| Ascorbic acid(AA)is a water-soluble ketolactone with two ionizable hydroxyl groups.Being an excellent reducing agent,ascorbate undergoes two consecutive readily,one-electron oxidations to form ascorbate radical and dehydroascorbic acid(DHA).The ascorbate radical is relatively unreactive due to resonance stabilization of the unpaired electron;it readily dismutes to ascorbate and DHA.AA is a vitamin because humans,higher primates,and a few other vertebrates such as guinea pigs have lost the ability to synthesize it from glucose.First took out from food in 1933 by Joseph L Svirbely and Albert Szent-Gyorgyi,the biological functions of AA is increasingly understood.Being one of the most common clinical drugs,AA has now been proved to be the deal that combine low cost with the promise of excellent outcomes.Reaching millimolar concentrations in most tissues,AA is involved in many important physiological and biochemical processes:acting as an antioxidant and cofactor of in at least eight enzymatic reactions.The positive effect of AA on synthesis of collagen and cholesterol metabolism operates by participating in hydroxylation reactions,thus the plasma triglycerides and cholesterol level were reduced and the artery atherosclerotic plaques were decreased accordingly.The relevance to immune system was also investigated and many results showed that AA could not only control infections but also inhibit the spread of virus by stimulating the immune system.The detoxicating ability of the liver was promoted by an increased protein synthesis capacities,which is boosted by AA.Scavenging oxygen free fadicals,AA was believed to have the effect on postponing the anile process of human body.Cancer occurs partly due to the excess exposure to nitrite,and AA could reduce the formation of nitrite in the body,thus reduce the occurrence of cancer;meanwhile,AA could also inhibite the proliferation and migration of cancer cell.AA is also available in the adjunctive therapy of anemia and acalcerosis for its promoted effect of absorption of magnesium and calcium.To be concluded,the benefits of AA to the human body are wide-raging and in a positive way.The neuroprotective efficacy of AA was also proved by the accumulating evidences.L-DOPA,for instance,was toxic for both DA and non-DA neurons,which could be completely prevented by AA thus reduce the damage of neurons.AA is neuroprotective againstglobal ischaemia in the striatum and that some of this action may be due to attenuation of ischaemia-induced DA release.Although the mechanisms underlying the involvement of AA in these processes remain to be clarified,lots of animal model studies have suggested the important role of AA in the nerve system.Systematically added into the culture medium of axon-Schwann cell co-culture experiment,AA has been proved to be absolutely necessary in the process of myelination in vitro.Competing with cAMP,AA may downregulate the expression of PMP22 and than corrects the phenotype of a mouse model of Charcot-Marie-Tooth disease by promoting the remyelination of the peripheral axon fibers.In addition to reduce the secondary damage-induced tissue necrosis in the acute phase post SCI,AA was suggested to be beneficial in the process of remyelination of the central nerve system for its improvement in the behavior function of SCI rats following high-dose of AA administration.Ascorbate must be transported into the neurons via the sodium-dependent Ascorbic Acid transporter 2(SVCT2).The function of AA in myelination and myelin maintenance in the peripheral nerve system was elucidated by the SVCT2+/-mice model,in which hypomyelination and defects in collagen formation in peripheral nerves were observed as consequences of the reduction of AA concentration.Although the effect of AA on the regeneration of nerve system was not mentioned,the functional recovery after SCI and the pain relief after peripheral nerve injury were closely tied to the regeneration of nerve tissues.To investigate the effect of AA on the regeneration of peripheral nerve injury,herein we prepare an in vivo standardized sciatic nerve crush model in mice and fed the mice with AA(200mg/kg body weight)for 28 days and an in vitro neuron culture model and supply the cells with a series concentration of AA.The results revealed that The results revealed that nerve regeneration and remyelination were promoted in respond to AA administration.Moreover,our data also showed that the gene expression of neurotrophic factors and their receptors were increased in AA-treated mice.The cell culture part showed that both the number and length of the neurite outgrowth were promoted in respond to AA administration.Moreover,the pseudopodium outstretched from the growth cone is also increased due to the AA treatment.In addition,the expression of RhoA is decreased by AA in a dose-dependent manner.Overall results of current study indicated AA has a prospective potential to improve the neurite outgrowth,which probably through regulating the RhoA expressions,may lead to a novel therapeutic strategy for treating peripheral nerve injuries.Materials and methods1.The effect of AA on the neurite outgrowth1.1 Cell culture48h-72h SD rat pups(provided by the Animal Center of the Southern Medical University)were deeply anaesthetized with 75%ethanol and then exsanguinated.The spinal cord was exposed and DRG from all spinal levels were harvested.Isolated ganglia were collected in a cold D-Hanks.Ganglia were washed three times with culture medium and enzymatically digested in Pancreatin(0.125%,Gibco,USA)dissolved in Ca2+ and Mg2+-free Hank's buffered salt solution(HBSS,Gibco,USA)for 30 min at 37?.DRG cells were dissociated by trituration using pipettes.Cells were centrifuged at 800 rpm for 10 min and resuspended in the culture medium,plated with a density of 1×105cell/ml on glass cover slips pretreated with 10mg/mL poly-D-lysine(Sigma).Cells were incubated at 37? in an incubator(95%O2 + 5%CO2 gas mixture)for 24 h.Considering from the concentration of AA in human nerve tissue and in the in vitro schwann cell and neuron co-culture system,the final concentrations of AA in this study are as follows:0,100,200,400 ?mol/L.The cells were fixed with 4%paraformaldehyde(PFA)for 15 minutes after 24 h.2.ImmunohistochemistryThe cells were washed with 0.01M PBS(pH 7.4)for 3 times,and each time lasted for 5 minutes.Then the cells were treated with 0.5%triton for 20 minutes followed by the treatment with 5%BSA for 1 hour.The incubations of specific antibodies are as follow.Double stained with ?-tubulin ? and phalloidin:The primary antibody used in this study was anti-?-tubulin ?(Sigma,1:500)and the sencondary antibody was Alexa 488(Life Technologies,1:500).The phalloidin(5?g/ml)was then incubated to identify the cytoskeleton microfilament.2.The effect of AA on the peripheral nerve regeneration2.1 AnimalsFemale C57BL/6 mice(6-8 weeks,provided by the Animal Center of the Southern Medical University)were randomly divided into three groups:(1)saline group:sciatic nerve-crushed(SNC)mice receiving saline intragastric administration(i.a.);(2)AA group:SNC mice receiving AA of 200 mg/kg/day i.a.;(3)sham group:non-crushed mice received neither saline nor AA.All mice were weighted before they got sciatic nerve crushed,and were excluded either weighted less than 18g or more than 20g.Animals were maintained in standard laboratory conditions with a 12 h/12 h light-dark cycle and were allowed free access to food and water.All efforts were made to minimize the number of animals used and their suffering.At the end of the studies,all mice were euthanized with an overdose of sodium pentobarbital.2.2 Sciatic nerve crushThe mice were anesthetized with 1%sodium pentobarbital(50 mg per kg),both side of the sciatic nerve at the mid-thigh region were exposed and crushed for 2 minutes with a fine,smooth,straight hemostat(tip width:1 mm).The sham group underwent surgery without crushing.The crushed site was inspected,no active bleeding was observed,the muscle and skin were then closed.Then the operated animals were kept in cages with soft bedding and ambient temperature maintained at 20-24?.Mice were killed 28 days after the crush surgery.2.3 Drug administrationAA(Guangdong Huanan Pharmaceutical Group Co.,Ltd,China)was dissolved in physiological saline solution and administrated intragastrically.The mice received intragastrical administration of AA or saline after they recovered from the anesthetic.Until the day they were killed,the mice of AA group received the normal daily dosage of AA(200mg/kg·day)and saline group received 0.3 ml/(mice·day)following the injured day.All mice received postoperative treatment including the intraperitoneal injection of penicillin for 3 days.The mice were maintained for 4 weeks under postoperative care.2.4 Behavior assessmentsAll behavioral tests and electrophysiological measurements were performed by a trained investigator blinded towards the groups of the mice.2.4.1 Toe out angle(TOA)TOA,first defined by Varejao,is the degree of foot rotation during normal walking.The test was performed on a glass pane which was 1 meter in length and 7.5 cm in width.The mice were trained to run down the runway the day before the testing day.During the training protocol,a digital camera was placed under the glass pane to record the gait patterns of the mice.If necessary,the animals were allowed to walk multiple times in order to obtain measurable prints.2.4.2 Rota Rod TestFirst described by Dunham and Miya to test neurological deficits in rats and mice.Briefly,mice were pre-trained on an automated 6-lane rotarod unit that could be set on fixed or accelerating speed.During the training protocol,the mice were placed on the rod with a speed of 5 RPM for 300s.For the testing protocol,the mice were placed on the rod that accelerated from 0 RPM to 40 RPM over a period of 240s.The length of time that each mouse stayed on the rod was recorded as the latency to fall,which registered automatically by a trip switch under the floor of each rotating drum.2.5 Sensation function analysis:Hot Plate TestMice were screened by placing them on a hot plate maintained at 55±1? and recording the reaction time in seconds for hindpaw licking or jumping.The mice reacted within 60 sec and did not show large variation when tested on four separate occasions(each 15 min apart)were selected for the test.The time(s)for hindpaw licking or jumping on the heated plate of the analgesiometer was taken as an index of algesic state of the animals.2.6 Electrophysiologic assessmentAt 28 days after injury(DPI28),electrophysiologic tests were performed in all animals.The animals were anesthetized with tribromoethanol,and the surgery sites were reopened.Compound muscle action potentials(CMAPs)were recorded via an electrode inserted into the facies palmaris of dorsal limbs.The difference in latency of CMAPs was computed for the determination of the conduction velocity across the regenerated nerve,and the amplitude of the CMAPs was measured for the quantity of regenerated neurons.2.7 Histological investigation of sciatic nerveThe sciatic nerves were immediately harvested after the electrophysiologic tests.The nerve specimens were fixed with 4%paraformaldehyde for 24 hours.The 2mm segments of regenerated nerve in each group were embedded with olefin and then cut into 4-?m thickness for Neurofilaments-200(NF-200)and Myelin Basic Protein(MBP)immunohistochemistry analysis.The other 2mm of dorsal end of regenerated nerves were fixed with glutaraldehyde for transmission electron microscopy tests.2.8 Transmission electron microscopyAt 4 weeks post-surgery,the distal segments(2mm),which are 2mm far away from sciatic nerve crushed site in each group were harvested and fixed with 2.5%glutaraldehyde for 24 hours.Then the samples were placed in 1%osmic acid in 4?for 24 hours before they were cut into superthin(<0.1?m)sections.The ultrastructure of axons and myelin in the regenerating nerves was observed with transmission electron microscope.The measurements were performed using Image-Pro Plus 6.0 software.2.9 Muscle histologyThe gastrocnemius muscles were fixed with 4%paraformaldehyde for 24 hours,and the mid-belly part were embedded with Tissue OCT-Freeze Medium and then cut into 10-?m transactions.The 10-?m transactions were prepared for the haematoxylin(H)staining to evaluate the area of gastrocnemius muscles.3.The mechanisms of AA promotes the nerve regeneration3.1 AA regulates the expression of RhoACultured DRG neurons incubated with or without AA were fixed after 24h.Then the cells were stained with anti-RhoA antibody:the cells were incubated with anti-RhoA(Santa Cruz,1:200)in in 4? overnight,then washed with O.Olmol/L PBS for 3 times before the sencondary antibody(Alexa 488,Life Technologies,1:500)was added and incubated in room temparature for 2 hours.3.2 AA regulates the neurotrophic factors secreted by Schwann cells3.2.1 Purification of Schwann cellsSciatic nerves harvested from P3 SD rat pups were digested with 0.25%trypsin(Sigma)for 30min.The cells were seeded onto the PLL-coated dishes.10?mol/L cytosine arabinoside was added into the culture medium to purify the Schwann cells for 4 days.The cells were fixed with 4%PFA and then stained with Schwann cell specific antibodis:GFAP,P75 and S100.Our results showed that most of the cultured cells were Schwann cells.3.2.2 AA increases the expression of neurotrophic factors secreted by Schwann cellsThe purified Schwann cells were incubated with or without 200?mol/L AA for 36h.The protein was harvested following the RIPA protocol.The Western blot results showed that the expression of BDNF and NT-3 were significantly increased by AA treatment.4.Statistical analysisAll data were expressed as mean±s.d.Statistical analysis were performed using Student's t-tests and one-way analysis of variance with least squared difference post hoc tests,as appropriate.All experiments were carried out at least three times independently.All P-values are two tailed.A value of P<0.05 was considered statistically significant.Statistical analyses were done using SPSS v.19.0.0.Results1.Effect of AA on the neurite outgrowth1.1 Effect of AA on the length and number of neurite outgrowthBeing the most abundant protein in the neuron cytoskeleton,the cellular distribution of ?-tubulin ? was observed by indirect immunofluorescence.We observed that both the number and length of the neurite outgrowth of control group are remarkably less than the AA treated groups.1.2 Effect of AA on the growth coneCombining with F-actin,the phalloidin is applied to show the distribution of microfilaments in the cytoskeleton.The number of pseudopodium is a common standard for ability of growth.Stained with phalloidin helped to observe the pseudopodium outstretched from the growth cone.The results showed that the number of pseudopodium of control and 100?mol/L AA group are significantly less than the 200 and 400?mol/L AA group.2.Effect of AA on the peripheral nerve regeneration2.1 AA administration accelerates both motor and sensory functional recovery after nerve injuryIn this study,our aim is to gain the outcome of administration of AA,a widely used vitamin that may play important role in the myelination or/and remyelination,after nerve injury.Therefore,we created in mice a model of sciatic nerve crush and received motor and sensory function defects,of which the functional recoveries after injury were exposed to footprint analysis,rota rod test and hot plate test.Footprint analysis focused on the abnormal foot rotation,the toe out angle(TOA)parameter,which was caused by sciatic nerve lesion.At DPI28,feet from sham group shared the smallest TOA,whereas the TOA of saline group is significantly larger than that of AA group.Rota rod test was performed to confirm the motor function recovery.In the testing protocol,we found that mice from sham group were able to stay on the accelerating rod for the maximum time(test was stopped after 300s),whereas the surgery groups could not hold up to 60s.In the DPI28,the latency at which the mice from saline group fell from the rod was shorter than that of AA mice.Afterward,we analyzed the sensory function by means of hot plate test.At DPI28,better recovery from injury was revealed in the AA group.Increased latency to respond to the hot plate assay was evidence and was observed in saline group.Taking together,we can consider that mice from AA group recovered better than those from saline group,both in motor and sensory function.2.2 AA administration enhances neuronal activity after nerve injuryTo more directly correlate the behavioral performance with neuronal activity,we then analyzed the conduction velocity and amplitude of compound muscle action potentials(CAMPs)by means of Clampfit device.Nerve conduction studies of sciatic nerves were performed at the end of week 4 after the injury.In the surgery groups(AA and saline group),characteristic findings caused by the demyelination were observed.These abnormalities contain delayed conduction velocity and dispersion of CMAPs.The amplitude of CMAPs of surgery groups(saline and AA group)did not fully recover over the time course of the study.The CMAPs in saline group were significantly reduced when compared to the AA and sham group.Accordingly,the NCV shows the same tendency.These results consistent with those in the motor and sensory functional recovery,which suggests that AA administration have benefits in the regeneration of sciatic nerves.2.3 AA administration enhances regeneration of axon and remyelination of sciatic nerve after nerve injuryData from behavioral,hot plate and electrophysiological tests suggested impaired axonal regeneration of motor and sensory nerve fibers after sciatic nerve crush.Therefore,we were interested to study the axon regeneration and sciatic nerve remyelination after crush injury on a histological basis.For this purpose,we next analyzed the histology changes of the sciatic nerve(0-2mm distal from the injured site)using EM.The crushed nerves from surgery groups showed a thicker sheath,together with the percentage of axon area distribution in figure 3B,which showed a thinner axon,suggesting nerve remyelination.Furthermore,the calculation of G-ratio(i.e.,axon diameter/myelin sheath diameter)suggested that nerves from AA group gain better remyelination than that from saline group.The G-ratio of saline group(0.7371±0.6663)is significant higher than of AA-treated group(0.7176±0.5696).Immunofluorescence was performed for the part of analysis of myelin associated protein expression.The mice were euthanized after the electrophysiology analysis was done,and the sciatic nerves were sagittally sectioned and double stained with anti-Myelin Basic Protein(MBP)antibody,and anti-Neurofascin(NF-200)antibody,and anti-Laminin antibody.As expected,more necroses of nerve fibers and less and weaker myelin sheaths were observed in the saline group comparing to the high-dose of AA group and the sham-group.Taken together,these data indicate that AA can enhance the regeneration of axons and remyelination of sciatic nerve after nerve injury.2.4 AA administration improve the gastrocnemius muscle amyotrophy after nerve injuryThe acinetatropbia of the gastrocnemius muscles could be trigged by the sciatic nerve crush injury.No significant difference was observed in the size or the wet weight of gastrocnemius muscle from saline and AA group(data not shown).The cross section of area of the muscle measured at DPI28 showed a significant decrease in saline group,while the area from AA group was similar but statistically different to the sham group.3.The mechanisms of AA promotes the nerve regeneration3.1 3.Effect of AA on the RhoA expressionThe small GTPase RhoA has been implicated in the formation of cell cytoskeleton.The immunohistochemistry results showed that RhoA was highly expressed in the neuron cell body of the control group.In the contrary,the expression of RhoA was decreased by AA treatment in a dose-dependent manner.In addition,we also observed that the size of high-RhoA expression cell was significantly smaller than that of low-RhoA expression cells.The size of neuron cell body of 200 and 400?mol/L were significantly larger than that of control and 100?mol/L AA groups.3.2.1 Purification of Schwann cellsThe cells were fixed with 4%PFA and then stained with Schwann cell specific antibodis:GFAP,P75 and S100.Our results showed that most of the cultured cells were Schwann cells.3.2.2 AA increases the expression of neurotrophic factors secreted by Schwann cellsThe Western blot results showed that the expression of SOX 10,c-Jun,BDNF and NT-3 were significantly increased by AA treatment.DiscussionIn this study,our in vitro results showed that AA can promote the outgrowth of neurite,whitch is the one of the most important progress in the nerve regeneration.Afterwards,we prepared a standardized sciatic nerve crush model in mice and fed the mice with AA(200mg/kg body weight)for 28 days.The results revealed that nerve regeneration and remyelination were promoted in respond to AA administration.In particular,AA administration accelerates both motor and sensory functional recovery after nerve injury and enhances neuronal activity after nerve injury.Moreover,AA administration promotes restoration of motor end plate innervation after nerve injury.Moreover,our data showed that the expression of RhoA was decreased in the AA-treated neurons,on the contrary,the expression of neurotrophic factors were increased in AA-treated Schwann cells.Overall results of current study indicated ascorbic acid has a prospective potential to improve the axon regeneration and remyelination in the PNS.which probably through regulating the gene expressions,may lead to a novel therapeutic strategy for treating peripheral nerve injuries. |
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