University of Groningen

Artificial nerve guidesMeek, Marcel Frans

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Functional nerve recovery after bridging a 15 mm rat sciatic nerve gapCHAPTER 8

Functional nerve recovery after bridging a 15 mm rat sciaticnerve gap with a biodegradable nerve guideMF Meek,1 F Klok,2 PH Robinson, FRCS,1 J-PA Nicolai,1 JFA van der Werff,1A Gramsbergen2

1 Department of Plastic Surgery, University Hospital Groningen, Groningen, The Netherlands2 Department of Medical Physiology, Groningen University, Groningen, The Netherlands

Submitted for publication, 2000

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Chapter 8AbstractRecovery of nerve function was evaluated after bridging a 15 mm sciatic nervegap in the rat with a biodegradable poly(DL-lactide-ε-caprolactone) nerve guide.Nerve recovery was investigated by analyzing the foot prints, by analyzingvideorecordings of gait, by electrically eliciting the withdrawal reflex, nerveconduction velocity and electromyography (EMG). Sensory nerve functionrecovered as measured by the electrostimulation. Motor nerve function partlyrecovered but electromyograms remained highly abnormal throughout the study.It is concluded that functional reinnervation by regenerating axons occurs afterbridging a 15 mm nerve gap with a biodegradable poly(DL-lactide-ε-caprolac-tone) nerve guide, but the walking patterns remain abnormal. The use of videoanalysis is a useful tool to record and analyze the walking patterns of the rat.Further studies are necessary to investigate the possibility of obtaining selectivereinnervation of specific muscles.IntroductionThe most commonly used technique tobridge a peripheral nerve gap is anautograft. One promising alternative toprevent a donor site is the use of artificialnerve guides. Several conduit materialshave been investigated.1 The use ofbiodegradable nerve conduits isparticularly promising. After functio-ning as a temporary scaffold for axonoutgrowth, they degrade in the courseof months or years.Den Dunnen et al. showed that a biode-gradable nerve guide composed of anamorphous copolymer of DL-lactideand ε-caprolactone [p(DLLA-ε-CL)] iseffective with regard to nerve out-growth.2 They also concluded that anerve guide with an internal diameterof approximately 1.6 mm and a wallthickness of 0.3 mm functioned best ina rat model. Previous studies with thistype of material demonstrated that tubedimensions and swelling of degradingbiomaterials are important parametersfor the final outcome of nerve regenera-

tion.3 Meek et al. showed that thin-walled nerve guides tend to collapse,4unless some mechanical support insidethe lumen is used,5 such as muscletissue.6 Thus far, p(DLLA-ε-CL) nerveguides were used to bridge 10 mmsciatic nerve gaps, which has beenshown to be a critical length to bridgein the rat model.7,8Because the results of microscopicalanalysis and functional nerve recoverymay be conflicting,9-12 conclusions aboutthe return of function cannot be drawnsolely on the basis of an analysis of thelight- or electron microscopy at therepair site. To establish sciatic nervefunction in the rat, often foot printanalysis is used to calculate the sciaticfunction index (SFI). According toDellon and Mackinnon, however, thegait pattern cannot be analyzed by footprint analysis when chronic footdeformities or automutilation occur.10The use of other evaluation techniques,therefore, is necessary to obtain reprodu-

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Functional nerve recovery after bridging a 15 mm rat sciatic nerve gapcible data on the recovery of nervefunction in order to understand theevents during recovery. In 1979, Hruskaet al. introduced footprint analysis usingthe video analysis technique andassessed the efficacy of measuringswing and stance duration of the normalwalking pattern.13 Westerga and Grams-bergen introduced the use of a mirror toshow both the plantar surface and theside-view of the rat’s hindpaw.14 It isimportant to note that by using the videoanalysis technique the movementsduring walking can be objectivated aswell.In this study we bridged a 15 mm gapin the sciatic nerve of the rat by abiodegradable p(DLLA-ε-CL) nerveguide. The recovery of nerve functionswas studied by using a combination ofevaluation methods (walking trackanalysis, video analysis, withdrawalreflex, nerve conduction velocity andelectromyography). The results werecompared with the non-operatedcontralateral side as well as a non-operated control group.Materials and MethodsSurgical ProceduresIn total, 57 Male Wistar rats weighingapproximately 250 g (225 - 275 g) werestudied. In group A (n = 51), the ratswere premedicated with atropine (0.25mg/kg body weight) and anaesthetizedwith 1% isoflurane (Forene®) and O2/N2O. The left sciatic nerve was exposedby splitting the left superficial glutealmuscle. A gap of 15 mm was made andbridged by a 18 mm biodegradablenerve guide (Polyganics BV, Groningen,The Netherlands), composed of a

copolymer of 50% DL-lactide and 50%ε-caprolactone [85% L-lactide (LLA)and 15% D-lactide (DLA)]. The internaldiameter of the nerve guide wasapproximately 1.6 mm (range 1.57 -1.67 mm) and the wall thickness 0.3 mm(range 0.27 - 0.37 mm). Both theproximal and distal ends of the sciaticnerve were telescoped into the nerveguide and fixed with a single 10-0 nylonepineural suture. The tube was prefilledwith phosphate buffered saline. In groupB (n = 6), the rats were not operated,received no anaesthesia and served as acontrol group.Surgical procedures were performedunder an operation microscope (magni-fication 25 x) and a sterile procedure wasused. Following surgery, the animalswere housed in a temperature- andhumidity-controlled room with 12 hrlight cycles and had access to standardrat food and water ad libitum. Goodlaboratory practice (GLP) was main-tained, according to the National Guide-lines for Animal Welfare, comparablewith the international rules for animalexperimentation (International Guide forAnimal Biomedical Research and EthicalCode for Animal Experimentation of theCouncil for International Organization ofMedical Sciences).Evaluation TestsWalking Track AnalysisAt 2, 4, 7, 15, 21 and 36 weeks, fingerpaint was applied onto the plantarsurface of the hind feet and it wasensured that all anatomical landmarkswere covered. The rat was allowed towalk down a track, leaving prints of itsfeet on the paper. From the footprints,

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Chapter 8the sciatic function index (SFI) wascalculated using the formula developedby Bain and Mackinnon.15,16 An SFI of0 is normal whereas an SFI of -100means total impairment.Next, the rats were placed in a Perspexrunway.14 The lateral view of the animalwas recorded directly with a videocamera, whereas the ventral view of theanimal was visualized by means of anadjustable mirror under the cage,positioned at an 45o angle. In thismanner, a split screen image wasobtained with the lateral view of the ratin the upper half and the ventral view inthe lower half (Fig. 1). The runway wasilluminated with two 120 W concentricbulbs to improve contrast and toenhance the point of foot-contact.Walking movements of the rat wererecorded with a video camera containinga stroboscobic shutter (25 frames persec), creating blur-free stills foranalyzing the footsteps, until at least 4consecutive and non-hesitant step cycleswere collected. The video-tape was thenreplayed frame by frame, until themaximal contact of the rat’s foot to thefloor was reached. The SFI wassubsequently measured from theseimages.The video recording technique was alsoused to obtain the stance factor, asdescribed by Walker et al.17 The stancefactor is the ratio between duration offloor contact (gait-stance duration)between the left operated and the rightnon-operated hind paw. Injured ratsgenerally show a walking pattern witha shorter gait-stance duration of theinjured leg than of the non-injured leg.

Withdrawal TestAt 2, 4, 7, 15, 21 and 36 weeks, anelectrostimulation test was carried outon the sole of the foot using a bipolarelectrode consisting of two copperwires. The lateral side of the left(operated) sole was stimulatedproximally, distally and in between inall rats, as described by De Koning.18 Ahealthy rat immediately withdraws itsfoot and spreads its toes afterstimulation. The threshold, i.e. thelowest current causing this reflex, wasrecorded. Then, at each evaluationperiod, the sciatic nerve was cut in 3 ratsand again the foot-sole was electricallystimulated in order to verify the role ofthe sciatic nerve in the withdrawal testat the respective ages.Conduction Velocity (CV)At 2, 4, 7, 15, 21, and 36 weeks, thesciatic nerve was stimulated proximallyand distally to the nerve guide using abipolar silver electrode under generalanaesthesia in 3 rats. During therecordings, the body temperature of theanimals was maintained at 37° C. Themuscle action potentials (MAP) fromthe gastrocnemius muscle (GC) and theanterior tibial muscle (TA) wererecorded with micro needle electrodesand the conduction velocity (CV) wasmeasured.19 The MAP was displayed onan oscilloscope at settings appropriateto measure the latency time fromstimulus to the onset of the first negativedeflection. A deflection in the form of acompound action potential in theoscilloscopic screen symbolized theneural response. All data were collectedon a data recorder. The distance betweenthe electrodes proximally and distally to

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Functional nerve recovery after bridging a 15 mm rat sciatic nerve gap

Fig. 1. Sketch and photograph showing the experimental set-up for the video analysis. A lateral viewas well as a ventral-view of the animal were simultaneously obtained.

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Chapter 8the nerve guide was measured and theCV was calculated.20 Finally the animalswere killed by an intracardiac overdoseof barbiturate.Electromyography (EMG)At 15, 21, and 36 weeks of implantationof the nerve guides, EMG electrodeswere implanted under anesthesia in thehindlegs of 9 rats (3 at each evaluationperiod). EMG electrodes were made ofmultistrand stainless steel wire(diameter 0.003 inch), insulated by ateflon coating except for a bare tip of 1mm. The electrodes were implanted inpairs and the interelectrode distance was1-1.5 mm with both bare tips orientatedfrom proximal to distal. A commonelectrode was placed in the lower backregion. Pairs of electrodes were implan-ted in the midbelly regions of the GCand the TA muscles of both hindlegs.The electrodes were sutured to thefasciae of the muscles and the electrodewires were led subcutaneously to theback and soldered to a miniatureconnector. EMG recordings were madeapproximately 8 hr after recovery fromthe anaesthesia on the day of operationas well as on consecutive days. Theanimals were allowed to walk freely ona flat surface and they were connectedto an amplifier system. The EMGsignals were stored and processed off-line on a personal computer. EMGrecordings were then rectified andaveraged and displayed for visualinspection (For further details seeGramsbergen et al.21).ResultsIn group A, 20 of the 51 rats sooner or

later showed automutilation of theoperated hindpaw. From these rats, theSFI could not be calculated. One of theserats showed automutilation of thecomplete lateral side of the sole of thefoot, and was therefore excluded fromthe electrostimulation test as well.Sciatic Function Index (SFI)SFI values obtained by video recordingdid not differ from those on paper inboth groups. Preoperative sciatic nervefunction indices for group A (nerveguides) did not differ from the controlgroup B (non-operated control rats). Ingroup A, the SFI significantly increasedwith time from -93 after 2 weeks to -43after 36 weeks of implantation (Fig. 2A).In group B, reproducible walking trackpatterns could be measured in all ratsand the mean SFI did not change duringthe study (Fig. 2A).Stance FactorIn group B no measurable differenceswere observed in gait-stance durationsof the left and right hindpaws throughoutthe evaluation period (Fig. 2B). In groupA, the gait-stance duration of the leftinjured leg was of a shorter duration thanthe right non-injured leg. The stancefactor increased with time, but did notrecover to control values (Fig. 2B).Withdrawal TestIn group A, a withdrawal reflex couldnot be elicited in the first weeks afterimplantation. From 7 weeks onwards,the first positive reflexes were obtainedbut with high thresholds. This recoverycontinued and after 36 weeks, the thres-hold had decreased to almost controlvalues (Fig. 2C). The values at the

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Functional nerve recovery after bridging a 15 mm rat sciatic nerve gap

Fig. 2. Graphs showing changes in Sciatic Function Index (SFI) (A), the gait-stance duration - ratioinjured : non-injured hindfeet (B), and the average current intensity necessary to elicit a withdrawalreflex (C) with time. Filled squares, rats with nerve guides; open diamonds, control rats.

A

B

C

= group A = group B

-120-100-80-60-40-20020

0 10 20 30 40Time (weeks)

SFI

00.20.40.60.8

1

0 10 20 30 40

Averagethreshold (mA)

00.20.40.60.8

1

0 10 20 30 40Time (weeks)

Gait Stance Duration

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Chapter 8contralateral side were not significantlydifferent from the non-operated controlrats.To verify the role of the saphenous nervein the reflex, the sciatic nerve was againcut (proximal to the implantation side)in 3 rats at 2, 4, 7, 15, 21, and 36 weeks.Until 7 weeks after implantation of thenerve guides the withdrawal reflex didnot disappear after the sciatic was cut.However, from 15 weeks and beyond,the reflex could not be elicited aftersecondarily cutting the sciatic nerve.Conduction Velocity (CV)The mean CV in the right non-operatedpaw was 53 ± 6 m/s. In the first weeksafter implantation of the nerve guides,MAP’s could not be obtained. After 7weeks, MAP’s could be obtained in oneout of three rats, and the CV was 9 m/s.Thereafter, the mean CV graduallyincreased to 27 ± 8 m/s after 36 weeks.Electromyography (EMG)In control rats, the swing phase startedwith a shortlasting, brisk burst in the TA.During the stance phase this muscle waselectrically silent (Fig. 3; asterisksindicate onsets of swing phases). EMGamplitudes in the GC muscle droppedduring the swing phase, whereas thestance phase was accompanied by atonic burst in the GC muscle.In the right non-operated side of theanimals in group A sometimes extrabursts (Fig. 4; see arrows) and irregularphasing of stance and swing phase couldbe observed (Fig. 4).At the left operated side, the EMGpatterns of the GC and TA muscles werehighly abnormal in all animals. Insteadof a brisk burst at the onset of the swing

phase in the TA, the muscle often wascontinuously active (Fig. 4 and 5), andsometimes even without a clear increasein activity during the swing phase (Fig.4). The bursts in the GC muscle wereirregular and activation during the swingphase often continued (Fig. 4 and 5).Often simultaneous activation of theantagonist (coactivation) was observed.These abnormalities persisted until theend of the study.DiscussionDespite several reports on excellentmorphometrical data on regrowth ofperipheral nerves after transection andreconstruction with nerve conduits, fullrestoration of sensory and motor nervefunction after peripheral nerve recon-struction often fails.9-10 Also clinicalresults after nerve repair often remaindisappointing. Reestablishment ofoptimal function following peripheralnerve lesions, therefore, continues to bea considerable (clinical) challenge. Theaim of the surgeon is not only to repaira damaged nerve so that the maximumnumber of axons cross the suture line,but also to ensure optimal return offunction. The latter, however, is oftendisappointing. A better understanding ofrecovery of nerve function and adapta-tion may help to achieve better functionafter repair in the future.Only a few articles described the use ofthe video analysis technique for theevaluation of functional nerve recoveryin the rat after sciatic nerve lesion andrepair.17,23-25 The use of video recordingsof gait in the rat model is therefore anunderestimated method. Recently,Dijkstra et al. evaluated the SFI in the

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Functional nerve recovery after bridging a 15 mm rat sciatic nerve gap

Fig. 3. Averaged EMG records of a normal rat.Records from the gastrocnemius (GC) and thetibialis anterior muscles (TA) of the right side.Time in seconds, amplitudes in µV; asteriscsindicate the onset of swing phases.

Fig. 4 and 5. Averaged EMG records after 21 (Fig. 4) and 36 (Fig. 5) weeks of implantation.Records from the gastrocnemius (GC) and the tibialis anterior muscles (TA) of the right side (above)and of the left side (below). Time in secs, amplitudes in µV; asterisks indicate onsets of swing phasesin the right and left hindpaws; arrows in Fig. 4 indicate abnormal burst activity at the right non-operated side.

rat after crush lesion, after autologousnerve grafting and in a non-operatedcontrol group; it was shown that nosignificant differences were foundbetween the SFI’s measured by thefinger paint and video analysistechnique.25 They concluded that the useof video analysis proved to be avaluable, non-invasive method for theevaluation of nerve function because thewalking pattern and the stance factor canbe studied simultaneously. Similar to theresults in that previous study, the SFIvalues obtained with video analysis inthe present study did not differ from thefinger paint technique.Chronic foot deformities may result in

4 5

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Chapter 8gait patterns that render walking trackassessment invalid.10 When auto-mutilation of the hindpaw occurs, theSFI can not be calculated either. Despitethis disadvantage, the SFI is a widelyused parameter because of its reliability.One recently published study of Shenand Zhu showed that the SFI had apositive correlation with musclestrength,26 muscle induced actionpotentials, nerve compound actionpotential, and motor nerve conductionvelocity. Nerve CV’s as obtained by theMAP’s are indicative for nerveregeneration and target reinnerva-tion.20,27 Recovery in CV was found inthe present study but control values werenever achieved. Good recovery of motornerve function depends on thereinnervation of the new target by theoriginal axons. Correct innervation ofthe motor endplates is also necessary.Furthermore, changes in the distributionof fibre types of the different musclesinnervated by the sciatic nerve may playa role in the functional outcome.De Koning et al. showed that the localapplication of electrical stimuli to thesole of the rat’s foot is a non-invasive,rapid, easy and very precise method toevaluate the return of sensory nervefunction after nerve injury.18 They alsodescribed that there is no conditioningduring the test, and it is therefore awidely used technique.28-30 As inprevious studies it was found that thereturn of sensory nerve innervation ofthe skin is faster than the return of motornerve function. The return of sensoryfunction, as measured by theelectrostimulation test, may not only beexplained by the outgrowth of regenera-ting axons from the sectioned proximal

sciatic nerve stump but also by collateralsprouts from intact fibers in the skinsurrounding the denervated zone.31-32Devor et al. showed that cutaneousreinnervation starts with collateralexpansion of afferents from intactneighboring saphenous nerve fibers.33Thereafter, with the return of sciaticnerve fibers, the expanded distributionof the saphenous nerve retracts to itsoriginal boundaries. They found thatapproximately 3 weeks after crushing,the regenerating sciatic nerve began toregain its function. This was concludedby the return of sensation to zones notinvaded by the saphenous nerve and bythe onset of sensation in rats in whichthe saphenous nerve had previouslybeen ligated. In the present study, thewithdrawal reflex could be elicited after7 weeks of implantation and also afterthe sciatic nerve was cut. From 15 weeksand beyond the withdrawal reflexdisappeared after secondarily cutting thesciatic nerve. We conclude therefore,that recovery of sensory nerve functionstarts with the expansion of the territoryof intact neighboring fibers. After 15weeks, fibers from the original nerveagain started to make functional contact.This is in accordance with the results ofDevor et al.,33 although only after alonger time period.EMG’s remained abnormal throughoutthe period of investigation. This can beexplained by the fact that musclesreceived aselective inputs from axonsoriginally connected to other muscles,e.g. cross-innervation. This has beendemonstrated to lead to dramaticchanges in activation patterns of themuscles.21 On the right non-operatedside, some irregularities could be

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Functional nerve recovery after bridging a 15 mm rat sciatic nerve gapobserved in the EMG’s, such as extrabursts and irregular phasing duringstance and swing. This may be explainedby a compensation for the rat’s inabilityto bear body weight and adequatelypropulse with the left hindleg. Theincrease in quality of the walking patternwith time in conjunction with a severelydisturbed EMG activity is confusing. Itmight be possible that despite aselectivereinnervation of the muscles, readjust-ments in the force recruitment of thesemuscles occur due to subtle readjust-ments by supraspinal motor systems.22These processes may be situated in thecerebellum. It is well known that thecerebellum is involved in motor learningand modulation of motor output on thebasis of ascending spinal cordinformation. Presently, ongoing researchaims at identifying the process of

functional recovery in spite of aberrantreinnervation.In conclusion, recovery of nervefunction over a 15 mm gap in the sciaticnerve of the rat is possible with ap(DLLA-ε-CL) nerve guide without theaddition of growth factors or extracellu-lar matrix molecules. Sensory nervefunction as measured by the electrosti-mulation test did recover well but motornerve function as measured by the SFI,NCV, stance factor only partlyrecovered. Electromyograms duringwalking remained highly abnormalthroughout the study. Investigation onhow to selectively reinnervateantagonist muscles, how to influence thedistribution of muscle fibre types, andfinding cues for specific axon guidanceare the main aims for further research.

ACKNOWLEDGEMENTSThis research was made possible by the MW-NWO (Dutch Organization for Sci-entific Research), Den Haag, The Netherlands. This project has been supportedby the Foundation “De Drie Lichten”, Hilversum, The Netherlands. Theassistance of H.L. Bartels with microsurgical techniques is greatly appreciated.

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Chapter 8References1. Doolabh VB, Hertl C, Mackinnon SE. The role of conduits in nerve repair: a review.

Rev Neurosci 1996;7:47-84.2. Den Dunnen WFA, Van der Lei B, Schakenraad JM, Stokroos I, Blaauw EH, Bartels

H, Pennings AJ, Robinson PH. Poly(DL-lactide-ε-caprolactone) nerve guides performbetter than autologous nerve grafts. Microsurgery 1996;17:348-357.

3. Den Dunnen WFA, Van der Lei B, Robinson PH, Holwerda A, Pennings AJ,Schakenraad JM. Biological performance of a degradable poly(DL-lactic acid-ε-caprolactone) nerve guide: influence of tube dimensions. J Biomed Mater Res1995;29:757-766.

4. Meek MF, Den Dunnen WFA, Bartels HL, Pennings AJ, Robinson PH, SchakenraadJM. Peripheral nerve regeneration and functional nerve recovery after reconstructionwith a thin-walled biodegradable poly(DL-lactide-ε-caprolactone) nerve guide. CellsMater 1997;7:53-62.

5. Meek MF, Den Dunnen WFA, Schakenraad JM, Robinson PH. Evaluation of functionalnerve recovery after reconstruction with a poly(DL-lactide-ε-caprolactone) nerveguide, filled with modified denatured muscle tissue. Microsurgery 1996;17:555-561.

6. Meek MF, Den Dunnen WFA, Schakenraad JM, Robinson PH. Evaluation of severaltechniques to modify denatured muscle tissue to obtain a scaffold for peripheral nerveregeneration. Biomaterials 1999;20:401-408.

7. Lundborg G, Dahlin LB, Danielsen N, Hansson HA, Johannesson A, Longo FM,Varon S. Nerve regeneration in silicone chambers: influence of gap length and ofdistal stump components. Exp Neurol 1982;76:361-375.

8. Francel PC, Francel TJ, Mackinnon SE, Hertl C. Enhancing nerve regeneration acrossa silicone tube conduit by using interposed short-segment nerve grafts. J Neurosurg1997;87:887-892.

9. Mackinnon SE, Dellon AL. Surgery of the peripheral nerve. New York: Thieme, 1988.10. Dellon AL, Mackinnon SE. Sciatic nerve regeneration in the rat: validity of walking

track assessment in the presence of chronic contractures. Microsurgery 1989;10:220-222.

11. Buehler MJ, Seaber AV, Urbaniak JR. The relationship of functional return to varyingmethods of nerve repair. J Reconstr Microsurg 1990;6:61-69.

12. Munro CA, Szalai JP, Mackinnon SE, Midha R. Lack of association between outcomemeasures of nerve regeneration. Muscle Nerve 1998;21:1095-1097.

13. Hruska RE, Kennedy S, Bergeld SK. Quantitative aspects of normal locomotion inrats. Life Sci 1979;25:171-180.

14. Westerga J, Gramsbergen A. Development of locomotion in the rat. Dev Brain Res1990;57:163-174.

15. Bain JR, Mackinnon SE, Hudson AR, Falk RE, Falk JA, Hunter DA. The peripheralnerve allograft: an assessment of regeneration across nerve allografts in rats immuno-suppressed with cyclosporin A. Plast Reconstr Surg 1988;82:1052-1066.

16. Bain JR, Mackinnon SE, Hunter DA. Functional evaluation of complete sciatic,peroneal and posterior tibial nerve lesions in the rat. Plast Reconstr Surg 1989;83:129-136.

17. Walker JL, Evans JM, Meade P, Resig P, Sisken BF. Gait-stance duration as a measure

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Functional nerve recovery after bridging a 15 mm rat sciatic nerve gapof injury and recovery in the rat sciatic nerve model. J Neurosci Methods 1994;52:47-52.

18. De Koning P, Brakkee JH, Gispen WH. Methods for producing a reproducible crushin the sciatic and tibial nerve of the rat and rapid and precise testing of return ofsensory function. J Neurol Sci 1986;74:237-246.

19. Hoppen HJ, Leenslag JW, Pennings AJ, Van der Lei B, Robinson PH. Two-plybiodegradable nerve guide: basic aspects of design, construction and biologicalperformance. Biomaterials 1990;11:286-290.

20. Yu LT, England J, Sumner A, Larossa D, Hickey WF. Electrophysiologic evaluationof peripheral nerve regeneration through allografts immunosuppressed withcyclosporin. J Reconstr Microsurg 1990;6:317-323.

21. Gramsbergen A, IJkema-Paassen J, Meek MF. Sciatic nerve transection in the adultrat: abnormal EMG patterns during locomotion by aberrant innervation of hindlegmuscles. Exp Neurol 2000;161:183-193.

22. Gramsbergen A, Van Eykern LA, Meek MF. Sciatic nerve transection at adult andyoung age: EMG patterns during locomotion in rats. Equine Vet J 2000;in press.

23. Walker JL, Evans JM, Resig P, Guarnieri S, Meade P, Sisken BS. Enhancement offunctional recovery following a crush lesion to the rat sciatic nerve by exposure topulsed electromagnetic fields. Exp Neurol 1994;125:302-305.

24. Lin FM, Pan YC, Hom C, Sabbahi M, Shenaq S. Ankle stance angle: a functionalindex for the evaluation of sciatic nerve recovery after complete transection. J ReconstrMicrosurg 1996;12:173-177.

25. Dijkstra JR, Meek MF, Robinson PH, Gramsbergen A. Methods to evaluate functionalnerve recovery in adult rats; walking track analysis, video analysis and the withdrawalreflex. J Neurosci Methods, 2000;96:89-96.

26. Shen N, Zhu J. Application of sciatic function index in nerve functional assessment.Microsurgery 1995;16:552-555.

27. Zellem RT, Miller DW, Kenning JA, Hoenig EM, Buchheit WA. Experimentalperipheral nerve repair: environmental control directed at the cellular level.Microsurgery 1989;10:290-301.

28. Van der Zee CE, Nielander HB, Vos JP, Lopes da Silva S, Verhaagen J, OestreicherAB, Schrama LH, Schotman P, Gispen WH. Expression of growth-associated proteinB-50 (GAP43) in dorsal root ganglia and sciatic nerve during regenerative sprouting.J Neurosci 1989;9:3505-3512.

29. Hoogeveen JF, Troost D, Wondergem J, Kracht AHW, Haveman J. Hyperthermicinjury versus crush injury in the rat sciatic nerve: a comparative functional,histopathological and morphometrical study. J Neurol Sci 1992;108:55-64.

30. Van Meeteren NLU, Brakkee JH, Hamers FPT, Helders PJM, Gispen WH. Exercisetraining improves functional recovery and motor nerve conduction velocity after sciaticnerve crush lesion in the rat. Arch Phys Med Rehabil 1997;78:70-77.

31. Lundborg G, Zhao Q, Kanje M, Danielsen N, Kerns JM. Can sensory and motorcollateral sprouting be induced from intact peripheral nerve by end-to-endanastomosis? J Hand Surg [Br] 1994;19:277-282.

32. Weddell G, Guttmann L, Guttmann E. The local extension of nerve fibers intodenervated areas of skin. J Neurol Neurosurg Psychiatry 1941;4:206.

33. Devor M, Schonfeld D, Seltzer Z, Wall PD. Two modes of cutaneous reinnervationfollowing peripheral nerve injury. J Comp Neurol 1979;185:211-220.

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