INTERNATIONAL JOURNAL OF LEPROSY^

Volume 62, Number 1Printed in the U.S.A.

Denatured Muscle Grafts for Nerve Repair in anExperimental Model of Nerve Damage in Leprosy.

2. Recovery of Peripheral Peptide-Containing NervesAssessed by Quantitative Immunohistochemical Study'

Luis Santamaria, Giorgio Terenghi, Jill Curtis,Gail E. de Blaquiére, Jerome H. Pereira, John L. Turk,

and Julia M. Polak 2

Several studies have indicated the presenceof peptide-containing nerves in differentstructures of the human skin (1,4, I I. 14, 19, 25, 30).

The most abundant cutaneous neuropep-tides include substance P (SP), calcitoningene-related peptide (CGRP), vasoactiveintestinal polypeptide (VIP), neuropeptidetyrosine (NPY) and its C-flanking peptide(CPON). Nerves immunoreactive for theseneuropeptides appear to be involved in avariety of cutaneous diseases where theyshow characteristic and quantifiable changes(18, 26, 28) .

Nerve damage is a regular feature of thepathology and clinical symptomatology ofleprosy (2,22,23). Previous studies haveshown marked depletion of neuropeptide-containing nerves in skin biopsies of leprosypatients ( 15 ), which reflects the cutaneoussensory and autonomic dysfunctions ob-served in this disease. Similar neuropeptidechanges were also observed in an animalmodel of leprosy ( 13 ).

Recently, a guinea pig model of leprosyneuropathy was established (3). Furtherstudies have shown that the nerve damagecould be repaired using muscle autografts,as assessed by the recovery of sensory and

' Received for publication on 24 July 1992; acceptedfor publication in revised form on 7 December 1993.

2 L. Santamaria, M.D.; G. Terenghi, Ph.D.; J. M.Polak, D.Sc., Department of Histochemistry, RoyalPostgraduate Medical School, Hammersmith Hospi-tal, London, U.K. J. Curtis, Ph.D.; G. E. de Blaquiêre,B.Sc.; J. H. Pereira, F.R.C.S.; J. L. Turk, M.D., De-partment of Pathology and Anatomy, Royal College ofSurgeons of England, London, U.K.

Reprint requests to Dr. G. Terenghi at his presentaddress: Blond Mclndoe Centre, Queen Victoria Hos-pital, East Grinstead, Sussex RH19 3DZ, U.K.

motor functions and by histology of thenerve distal to the graft ( 20). In view of themorphometric and functional results foundafter muscle grafting for nerve repair ( 6 ), itwas of interest to determine whether thesefindings might be paralleled by neuronalchanges in the skin, particularly in relationto the repair of sensory and autonomic fi-bers.

Since there are no immunohistochemicalstudies of the alterations of neuropeptide-containing nerves in this experimentalmodel, the aim of this investigation was toassess by image analysis quantification therecovery of neuropeptide-containing nervesin the guinea pig foot-pad skin at differenttimes after granuloma excision and graftingwith denatured autologous muscle. Anti-bodies for the general neuronal marker pro-tein gene product 9.5 (PGP) (5.9 ) were usedto define the distribution of all cutaneousinnervation. Specific subpopulations ofnerve fibers were identified by their im-munoreactivity to the sensory neuropep-tides CGRP and SP, and to the autonomicneuropeptides VIP and CPON.

MATERIALS AND METHODSAnimal model

Cobalt - irradiated Mycobacterium lepraeorganisms (10 8 ) suspended in 0.01 ml salinewere injected into the tibial fascicle of theright sciatic nerve of 27 guinea pigs (250-300 g) as described previously (6). Autolo-gous muscle grafting was carried out 5 weeksafter granuloma induction, as previouslydescribed. Groups of animals (at least sixper group) were killed at 8, 12, 16 and 20weeks after muscle grafting.

64

62, 1^Santamaria, et al.: Recovery After Muscle Grafts for Nerve Repair^65

THE TABLE. Antibodies used to quantify neuropeptide-containing nerves in the guineapig foot-pad skin.

Antigen Species No. Dilution Source

PGP 9.5 Rabbit 1648 1/1000 UltracloneCGRP Rabbit 1204 1/1000 Hammersmith HospitalSP Rabbit 1651 1/1000 Hammersmith HospitalVIP Rabbit 652 1/2000 Hammersmith HospitalCPON Rabbit 1555 1/400 Hammersmith Hospital

The second layer antiserum used was an anti-rabbit immunoglobulin-FITC complex (Tago, n. 2198) raisedin goat and diluted at 1:100.

ImmunohistochemistrySkin from the area of the foot pad inner-

vated only by the tibial nerve was collectedfrom the grafted limb (experimental speci-mens) and from the contralateral controlside. The tissues were fixed by immersionin Zamboni's fluid for 18 hr at 4°C, washedin phosphate buffered saline (PBS; pH 7.2)containing 15% sucrose and 10 pg/ml so-dium azide, and stored at 4°C prior to thepreparation of cryostat blocks.

Frozen sections (10-pm thick) were col-lected onto poly-L-lysine-coated slides andallowed to dry for 1 hr at room temperature.Skin sections were immunostained by anindirect immunofluorescence method (i 5 )using a variety of antibodies (The Table).Serial sections were cut and collected se-quentially onto seven slides such that everyseventh section was stained for the sameantigen. At least four sections were stainedwith each antiserum.

Quantitative evaluation of immunoreactivefibers

Quantification was carried out on thecontrol and experimental tissue of animalskilled at 8 and 20 weeks after grafting, asthese were the first point of electrophysiol-ogy and morphometrical analysis, and thelongest time point, respectively ( 6 ). In allanimals, samples from both experimentaland contralateral (control) sides were eval-uated.

Immunoreactive nerves were quantifiedin the different skin areas according to theirdistribution and functional significance. Theselected skin compartments were as follows:

Epidermis and papillary dermis. CGRPand SP immunoreactivities were evaluatedin these areas since these peptides are known

to subserve sensory functions at the dermo-epidermal junction and control the regula-tion of papillary blood vessel tone.

Sweat glands. Imunoreactivities forCGRP and VIP were quantified in the nerveplexus around the sweat glands.

Hypodermal blood vessels. Nerves im-munoreactive for CGRP and CPON, whichare known to modulate vasomotor tone,were evaluated in the adventitial plexus.

Hypodermal nerve bundles. To evaluatethe growth of regenerating nerve fibersCGRP, SP, CPON, and VIP immunoreac-tivities were measured in nerve bundles.

General topography of nerves. To evalu-ate the general topography of the nerves,PGP antibodies were used and the stainingquantified in all areas of the skin.

Quantification was carried out using amethod previously described ( 26). In brief,a low-light video camera (PanasonicMV 1900) was mounted on a Vanox fluo-rescence microscope (Olympus, U.K.) andlinked to a VIDAS analyzer (Kontron, U.K.).A long pass filter around 560 nm was usedto reduce autofluorescence. The field imagewas defined by use of interactively definedframes in order to establish precisely thearea to be measured while deleting any un-wanted part of the image present on thescreen. The image of immunostained struc-ture was enhanced to increase the signal/noise contrast. A background substractionprocedure was carried out by taking themaximum grey value of background as thethreshold at which to segregate the image.The binary image obtained in this way wasthen measured according to the selected pa-rameters.

The chosen parameters of measurementwere total field fluorescence area (pm 2 ), in-tercept counts, and field counts of the im-

66^ International Journal of Leprosy^ 1994

FIG. 1. Dermo-epidermal nerve plexus from foot-pad skin of a control specimen showing (a) abundant PGPimmunoreactive fibers and (b) fewer CGRP-immunoreactive nerves. The same skin area at 8 weeks after musclegraft: (c) few POP immunoreactive fibers are seen; (d) the CGRP-immunoreactive fibers have disappearedcompletely. At 20 weeks after grafting, a significant recovery was found for (e) PGP-immunoreactive and (f)CGRP-immunoreactive nerves. Indirect immunofluorescence method (all x 320).

62, 1^Santamaria, et al.: Recovery-11.ter Muscle Grafts for Nerve Repair^67

munoreactive nerves. For each immuno-staining in every specimen, two randomsections were analyzed and in each sectionthree fields ( x 20 objective magnification)were measured for every area as definedabove. The data are presented as the meanvalues of the total counts for each skin com-partment. The statistical analysis of the re-sults was carried out using analysis of vari-ance with logged data to ensure that thenormality assumption of the ANOVA wasvalid. Multiple t tests were used to comparethe values at different time points for eachnerve population. The p values quoted havebeen adjusted using the Bonferroni method.

RESULTSImmunohistochemistry

The quantitative results of immunofluo-rescence area, intercept counts and fieldcounts gave consistent results when controland experimental tissues were compared.Hence, a single description of the quanti-tative results which refers to all parametersis given. A full statistical analysis was car-ried out between the mean values of PGPimmunoreactivity of the controls, 8-weekand 20-week timepoints, and the analysisof variance showed significant heterogene-ity (p < 0.001). For the neuropeptide-im-munoreactive nerves, a statistical compar-ison could be carried out only between thecontrols and experimental sides at 20 weeks.In experimental tissue at 8 weeks, only afew immunoreactive fibers were observedin some sections, while most of the mea-sured fields gave a negative result.

Epidermis/papillary dermisNumerous PGP-immunorcactive fibers

were seen in the epidermis and papillarydermis of control samples (Fig. la) while amoderate amount of CGRP- and SP-im-munoreactive nerve were observed, partic-ularly around the capillaries of the papillaryplexus (Fig. lb).

At 8 weeks after grafting, few PGP-im-munoreactive nerves were present in theepidermis and papillary dermis (Fig. lc),and quantitative assessment showed a sig-nificant decrease of PGP-immunorcactivenerves (p < 0.01) (Fig. 2a). CGRP- and SP-immunoreactive fibers were sharply de-creased in the epidermis and around the

microvessels in the papillary dermis (Fig.1 d).

At 12 weeks postoperatively, some degreeof recovery was seen for all immunoreac-tivities when compared to samples at 8weeks, indicating that at this point reinner-vation of the skin can be detected by im-munohistochemistry. The number of im-munoreactive nerves increased progressivelywith time, and at 20 weeks all immuno-reactive nerves appeared widely distributedto both epidermis and papillary dermis (Fig.1, e and f). Quantitative results showed nosignificant difference for PGP, CGRP andSP immunoreactivities between the con-trols and the 20-week samples (Fig. 2a).

Sweat glandsIn control samples, numerous nerves im-

munoreactive for PGP, CGRP and VIP werefound around the sweat glands (Fig. 3a).Following muscle graft, CGRP- and VIP-immunoreactive nerves were absent at 8weeks postoperatively (Fig. 3b). The situa-tion remained unchanged up to 12 weeks,followed by a rapid increase, particularly forVIP-immunoreactive nerves, to nearly nor-mal levels by 20 weeks (Fig. 3c).

Quantification showed a difference forPGP when comparing controls versus 8weeks and controls versus 20 weeks (p <0.01) (Fig. 2b), and for CGRP between thecontrols and 20-week groups (p < 0.05). Nostatistically significant difference was foundfor VIP-immunoreactive nerves between thecontrols and the 20-week groups (Fig. 2b).

Blood vesselsIn control samples, moderate amounts of

PGP-, CGRP- and CPON-immunoreac-tive nerves were observed around dermaland hypodermal blood vessels (Fig. 4a). At8 weeks postoperatively no immunoreac-tivity for CGRP or CPON was observed(Fig. 4b). A certain amount of recovery wasseen at 20 weeks (Fig. 4c), but the immu-noreactive fibers were not as numerous asin the control samples. These results wereconsistent with those of quantification,which showed a difference between the con-trols and 20-week muscle grafts for CGRP(p < 0.01) and CPON (p = 0.003), but nosignificant difference between the two groupsfor PGP (Fig. 2c).

PGP^CGRP^SP PGP^CGRP^VIP

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CPON^VIPPGP^CGRP^SPPGP^CGRP^CPON

C) BLOOD VESSELS^ D) NERVE BUNDLE

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68^ International Journal of Leprosy^ 1994

A) PAPILLARY DERMIS^ B) SWEAT GLANDS

FIG. 2. Diagrams showing the variations of PGP, CGRP, SP, CPON and VIP immunoreactive area (t.tm 2 )per field in different skin compartments. All data are expressed as mean values with the S.E.M. represented byerror bars. (A) Epidermis and papillary dermis: no measurement could be made for CGRP and SP at 8 weeksafter grafting since these types of nerves were almost completely absent (**p < 0.01). (B) Sweat glands: nomeasurement could be made for CGRP and VIP at 8 weeks after grafting (*p < 0.05; **p < 0.01). (C) Dermalblood vessels: no measurement could be made for any of the immunoreactivities at 8 weeks (**p < 0.01). (D)Dermal nerve bundles: no peptide immunoreactivity could be measured at 8 weeks after grafting (**p < 0.01).

Hypodermal nerve bundlesThese structures showed a strong im-

munoreactivity for PGP (Fig. 4d) in the con-trol samples. A moderate number ofall typesof immunoreactive fibers was observedwithin nerve bundles in the dermis. Follow-ing muscle grafting, the immunoreactivitiesfor the markers and the neuropeptidesshowed a similar pattern of low levels at 8weeks (Fig. 4e) followed by an increase.However, their recovery was faster thanthose observed in other skin areas, beingalready evident at 16 weeks. At 20 weeksafter muscle graft the immunoreactivenerves showed no major difference to thosein the control tissues (Fig. 4f). This patternof change was reflected in the quantificationresults for all immunoreactivities whichshowed a difference for PGP between thecontrols and 8-week groups (p < 0.01) but

no significant difference between the con-trols and the 20-week groups for all im-munoreactivities (Fig. 2d).

DISCUSSIONThis study shows low numbers of PGP-

and neuropeptide-immunoreactive fibers inguinea pig foot-pad skin at the first time-point following muscle grafting, which is in-dicative of an incomplete reinnervationprocess in the peripheral tissues. Neuropep-tide changes were particularly evident forCGRP- and SP-immunoreactive nerves inthe papillary dermis, for CGRP- and VIP-immunoreactive fibers around sweat glands,and for CGRP- and CPON-immunoreac-tive fibers innervating blood vessels. At lat-er timepoints, there was a gradual reinner-vation by all nerve types in most skin areas,consistent with the results of morphometri-

62, 1^Santamaria, et al.: Recovery After Muscle Grafts for Nerve Repair^69

FIG. 3. (a) = Abundant VIP immunoreactive fibersarc observed around the secretory portions of sweatglands in control specimens. (b) = At 8 weeks after

cal analysis, electrophysiology and func-tional recovery (6 ).

In this study, quantitative immunohis-tochemistry was of value since it allowedan objective determination of the neuralchanges at the different timepoints. This wasparticularly important when assessing theextent of immunoreactive nerve recovery inthe different skin compartments since it canbe difficult to assess, by subjective micro-scopical examination, possible differencesbetween samples with abundant numbers ofnerves, such as are observed in control andexperimental tissues at 20 weeks after graft-ing.

Interestingly, the distribution pattern ofneuronal terminals after reinnervation issimilar to that of the control tissue. At pres-ent it is not entirely clear what mechanismis regulating this phenomenon, but it hasbeen suggested that the reinnervation pro-cess in denervated tissue might follow theexisting pathway of degenerated nerves ( 8 ).At 8 weeks after grafting, some recovery ofimmunoreactivity was evident for PGP,while only a few neuropeptide-immuno-reactive nerves were observed. This is prob-ably due to the fact that a recovery of thenerve terminal structure, as indicated byPGP immunostaining, might have startedat this time, although the transport of neu-ropeptides from the neuronal cell body tothe terminal was still affected. Hence, it islikely that nerve function in relation to thestudied neuropeptides may still be im-paired. However, it cannot be excluded thatother neuropeptides/neurotransmitters,which were not identified in this study, maybe present in the nerve terminals and mightbe responsible for some functional responseas observed in some animals at 8 weeks ( 6 ).

The recovery of immunoreactivities forneuronal markers and neuropeptides did notoccur within the same timeframe in all skincompartments. At 20 weeks after musclegrafting, quantification of perivascular PGP-immunoreactive fibers showed consider-able recovery with all measured parameters,

muscle graft the VIP immunoreactive fibers are totallydepleted. (c) = A significant recovery of VIP immu-noreactive fibers around sweat glands was observed 20weeks after muscle grafting. Indirect immunofluores-cence method (all x 310).

70^ International Journal of Leprosy^ 1994

fFla. 4. (a) = Control specimen: CPON immunoreactive fibers in the adventitial plexus from a hypodermal

blood vessel. (b) = Complete disappearance of adventitial CPON immunoreactive nerves is found at 8 weeksafter muscle grafting. (c) = At 20 weeks after graft, the CPON immunoreactive fibers innervating a hypodermalblood vessel show a significant recovery. (d) = Hypodermal nerve bundles in the dermis with intense PGPimmunoreactivity (arrows) are seen in a control specimen. (e) = Marked decrease (arrows) can be seen in thenumber of PGP immunoreactive fibers in hypodermal nerve bundles at 8 weeks after muscle grafting. (f) = A

62, 1^Santamaria, et al.: Recovery After Muscle Grafts for Nerve Repair^71

and no statistical difference was seen be-tween control and experimental sides. How-ever, the recovery of CGRP- and CPON-immunoreactive nerves was not completeat 20 weeks, as demonstrated by quantifi-cation. This would indicate complete struc-tural recovery, but a slower recovery relatedto neuropeptide functions. By contrast,nerve bundles in the dermis and nerve fibersin the epidermal/subepidermal layer showeda similar degree of recovery for all immu-noreactivities. The recovery of nerve bun-dles appeared at an earlier time than fornerves in other areas, since the bundles rep-resent the initial pathway of nerve regen-eration before the fibers branch out into sin-gle terminal fibers.

It is interesting to note that 20 weeks aftergrafting a more complete nerve recovery wasfound for sensory nerves at the dermo-epi-dermal junction. This might be related tothe possible action of neurotrophic factorssecreted by the keratinocytes (10, 24, 29, 31)

since these substances may improve thenerve fiber growth in this area. All of theguinea pigs harvested at 20 weeks in thisexperiment showed a normal response tothe crude sensory test used (6 ). Autonomicimmunoreactive nerves related to sweatglands and blood vessels showed a slowerrecovery, which is parallel to the slow re-covery of motor nerve functional tests aftermuscle grafting (6, 20s .) It also has been notedin other reinnervation experiments thatsensory CGRP-immunoreactive nerves arethe first to appear (12, 17 , .) Similarly, in fetalskin CGRP-immunoreactive nerves are thefirst neuropeptide-containing fibers to bedetected, soon after the appearance of PGP-immunoreactive nerve (27 ). It is thought thatCGRP may have a trophic role ( 7 ) and con-tribute to the healing and repair processes( a ), hence actively contributing to the rein-nervation process.

A recent pilot study has shown that themuscle-grafting technique applied to lep-rosy patients produces a considerable im-provement of sensory perception ( 21 ). Theseresults in man are consistent with the ex-

perimental evidence in the animal modelshowing a return to normal amounts of sen-sory neuropeptide-containing nerves in der-mo-epidermal junction after long-termmuscle grafts. The partial recovery ofCGRP,CPON and VIP immunorcactive fibers, in-nervating blood vessels and sweat glands,indicates that the vasomotor and secretoryfunctions also can be improved by musclegrafting, although a longer time than for sen-sory nerves might be needed for full recov-ery.

SUMMARYA marked depletion of neuropeptide-im-

munoreactive nerves, a consequence of thenerve damage which is commonly found inleprosy, has been reported in peripheral tis-sues of leprosy patients and of a leprosyanimal model. The aim of this study was toinvestigate peripheral reinnervation follow-ing a denatured autologous muscle graft inan animal model of leprosy nerve damage.Possible reinnervation of the foot-pad skinwas studied by immunohistochcmistry us-ing antisera to the neuronal marker proteingene product 9.5 (PGP), the neuropeptidescalcitonin gene-related peptide (CGRP),substance P (SP), vasoactive intestinal pep-tide (VIP), and the C-flanking peptide ofneuropeptide Y (CPON). The extent of thereinnervation process was assessed by im-age analysis quantification at different timepoints. At 8 weeks after muscle grafting,there were small numbers of immunoreac-tive nerves (p < 0.05). At 12, 16, and 20weeks postoperatively there was a gradualincrease in all immunostaining. At 20 weeks,no significant difference was found for PGP-,CGRP-, and SP-immunoreactive nerves inthe epidermal and subepidermal layerscompared to control (contralateral) tissue.In experimental tissue the recovery of im-munoreactive nerves around sweat glandstook longer (up to 12 weeks) than in otherskin compartments, but after that time therecovery was rapid and at 20 weeks no dif-ference was measured for VIP-immuno-reactive nerves in comparison with con-

4—

significant recovery of PGP immunoreactive fibers from hypodermal nerve bundles (arrows) is found at 20weeks after graft. Indirect immunofluorescence method (a, b, c x 380; d, e, f x 300).

72^ International Journal of Leprosy^ 1994

trols. Around blood vessels, the recovery ofCGRP- and CPON-immunoreactive fiberswas slow, and at 20 weeks a difference withcontrol samples (p < 0.01) was noted. Inthe same area, there was no significant dif-ference for PGP immunoreactivity betweencontrols and tissues at 20 weeks. In contrast,the immunoreactive nerve bundles in thedermis showed a faster recovery than nervesin other skin areas, with amounts similar tocontrols at 20 weeks. The significant recov-ery of immunoreactivc nerves, in particularof those containing sensory neuropeptide,is consistent with the described functionalrecovery.

RESUMENSc ha reportado una marcada depleciOn do nervios

inmunoreactivos a neuropeptidos (una consecuencia

del dafio ncrvioso comunmente encontrado en la lepra)

tanto en los tejidos perifericos de los pacientes con

lepra como en un modelo animal de Ia enfermedad. Elobjetivo de este estudio fue el investigar la reinervaciOn

periFerica subsecuente a un injerto autOlogo de miisculo

desnaturalizado en un modclo animal en el que la lepraindujo un claim nervioso. La reinervaciOn de la piel de

la almohadilla plantar se estudiO por inmunohisto-quimica usando antisueros contra las siguientes subs-

tancias: el marcador neuronal PGP 9.5 (PGP), los neu-

ropepetidos rclacionados con la calcitonina (CGRP),Ia substancia P (SP), el peptido vasoactivo intestinal

(VIP), y el peptido C que flanquca al neuropeptido Y(CPON). El grado del proceso de reinervaciOn se es-tableciO "cuantificando" las imagenes a diferentes in-

tervalos de tiempo. Ocho semanas despues del injertohubieron mimeros muy pequefios de nervios inmu-

noreactivos (p < 0.05). A las 12, 16, y 20 semanas se

note, el incremento gruadual en todas las inmunotin-

clones. A las 20 semanas no se encontraron diferencias

significativas con el tejido control contralateral encuanto a Ia reactividad para PGP, CGRP, y SP de los

nervios de las capas epidermica y subcpidermica. Enel tejido experimental, la recuperaciOn de los nervios

inmunoreactivos alrededor de las glandulas sudori-

paras tome, mds tiempo (hasta 12 semanas) que en losotros compartimentos de la piel pero despues de ese

tiempo la recuperaciOn fue rapida y a las 20 semanasno se encontraron diferencias con los controles en cuantoa los nervios VIP-inmunoreactivos. Alrededor de los

vasos sanguineos, la recuperaciOn de las fibras CGRP-y CPON-inmunoreactivos fue lenta, y a las 20 semanas

se note, una diferencia con las muestras control (p <0.01). En la misma area, a las 20 semanas no hubo una

diferencia significativa de la inmunoreactividad a PGPentre los controles y los tejidos experimentales. En con-trasts, los haces de nervios inmunoreactivos en la der-mis mostraron una recuperaciOn mas rapida que losnervios en otras areas de la piel con mimeros similares

a los de los controles a las 20 semanas. La significanterecuperaciOn de los nervios inmunoreactivos, en par-

ticular de aquellos que conticnen neuropeptidos sen-soriales, es consistente con Ia recuperaciOn funcional

dcscrita previamente.

RESUME

Une perte marquee de nerfs immunoreactifs aux

neuropeptidos, une consequence des lesions nerveusesfrequemment retrouvee dans Ia lepre, a etc rapporteedans les tissus peripheriques de patients lepreux et d'un

modele animal de la lepre. Le but de setts etude etaitd'êtudier la reinnervation peripherique apres une greffe

denatures d'un muscle autologue sur un modele animal

ayant tine lesion ncrvcuse de lepre. La reinnervationdu coussinct plantaire a etc êtudiee par immunochimic

en utilisant des antisera vis-a-vis du produit geniqueproteique 9.5 du marqueur neuronal (PGP), du peptide

genique de Ia calcitonine des neuropeptides (CGRP),

de la substance P (SP), du peptide intestinal vasoactif

(VIP), et du peptide C du neuropeptide Y (CPON).

L'etendue du processus de reinnervation a etc evalueepar quantification analytiquc d'imagcs a difierents mo-

ments. Iluit semaines apres la greffe de muscle, it y

avait un petit nombre de nerfs immunoreactifs (p <0.05). Aux douzieme, seizie.me et vingtieme semaine

apres ('operation, it y avait une augmentation pro-

gressive de toutes les reactions immunologiques. Apres

20 semaines, aucune difFerence significative n'a ete ob-serves pour Ics nerfs reagissant au PGP, CGRP et SP

dans les couches épidermiques et sous-epidermiques

par rapport au tissu de controle (contralateral). Dansle tissu de l'experimentation, Ia recuperation des nerfs

immunoreactifs prit plus longtemps autour des glandessudoripares (jusqu'a 12 semaines) que dans les autresregions de la pcau, mail apres cc moment, la recupe-

ration a etc rapide, et apres 20 semaines, on n'a mesureaucune difference entre Ics nerfs immunoreactifs au

VIP et les controles. Autour des vaisscaux sanguins, Ia

recuperation des fibres immunoreactives au CGRP etCPON etait iente, et apres 20 semaines, on a note une

difference avec Ics echantillons de controle (p < 0.01).

Dans Ia memo zone, il n'y avait pas de difFerence sig-nificative apres 20 semaines pour l'immunoreactivite

au PGP entre les controles et les tissus. Par contrasts,

les paquets de nerfs immunoreactifs dans le derme

montraient une recuperation plus rapide que les nerfsdans les autres regions de la peau, avec une intensitó

similaire aux controles apres 20 semaines. La recu-peration significative des nerfs immunoreactifs, en par-

ticulier ceux contenant du neuropeptide sensoriel, est

coherente avec la recuperation fonctionnelle decrite.

Acknowledgment. This work was supported byLEPRA, The Colt Foundation, and The Grand Charity

of Freemasons. LS was supported by Grant BE90-062from the Ministerio de Educacion y Ciencia, Spain.

The authors arc grateful to Deborah Price, MedicalPhysics Department, Hammersmith Hospital, for sta-

tistical analysis of the data.

62, 1^Santamaria, et al.: Recovery After Muscle Grafts for Nerve Repair^73

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