| Recent studies have shown that locally applied neurotrophins canenhance survival of damaged neurons and regrowth of lesioned axons in thecentral and peripheral nervous systems in rats. However,the beneficialeffects of systemically administered neurotrophins on axonal regeneration arelargely limited by enzymatic degradation as well as by their poor ability topenetrate into the central nervous system through the blood–brain barrier(BBB) . In addition , systemically delivered neurotrophins can haveunexpected side effects. For example,human clinical trials employingsystemic administration of ciliary neurotrophic factor to treat amyotrophiclateral sclerosis were unsuccessful because of the toxicity of the circulatingprotein , which required high-dose , frequent administration for 9months . Therefore,it is important to find chemicals that can easily passthrough the BBB when systemically delivered to produce neurotrophin-likeeffects on axonal regeneration without enzymatic degradation and toxicityproblems. Valproic acid (VPA) possesses the natural characteristic of penetratingrapidly through the BBB because of its small molecular weight . In additionto its widespread clinical use as an effective anti-epileptic drug and moodstabilizer,VPA has also been found to exert neuroprotective effects. Thus,VPA has been shown to protect cultured rat hippocampal neurons againstamyloid and glutamate neurotoxicity and can protect cultured cerebellargranule cells against low-K+-induced apoptosis by acting on thephosphatidylinositol 3-kinase pathway . Recently,Yuan et al. have furthershowed that VPA,at a clinically relevant therapeutic concentration,produceseffects similar to those of neurotrophic factors for SH-SY5Y cells,namely,theactivation of the extracellular signal regulated kinase (ERK) pathway andpromotion of neurite growth and cell survival .Based on the results of these in vitro studies,we initially hypothesizedthat systemic administration of VPA in adult rats could enhance peripheralnerve regeneration. To test this hypothesis,in the present study we examinedthe effect of daily systemic VPA treatment at a clinically relevant dosage onaxonal regeneration and the recovery of motor function after a single axotomyand subsequent repair of the sciatic nerve. It has long been known that a'test'injury performed 1 week after a 'conditioning' lesion significantly acceleratesthe regeneration speed of the injured peripheral axons,stimulates generation ofmore axonal sprouts at the injury site ,and enhances functional recovery ofinjured peripheral nerve . It is unknown,however,whether VPA andconditional lesion can provide additive effects on peripheral nerveregeneration. Therefore,in this study we also examined the effect of dailysystemic VPA treatment on axonal regeneration and the recovery of motorfunction after conditional sciatic nerve axotomy in adult Wistar rats.Animals and animal treatmentAdult male Wistar rats initially weighing 250–300 g were used for thisstudy. They were housed under temperature and light-controlled conditions(12:12 h light–dark cycle),with food and water available ad libitum. Allprocedures used were in strict accordance with the guide-lines established bythe Canadian Council on Animal Care and approved by the University ofSaskatchewan Animal Care Committee. Twenty-five rats were randomlydivided into five groups (five rats per group). Anesthesia was induced in therats with isoflurance (5% initial;2.5% maintenance). All procedures wereperformed using aseptic techniques. The right sciatic nerve was exposedthrough a dorsal/gluteal splitting incision. In the sham (S) group,ratsreceived a sham surgical procedure in which the right sciatic nerve wasisolated and mobilized. The right sciatic nerves of rats in the other groupswere sharply transected 10 mm distal to the sciatic notch and reconnected withtwo epineurial 9/0 nylon sutures. In groups A (single axotomy) and AV(single axotomy plus VPA),rats received a transection and immediateanastomosis to the right sciatic nerve. In groups C (conditional axotomy) andCV (conditional axotomy plus VPA),a priming axotomy was created in theright sciatic nerve of each rat,followed 7 days later by re-axotomy andimmediate anastomosis of the proximal stumps of the sciatic nerves. In allgroups the skin was closed with 4/0 nylon sutures. In groups AV and CV,thesurgical procedures were followed by oral administration of VPA (Sigma,St. Louis,USA) dissolved in drinking water at a dose of 300 mg/kg daily for16 weeks following nerve repair. The dose of VPA was determined based onthe findings that while the antimanic action of VPA appears at blood levelabove 50 mg/ml,and clinical toxicity appears at blood levels above 200mg/ml ,rats receiving treatment of VPA in drinking water at a dose of 300mg/kg daily showed a plasma level of about 50ug/ml. All rats received aweekly evaluation of motor function from weeks 4 to 16,the rats were thenkilled for histomorphometric examination of nerves and muscles.Motor functional assessmentTo assess the recovery of motor function,a brightly lit walkway (9.5 cmwide,42 cm long with 15-cm-high walls) with a dark box at one end wasconstructed. The rats had been allowed to 'practice' moving through thewalkway to the dark box several times to become familiar with theapparatus. The hind limbs of tested rats were dipped in a 'paste' made ofgreen food dye dissolved in 2% carboxymethyl cellulose. The rats were thenallowed to walk along the walkway,leaving footprints on white paperstrips. Paw length and toe spreads were measured and a sciatic function index(SFI) was calculated according to the Bain–Mackinnon–Hunter SFI formula :SFI = -38.3 ([EPL – NPL] / NPL) + 109.5 ([ETS – NTS] / NTS)+ 13.3 ([EIT – NIT] / NIT) – 8.8where PL=print length in mm,TS=spread in mm between 1st and 5th toes,IT=spread in mm between 2nd and 4th toes , and E and N indicateexperimental and normal hind foot,respectively.In some cases,it was impossible to obtain satisfactory footprints becauseof a markedly impaired walking track due to poor sciatic function or a markedeversion or inversion deformity resulting from imbalance of the posterior tibialand peroneal fascicles. In these instances,manual measurements were used tocalculate the SFI. The maximal toe spread was measured by holding the ratvertically by the skin on the back of the neck while the rat's hind feet restedlightly on a sheet of paper. The maximal spread between the first and fifthtoes was marked using a pen. The maximal toe spread index (TSI) wascalculated according to the Brown TSI formula :TSI = (ETS – NTS) / NTSHistochemistry of nerve and muscleSixteen weeks after the final surgery,all rats were killed by anesthesiawith isoflurane. A 4-mm length of sciatic nerve 5 mm distal to the repair sitewas removed by dissection and fixed in Baker's solution overnight,and thecorresponding section of nerve from the contralateral side in the same rat wasused as a control. The fixed sciatic nerves were postfixed with 1% osmiumtetroxide in sodium cacodylate buffer for 1 h. After three incubations in100% ethanol (5 min each),the sciatic nerves were incubated in propyleneoxide for 5 min and then in a 50:50 mixture of propylene oxide/epon aralditeovernight. The tissues were then transferred to pure epon araldite in plasticmolds and placed at 82oC overnight. The blocks were trimmed and semi-thincross-sections (1 mm) were cut and placed on glass slides. The sections werestained in a 1% solution of toluidine blue and then observed under a lightmicroscope.After harvesting the sciatic nerves,both sides of the tibialis anterior (TA)muscles of all rats were quickly removed,cut into three blocks,rapidly frozenin isopentane,cooled with liquid nitrogen,and stored at -700C. Serialcross-sections (10 mm thick) were cut and stained for myofibrillar ATPasewith acid (pH 4.5) preincubations.Data analysisUsing the Bain–Mackinnon–Hunter formula,the SFI was obtained. Ascore of '0' on the index indicated normal function,whereas a score of '-100'corresponded to a total loss of function. For the TSI,a score of '0' on theindex indicated normal function,whereas a score of '0.6' corresponded to atotal loss of function.To obtain the density of myelinated nerve fibers,six fields per nerve werecounted under high magnification (40×) and analyzed using an automateddigital image analysis system linked to morphometric computersoftware. The density was multiplied by the area of the nerve tissue cable toobtain the total number of myelinated fibers per nerve. In addition,a totalarea of myelinated nerve fibers was measured for each nerve with themorphometric computer software. The size of an individual myelinated nervefiber equals the total area divided by the number of fibers. The recoveryvalues of the total number of myelinated nerve fibers and the mean size ofmyelinated nerve fiber were expressed as a percentage of the control in allgroups.For measurement of muscle fiber size,400 myocytes from 10 fields ofthree muscle cross-sections were measured and the size of an individualmuscle fiber was calculated. The experimental value was expressed as apercentage of control. Muscle cross-sectional area was measured on threecross sections that contained the maximum number of muscle fibers. Themuscle cross-sectional area was divided by the size of an individual musclefiber in order to obtain the total number of muscle fibers. The recovery valueof the total number of muscle fibers was expressed as a percentage.Statistical analyses of the data were performed using a t-test and one-wayANOVA,followed by Hochberg post hoc tests using SPSS 9.0 software.ResultsEffect of VPA on axonal regeneration of axotomized sciatic nerveHistochemical staining of cross-sections of rat sciatic nerve with toluidineblue is a very efficient method for measuring myelinated nerve fiber area aswell as for counting myelinated nerve fibers. The sizes of the individualmyelinated nerve fibers of regenerated nerves in groups A,AV,C and CVwere significantly smaller than that in normal control rats (i.e. group S)(P<0.001). There was no statistical difference between the sizes of individualmyelinated nerve fibers in rats with (i.e. groups AV and CV) or without VPAtreatment (i.e. groups A and C). Although the sizes of the individualmyelinated nerve fibers of the conditional axotomized rats (i.e. groups C andCV) appeared larger than those of single axotomized rats (i.e. groups A andAV),there was no statistical difference. The number of myelinated fibers ofregenerated nerves in groups A,AV,C and CV was three times higher thanthat found in the normal nerves of group S (P<0.001). In the singleaxotomized sciatic nerve model,VPA treated rats (i.e. group AV) showedsignificantly more myelinated nerve fibers in comparison with rats withoutVPA treatment (i.e. group A) (P<0.05). The same results were also foundin the conditional axotomized sciatic nerve model. That is,the number ofmyelinated nerve fibers of VPA treated rats (i.e. group CV) was significantlyhigher than that of rats without VPA treatment (group C) (P<0.05). Therewas no significant difference of the numbers of myelinated nerve fibersbetween rats receiving single and conditional axotomy.Effect of VPA on the reinnervated musclesType I muscle fibers were individually distributed over the inner half ofthe normal TA muscles,whereas clusters of type I muscle fibers were found inthe reinnervated TA muscles. The muscle fiber size of the inner half of thenormal TA muscle was significantly smaller than that of exterior half of thenormal TA muscle (P<0.05). However,there was no regional difference inmuscle fiber size of the reinnervated TA muscles. The individual muscle fibersize of the reinnervated TA muscles in rats receiving axotomy and anastomosis(i.e. groups A,AV,C and CV) was significantly smaller than that of normalrats (P<0.05),but there were no significant differences among groups A,AV,C and CV. The numbers of the reinnervated TA muscle fibers in groups A,AV , C and CV were significantly smaller than that in group S(P<0.05). However,there were more reinnervated TA muscle ?bers in ratsreceiving VPA treatment than in rats without VPA treatment (P<0.05)(i.e. AV vs. A and CV vs. C). The number of the reinnervated TAmuscle ?bers of rats receiving conditional axotomy (group C) was signi?cantlyhigher than that in rats receiving single axotomy (group A) (P<0.05).Motor functional recovery of the axotomized sciatic nervesThe tests for motor function were equivalent for both hind limbs of shamcontrol rats (group S);therefore,the hind limb that received a sham operationto the sciatic nerve was used as a control.The serial changes in SFI and TSI are depicted. The SFI and TSI of ratsreceiving single axotomy (group A) significantly improved starting from week8 (P<0.05) and reached plateau levels at approximately week 11. The SFIand TSI of rats receiving conditional axotomy,conditional axotomy plus VPAand single axotomy plus VPA (i.e. groups C,CV and AV) began tosignificantly improve starting from week 4 (P<0.01),reaching plateau levelsat approximately week 8. Analysis by repeated one-way ANOVA showed asignificant group effect and significant interaction of groups over time in SFI(group effects:F3,19=14.699,P<0.001;interaction of groups over time:F36,19=3.552,P<0.001) and TSI (group effects:F3,19=28.337,P<0.001interaction of groups over time:F36,19=3.123,P<0.001). The functionalrecovery of rats receiving conditional axotomy plus VPA (group CV) appearedbetter than that of rats receiving conditional axotomy alone (group C) fromweek 11 to week 16,but there was no significant difference in SFI (P=0.546)and TSI (P=0.32). There was no statistical difference between rats receivingsingle axotomy plus VPA (group AV) and rats receiving conditional axotomyor conditional axotomy plus VPA (group C or CV) in SFI (P=0.282 orP=0.954) and TSI (P=0.052 or P=0.72) from week 4 to week 16.DiscussionIt has recently been reported that VPA robustly promotes neuriteoutgrowth and activates the ERK mitigen-activated protein kinase pathway ,asignaling pathway utilized by many endogenous neurotrophic factors. VPAhas also been demonstrated to increase the expression of the cytoprotectiveprotein bcl-2 and growth cone-associated protein 43 in the central nervoussystem and human neuroblastoma SH-SY5Y cells. The present study showedthat VPA,administered at a clinically relevant dosage,produced a significantpositive effect on the regeneration of myelinated nerve fibers in adultrats . The total numbers of regenerated myelinated nerve ?bers weresignificantly increased in both the single and conditional axotomized sciaticnerves after VPA treatment. However,there was no significant difference innerve fiber size between rats with or without VPA treatment. These resultssuggest that VPA is able to enhance sciatic nerve regeneration by increasingthe total number of regenerated myelinated nerve fibers in adult rats. Sinceloss of muscle fibers and a decrease in muscle fiber size are most prominentfollowing denervation and are characteristics of muscle atrophy,we examinedthe number and size of reinnervated muscle fiber. We found that the totalnumber of reinnervated muscle fibers increased significantly in VPA-treatedrats receiving either a single or conditional axotomy,although VPA exerts noobvious effect on the muscle fiber size of the reinnervated muscle. Inaddition,we also observed that the total number of reinnervated muscle fibersin rats receiving conditional axotomy was significantly higher than that in ratsreceiving a single axotomy,thus confirming the results reported in previousstudies.In this study,we further observed that VPA treatment significantlyaccelerated the recovery of motor function in the reinnervated hind limbs,asdemonstrated by the better SFI and TSI values,from weeks 4 to 8 after nerverepair , in rats receiving VPA treatment than in rats without VPAtreatment. Furthermore,we also found that conditional axotomy acceleratedmotor function recovery,and these results are in agreement with previousstudies. These results suggest that VPA may enhance the regeneration rate ofaxotomized sciatic nerve in adult rats. However,we failed to detect asignificant additive effect of VPA on the recovery of motor function inreinnervated hind limbs after conditional axotomy. It is unlikely that theabsence of an additive effect was produced by the interaction between VPAand conditional axotomy. It is more likely that conditional axotomy mayalready provide a 'ceiling effect' on the rate of axonal regrowth of lesionedneurons as implied in previous studies.The degree of recovery of motor function in reinnervated limbs reachedplateau levels between 8 and 11 weeks;therefore,it is unlikely that the degreeof recovery of motor function would be significantly improved if rats wereallowed to survive for longer periods. The incomplete recovery of motorfunction (i.e. 50–63.38% of normal motor function) in these rats may becaused by the misdirected reinnervation and/or a certain degree of atrophy ofthe denervated muscle,because these two factors have been found tocompromise functional recovery.The mechanisms responsible for the enhancement effect of orallyadministered VPA on sciatic axonal regeneration are not clear. It is possiblethat the effect is produced by the direct activation of ERK signal transductionpathway by VPA,or indirect activation of ERK pathway following enhancedsynthesis of neurotrophins by VPA. This is because VPA at clinically relevanttherapeutic concentrations produces effects similar to those of neurotrophicfactors by activation of the ERK pathway and increase of the expression ofgrowth cone-associated protein 43 and neurotrophins. The exact location ofthe action of VPA in enhancing sciatic nerve regeneration is not clear. It ispossible that VPA can act in the spinal cord and/or injured sciatic nerve,because VPA possesses the natural characteristic of penetrating rapidlythrough the BBB.In summary,the present study has demonstrated that VPA is able toenhance sciatic nerve regeneration in adult rats. Although locally appliedneurotrophins can also enhance axonal regeneration in rats,the beneficialeffects of systemically administered neurotrophins on axonal regeneration arelargely limited by enzymatic degradation of neurotrophins in the blood,bytheir poor ability to pass through the BBB,and by the obvious toxic effectsproduced by high-dose,frequent administration of neurotrophins. Becausesystemically administered chemical VPA can easily pass through the BBB andhas been safely used clinically as an effective anti-epileptic drug and moodstabilizer for many years,the present finding that VPA is able to enhancesciatic nerve regeneration in adult rats indicates that VPA may be used toreplace the previously suggested application of neurotrophins to promotion orenhancement of axonal regeneration in both central and peripheral nervoussystems in humans.
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