein.It plays a neuroprotective role in many nervous system diseases that Rapamycin activate autophagy function.In recent years,rapamycin is widely used in the studies of nervous system diseases.The research shows that in the traumatic brain injury model,the mTOR inhibitor rapamycin can enhance autophagy,reduce the central nervous system inflammatory response,promote the recovery of the central nervous system function.Application of rapamycin in Huntington,Parkinson and other neurodegenerative diseases shows that it can enhance the function of autophagy and protect nerve cells.Autophagy is a universal phenomenon in eukaryotes,through the lysosomal degradation of cytoplasmic components,it's main function is to remove the cells within the abnormal proteins and damaged organelles,plays an important role in cell reconstruction.It is one of the hot research field in life science in recent years.Research shows that autophagy is related with the physiological and pathological process of many diseases(including infection,cancer,neurodegeneration and trauma).Among them,the autophagy dysfunction is an important cause of occurrence and development of neurodegenerative diseases and tumor.The nerve injury models(including brain injury and spinal cord injury)study shows that autophagy can reduce the damage of oxidative stress.In the conditions of disease and injury,autophagy can provide the necessary energy for cells,maintaining cell survival.Peripheral nerve fibers mainly formed by axons and dendrites which issued by glial cells and neurons of the CNS.At present,the regulatory roles in peripheral nerve system of autophagy is not yet clear.The peripheral nervous system(PNS)and central nervous system(CNS)form the body nervous system in uniform,and they have much in common.oligodendrocyte of CNS and Schwann cells of peripheral nervous system encapsulated nervous constitute myelin,regulating fast nerve conduction of myelinated fiber.Among them,central nervous system disease and peripheral neuropathy directly cause nerve damage immediately,accompanied with the lost of axons and dendrite,result of the contact of nervous damaged.Autophagy may also play a neuroprotective role in the condition of peripheral nerve injury stress,so as to promote the recovery of nerve regeneration and it's function.Objective:Autophagy maintains cellular homeostasis by stimulating the lysosomal degradation of cytoplasmic structures,including damaged organelles and dysfunctional proteins.The role of autophagy in the renewal and regeneration of injured peripheral nerves remains poorly understood.The current study investigated the role of autophagy in peripheral nerve regeneration and motor function recovery following sciatic nerve crush injury in rats by stimulating or suppressing autophagy and detecting the presence of autophagosomes and LC3-II expression by electron microscopy and western blotting,respectively.Neurobehavioral function was tested by Cat Walk gait analysis 1,2,3,and 6 weeks after injury,and the expression of neurofilament(NF)-200 and myelin basic protein(MBP)at the injury site was examined by immunocytochemistry.Schwann cell(SC)apoptosis at the lesion site was determined by the terminal deoxynucleotidyl transferase dUTP nick end labeling assay.Methods:1.Animals and surgical procedures All protocols were approved by the animal experimental ethics committee of Southern Medical University.Adult female Sprague-Dawley rats weighing 180-220 g were purchased from the Laboratory Animal Centre at the Southern Medical University.Rats were subjected to sciatic NCI as previously described.Briefly,rats were deeply anesthetized with sodium pentobarbital(50 mg/kg body weight by intraperitoneal injection),and the sciatic nerve in the right mid-thigh was exposed and clamped with a pair of forceps three times for 10 s each at 10-s intervals.Animals were housed in controlled conditions on a 12:12 h light/dark cycle,with free access to food and water.2.Drug treatment Animals were divided into four groups:sham + vehicle(v),crush + v,crush+ rapamycin(rapa),and crush + 3-methyladenine(MA).Rapamycin and 3-MA were obtained from Sigma-Aldrich(St.Louis,MO,USA)and dissolved in 50%dimethyl sulfoxide(DMSO).Animals received daily intraperitoneal injections of rapamycin at a dose of 1 mg/kg(crush + rapa group),3-MA at a dose of 50 mg/kg(crush + 3-MA group),or DMSO(crush + v group)for 5 days after the surgery.Sham-operated rats(sham + v group),in which the sciatic nerve was exposed but not subjected to crush injury,received daily intraperitoneal injections of an equal volume 50%DMSO for 5 days.Drug dosages were selected based on data from previous studies and preliminary experiments.3.Tissue preparation Sciatic nerves were dissected and harvested 1 and 6 weeks post-injury(n?6 for each time point and group).Animals were decapitated and perfused via the left ventricle with cold saline followed by 4%paraformaldehyde in 0.01 M phosphate-buffered saline(PBS;pH 7.35).The injured sciatic nerve was then removed and frozen at-80? for histological or western blot analysis.4.Immunocytochemistry Transverse frozen sections(10?m thickness)of sciatic nerve were dried,mounted onto slides,and washed in 0.1 M PBS,then blocked in 10%goat serum and 3%bovine serum albumin(BSA)for 1 h at 37?.After two washes with PBS,the slides were incubated with primary antibodies against MBP(1:1000;Sigma),NF-200(1:500;Sigma),or LC3B-II(1:500;Cell Signaling Technology,Danvers,MA,USA)at 4? for 12 h,followed by repeated washes with PBS,and then incubated with Alexa 488-or Cy3-conjugated secondary antibodies(1:1000;Invitrogen,Shanghai,China)for 2 h at room temperature.Sections were visualized under an epifluorescence microscope and imaged at 40×magnification.5.Western blot analysis LC3-II protein expression at the lesion site following sciatic NCI was analyzed by western blotting.Nerve tissue samples were homogenized in tissue lysis buffer(20 mM Tris-HCl,pH 7.5;150 mM NaCl;1%Triton X-100;25 mM NaPPi;80 mM[3-glycerophosphate;2 mM EDTA;0.2 mM Na3V04;and protease inhibitor cocktail)and centrifuged at 12,000 rpm for 15 min at 4?.The protein concentration in the supernatant was measured with a bicinchoninic assay kit(Beyotime Institute of Biotechnology,Shanghai,China),and the supernatant was mixed with gel loading buffer(50 mM Tris-HCl;10%sodium dodecyl sulfate;10%glycerol;10%2-mercaptoethanol;and 2 mg/ml bromophenol blue)at a 1:1 ratio and boiled for 5 min.A total of 20?g of each sample was separated on a 15%acrylamide gel and transferred to a nitrocellulose membrane(BioScience,Shanghai,China)by electroblotting(Bio-Rad,Hercules,California,USA)at 120 V for 1.5 h followed by 70 V for 0.5 h at 4?.Membranes were blocked with 3%BSA and incubated with rabbit anti-LC3B-II antibody(1:000)overnight at 4?,followed by incubation with an anti-rabbit secondary antibody(1:2000;Biosynthesis Biotechnology Co.Ltd.,Beijing,China)for 1.5 h at room temperature.The blots were visualized using Scanmaker 3836(Microtek Technology Co.Ltd.,Shanghai,China)and quantified with Quantity One software(Bio-Rad).The expression level of LC3-II was determined by calculating band intensity relative to ?-actin.6.Electron microscopy Sciatic nerve segments were removed 1 week after NCI and fixed with 2.5%glutaraldehyde overnight at 4?.The segments were cut with a sharp razor blade into serial 1-mm transverse sections around the site of injury,which were postfixed in 1%osmium tetroxide for 1 h at 4?,rinsed in PBS,dehydrated in a graded series of alcohol and propylene oxide,and embedded in Epon.Blocks showing a predominantly transverse orientation of the injured sciatic nerve were selected from toluidine blue-stained thick sections.Ultrathin(70-nm)sections were prepared on an ultramicrotome(Ultracut R,Leica,Heerbrugg,Switzerland)with a diamond knife,stained with uranyl acetate and lead citrate,and visualized using an a JEM-1010 electron microscope(Jeol,Tokyo,Japan)at 1500×or 30,000×magnification.7.Terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)DNA fragmentation resulting from cell death was detected in transverse frozen sections(10?m)of sciatic nerves at the lesion site by TUNEL staining using the TUNEL apoptosis assay kit(Beyotime Institute of Biotechnology)according to the manufacturer's protocol 1 week post-injury.Samples were counterstained with DAPI and imaged at 400× magnification and the number of TUNEL-positive cells in each section were counted.8.Behavioral analysis Experimenters performing the behavioral tests were blinded to the drug treatment conditions.Motor function was determined by stand time and footprint intensity using the CatWalk system(Noldus Inc.,Wageningen,the Netherlands).Animals crossed a walkway with an illuminated glass floor,and a GP-3360 high speed video camera(Gevicam,Milpitas,CA,USA)equipped with an 8.5-mm wide-angle lens(Fujicon Corp.,Shenzhen,China)positioned underneath the walkway automatically recorded paw prints as the animals moved across.The test was performed 1,2,3,and 6 weeks after injury.Statistical analysisData are presented as the mean±SEM.Comparisons within groups were made by one-way analysis of variance(ANOVA),and between-groups comparisons were made by one-way ANOVA and,if significant,were followed by discrete comparisons using a post hoc test with a Bonferroni correction for multiple comparisons.A P value<0.05 was considered statistically significant.All analyses were performed using GraphPad Prism 5 software(GraphPad Software,Inc.,San Diego,CA,USA).Results:1.Autophagy is enhanced by rapamycin and suppressed by 3-MA at the early stages of PNIAutophagosomes were visualized by electron microscopy 1 week after sciatic NCI.A greater number of autophagosomes were present in the rapamycin-treated(Fig.1C)and crush + v groups(Fig.1B);the numbers were 8-and 3-fold higher,respectively,than in the sham(Fig.1A)group and were also higher than in the 3-MA-treated group(Fig.ID).These data are consistent with an increase of autophagy after NCI.Western blot(Fig.2B)and immunocytochemical(Fig.2A)analyses of the expression of the autophagy marker LC3-? revealed higher protein levels in rapamycin-treated animals,and lower levels in the 3-MA group(P<0.05)compared to controls.These results indicate that autophagy is enhanced by rapamycin and suppressed by 3-MA in the early stages after sciatic NCI.2.Autophagy promotes motor function recovery following PNIThe recovery of motor function was assessed using the CatWalk system.The stand time and footprint intensity of the operated limbs were used to analyze motor function 1,2,3,and 6 weeks post-injury(Fig.3E,F).The mean stand time in the rapamycin-treated group was 2-fold higher than in the crush + v group at 1 and 2 weeks post-injury(P<0.05),while the mean footprint intensity was comparable between the two groups at 2 weeks.These results indicate that animals treated with rapamycin were better able to support their body weight on injured limbs starting 2 weeks after injury,possibly as a result of the enhanced autophagy at early stages.In contrast,in the 3-MA group,the mean stand time was significantly lower 1 and 2 weeks post-injury(45%and 15%,respectively),while the mean footprint intensity was about 20%-30%lower at 1,2,and 3 weeks post-injury as compared to sham-operated animals(P<0.05).3.Autophagy promotes axonal regeneration and SC remyelinationMBP is a major constituent of the myelin sheath produced by SCs in the peripheral nervous system,while the expression of NF-200—an axon-specific intermediate filament—is critical for axon stabilization during their maturation.To investigate the effects of autophagy on nerve regeneration,the expression of MBP and NF-200 was evaluated by immunocytochemistry.At 1 week post-injury,at the peak of nerve regeneration,MBP and NF-200 immunoreactivity was 1.4-and 2-fold higher,respectively,in rats treated with rapamycin as compared to those treated with 3-MA or were subjected to injury but untreated(P<0.001;Fig.4).In addition,MBP and NF-200 expression was lower in the 3-MA-treated group relative to the crush + v group(P>0.05).The lesion site of the sciatic nerve was examined 6 weeks post-injury by electron microscopy(Fig.5).Axon number and G-ratio—which is the ratio of inner to outer diameter of the myelin sheath—differed in the four groups(Fig.4B).There was a greater number of smaller axons(diameter ?4?m)while larger axons(diameter>4?m)were more abundant in the crush + v and 3-MA groups than in the rapamycin-treated and sham-operated groups,indicating that axon remyelination was delayed in the former two groups.The G ratio of large axons was 0.59-0.68 in the rapamycin-treated and sham-operated groups,suggesting that axonal maturation was inhibited,whereas in the crush + v and 3-MA groups the G ratio was 0.73-0.83,indicating that axonal maturation was enhanced by activating and suppressed by inhibiting autophagy.Thus,the activation of autophagy stimulates the remyelination function of SCs as well as axon regeneration.5.Autophagy enhances SC survival following sciatic PNI The cytoprotective role of autophagy was investigated by TUNEL staining(Fig.6).A marked decrease in the number of apoptotic cells was detected in rapamycin-treated animals 1 week post-injury as compared to the crush + v and 3-MA groups,indicating that the activation of autophagy promotes SC survival following sciatic NCI.Conclusion:The results of the current study indicate that activating autophagy in injured peripheral nerves by mTOR inhibition serves a protective function in the early post-injury stages that can ensure the recovery of mo tor function later on.the findings presented here provide evidence supporting a role for autophagy in nerve regeneration and motor function recovery in the PNS,and can serve as a basis for the development of therapeutics that improve the outcome of PNI via pharmacological induction of autophagy.