Immunohistochemical study of skin reinnervation by regenerative axons

Immunohistochemical Study of SkinReinnervation by Regenerative Axons

X. NAVARRO,1 E. VERDU,1 G. WENDELSCHAFER-CRABB,2 AND W.R. KENNEDY2*1Department of Cell Biology and Physiology, Universitat Autonoma de Barcelona, E-08193,

Bellaterra, Spain2Department of Neurology, University of Minnesota, Minneapolis, MN 55455

ABSTRACTThe time sequence of sensory and sudomotor nerve regeneration to the mouse footpad

was studied between one and seven weeks after crush or section of the sciatic nerve. Proteingene product 9.5, vasoactive intestinal peptide, substance P, and calcitonin gene-relatedpeptide were localized in thick sections by using indirect immunofluorescence techniques andimaged by confocal microscopy. Nerve regeneration was visually assessed in all nervesand quantified in sweat glands. After denervation, protein gene product 9.5 immunoreactivityremained as dim fluorescence within thick fibers of dermal nerve trunks, whereas thin nervefibers to sweat glands and to epidermis disappeared. By 14 days postcrush and 35 dayspostsection, the first protein gene product 9.5 immunoreactive regenerating axons appearedin large nerve trunks, quickly extending to epidermis and sweat glands. Reinnervation ofMeissner’s corpuscles occurred nearly simultaneous with return of epidermal free nerveendings and sudomotor network. Calcitonin gene-related peptide, vasoactive intestinalpeptide, and substance P immunoreactivity disappeared completely one week after denerva-tion, then reappeared at 17–18 days postcrush and 35 days postsection. Fewer nerve fiberswere immunoreactive to these peptides than to protein gene product 9.5. The overall densityof reinnervation, although reduced, more closely resembled normal in the sweat glandsand Meissner’s corpuscles than in the epidermis. Reinnervation was more successfulafter crush than after nerve section. The time course for functional return of sweatingparalleled the return of protein gene product 9.5 immunoreactivity, whereas reappearance ofvasoactive intestinal peptide was delayed by several days. J. Comp. Neurol. 380:164–174,1997. r 1997 Wiley-Liss, Inc.

Indexing terms: nerve regeneration; epidermis; sweat glands; neuropeptides; protein gene product

9.5

The most clinically significant measure of successfulnerve regeneration after injury is return of function. Themorphological equivalent is the number of axons thatreach the correct end organs and the latency of return. Wepreviously investigated the latency and degree of return ofsweating, pain sensitivity, and neuromuscular functionin the mouse hindpaw after sciatic nerve injuries (Navarroand Kennedy, 1989; Navarro et al., 1994). We now reporton the corresponding morphological return of sudomotorand sensory nerve fibers using immunohistochemicalmethods.Immunohistochemical techniques provide more consis-

tent staining of all nerve fibers than do the classical gold,silver, and methylene blue methods, and they allowfor simultaneous localization of different types of neuro-peptide immunoreactive (-ir) nerves to the epidermis,dermis, and appendages of normal skin in mammals(Bjorklund et al., 1986; Dalsgaard et al., 1989; Wang et al.,

1990; Karanth et al., 1991; Kennedy and Wendelschafer-Crabb, 1993; Navarro et al., 1995). These newer methodshave also been used to characterize reinnervation ofdenervated skin, either by collateral sprouting from neigh-boring intact nerves or by expansive regeneration (Kishi-moto, 1984; Karanth et al., 1990; Kinnman et al., 1992;Kinnman andWiesenfeld-Hallin, 1993; Manek et al., 1993;Ann et al., 1994; Santamaria et al., 1994). The methods

Contract grant sponsor: FIS; Contract grant number: 95-0033-02; Con-tract grant sponsor: CIRIT; Contract grant number: EE93-2-219; Contractgrant sponsor: U.S. National Institutes of Health; Contract grant numbers:NS 26348, NS 31397; Contract grant sponsor: Toray Industries, Inc., Japan.*Correspondence to: Dr.W.R. Kennedy, Department of Neurology, Univer-

sity of Minnesota, Box 187 UMHC, 420 Delaware St. SE, Minneapolis, MN55455. E-mail: [email protected] 13 October 1995; Revised 28 October 1996; Accepted 20

November 1996

THE JOURNAL OF COMPARATIVE NEUROLOGY 380:164–174 (1997)

r 1997 WILEY-LISS, INC.

have potential for quantitation of nerve fiber in skinbiopsies as a method for diagnosis of diseases affectingperipheral somatic and autonomic nerves, and for evaluat-ing the effects of treatment (Karanth et al., 1989; Levy etal., 1992; McCarthy et al., 1995; Kennedy et al., 1996).Such uses will rely upon prior detailed knowledge of thedegenerative and regenerative events gained from con-trolled nerve injuries in animal models. We are using themouse model as an adjunct to our studies on cutaneousnerves in human subjects (Kennedy and Wendelschafer-Crabb, 1993; Kennedy et al., 1994; Kennedy et al., 1996).The purpose of the present study was to use immunohis-

tochemical methods with confocal microscopy to investi-gate the time course of reinnervation of epidermis, dermis,and sweat glands (SGs) in the mouse hindpaw. We previ-ously studied return of function after different types ofsciatic nerve injuries and repairs (Navarro and Kennedy,1989; Navarro et al., 1994), and chose crush and unre-paired section lesions because they are followed by signifi-cantly different degrees of reinnervation in the mouse.These two lesions also represent opposite ends of thespectrum of clinically significant nerve lesions in humans.Regenerating nerves were visualized by immunolocaliza-tion of the pan-neuronal marker protein gene product 9.5(PGP 9.5), that reveals the overall innervation because itreacts with all peripheral nerves and demarcates morenerve fibers than any previously examined nerve stain(Thompson et al., 1983; Wilson et al., 1988). We alsostudied immunoreactivity to other neuropeptides that arenormally present in nerves to cutaneous structures, suchas substance P (SP) and calcitonin gene-related peptide(CGRP) in small sensory neurons (Gibbins et al., 1985;Lindh et al., 1989; Kruger et al., 1985), and vasoactiveintestinal peptide (VIP) in sympathetic nerve fibers to SGs(Bjorklund et al., 1986; Landis and Fredieu, 1986).

MATERIALS AND METHODS

Surgical procedure

Two groups of Swiss female mice, aged 2.5 months, wereoperated on under pentobarbital anesthesia (60 mg/kg.,i.p.). In 38 mice, the right sciatic nerve was exposed inmid-thigh and crushed for 30 seconds three times insuccession with a Dumont No. 5 biological forceps. In 12mice the right sciatic nerve was sectioned with microscis-sors and left unrepaired. Crush and section weremade at apoint 45 mm proximal to the tip of the third toe. The rightsaphenous nerve of all mice was cut below the femoraltriangle and a long segment of the distal stump removed toprevent regeneration. The effectiveness of these surgicalprocedures to produce complete denervation of the oper-ated hindpaw was proved by the absence of nociceptiveresponses to pinprick and of plantar muscle response toelectrical stimulation of the proximal sciatic nerve, and thecessation of sweating (for details of methods see Navarroet al., 1994).At each selected time between 7 and 46 days postsur-

gery, one or two mice were re-anesthetized and the plantarpads of operated and unoperated hindpaws were carefullyremoved under a dissecting microscope, fixed in Zamboni’ssolution, then cryoprotected in 0.1 M phosphate bufferedsaline (PBS) (pH 7.4) containing 20% sucrose and stored at4°C. After collection of the samples, the mice were given alethal dose of pentobarbital and after cessation of heart-beat an open thoracotomy was performed. The protocols

were approved by the University of Minnesota’s institu-tional animal care and use committee.

Immunohistochemical staining

Samples were processed as previously described (Na-varro et al., 1995). Briefly, cryotome sections 50–60 µmthick were washed free-floating in 0.1 M PBS with 0.3%Triton X-100 and 5% normal donkey serum for 1 hour, thenincubated in primary antisera overnight at 4°C. Rabbitantisera to PGP 9.5, VIP, CGRP, and SP diluted in PBSwith Triton X-100 and normal donkey serum were used.Different foot padswere immunostained for each neuropep-tide (see Table I). Non-immune serum controls were pro-cessed concurrently. After three washes with the samediluting solution, sections were incubated in secondaryantisera (donkey anti-rabbit cyanine 3.18 labeled IgG1:200; Jackson Immunoresearch, West Grove, PA) over-night at 4°C. Following additional washes, sections wereadhered to coverslips, dehydrated in ethanol, cleared withmethyl salicylate, andmounted on slides with DPX (Fluka,Ronkonkoma, NY). Samples were observed with NikonMicrophot-SA and Olympus BX-40 microscopes equippedfor epifluorescence using appropriate filters. Selected im-ages were collected with a MRC-1000 Confocal ImagingSystem (BioRad, Boston, MA).

Quantitation of SGs innervation

We performed quantitative analysis of SGs reinnerva-tion because both functional and morphological methodsare reliable and repeatable and they can be correlated.Pilocarpine-induced sweating activity in the operated hind-paw was assessed by the silastic mold method (Navarroand Kennedy, 1989). Silastic impressions of sweat dropletsweremade from a subset of mice at different intervals afternerve crush, preceding removal of the foot pads for immu-nohistochemical staining. The number of sweat-dropletimpressions in the plantar pads was determined by count-ing under a dissecting microscope with transillumination.Nerves to SGs were quantified from laser-scanning confo-cal microscopy (LSCM) image sets of PGP 9.5 and VIPimmunostained sections. Image sets were collected using a40X plan apochromat objective (N.A. 0.95) at focal incre-ments of 2 µm. Three image sets from each time point andfor each antibody were used for analysis. Image Volumesysoftware (Minnesota Datametrics, Inc., St. Paul, MN),running on a Silicon Graphics Indigo 2 system, was used todetermine the volume of nerve per volume of SG fromvolume-rendered images.

RESULTS

Normal innervation and reinnervationafter nerve crush

Control mouse foot pads from the unoperated left hind-paw immunostained with antibody to PGP 9.5 revealed a

TABLE 1. Sampling schema and antisera used in this study

Foot pads1 Antiserum to Dilution Source

a, D, B PGP9.5 1:800 UltracloneA VIP 1:1000 AmershamC CGRP 1:1000 Amershamb SP 1:1000 Amersham

1 According to the nomenclature proposed by Kennedy et al. (1984) PGP 9.5: Pan-neuronal marker protein gene 9.5; VIP: vasoactive intestinal peptide; CGRP: calcitoningene-related peptide; SP: substance P.

REINNERVATION OF SKIN 165

complex network of nerves (Fig. 1A), as previously de-scribed in detail in foot pads of unoperated mice (Navarroet al., 1995). Nerve trunks entering from the base of thepad divided into nerve bundles. Some bundles progressedalong the periphery of the pad and formed a subepidermalnerve plexus (SNP) containing many thin and a fewlarge-diameter nerve fibers. Other nerve bundles passedthrough or swept around a central cluster of SGs to formthe SNP at the apex of the pad. En route, many thin nervefibers left the central bundles and innervated SGs. Largenerve fibers, known by their location and termination to bemyelinated (Navarro et al., 1995), terminated withindermal papilla as Meissner’s corpuscles, predominantlylocated at the apex of the pad. Other thin nerve fibersentered the epidermis and terminated in different configu-rations, varying from simple free endings to branchedcomplex endings. Brightly stained rounded cells near thebasement membrane were Merkel cells.At seven days postcrush (pc), residual diminished reac-

tivity for PGP 9.5 was present within the remainingendoneurial sheaths of large nerve bundles as distal as thepapillary dermis, ending abruptly at the site of Meissner’scorpuscles (Fig. 1B). Thin nerve fibers were no longervisible in nerve bundles, SGs, SNP, or epidermis. Becauseof the absence of bright-staining nerve fibers, low-levelbackground fluorescence was visible in SGs in a patterncharacteristic of capillaries. Bright immunoreactivity toPGP 9.5 reappeared in axons at 14–15 days pc within thelarge nerve trunks at the base of the pad, and extendednearly to the dermal papilla within existing endoneurialsheaths. No staining was present in SGs or epidermis (Fig.1C). Merkel cells were brightly stained. By 17–18 days,increased numbers of newly regenerated PGP-ir axonswere present, usually with varicosities along their length.Nerves entering the dermal papilla exhibited some spiral-ing consistent with reformation of Meissner’s corpuscles.Some thin nerve fibers penetrated a variable distance intothe apical epidermis. Much of the SNP had returned alongthe sides of the pad. Branches from the SNP, some withterminal enlargements, entered into the epidermis for ashort distance (Fig. 1D). There were few nerves in the SGsat this time.Early in the fourth week many brightly stained nerves

were present throughout the pad (Fig. 1E). Several Meiss-ner’s corpuscles had formed but the coiled endings had anabnormal configuration. Occasional axons within papil-lary tufts appeared swollen. Clustered trapezoid-shapedcells, perhaps Merkel cells, were interconnected by whatappeared to be brightly stained fibers. The number ofepidermal nerves appeared to have increased; severalextended about half-way to the corneum and had terminalvaricosities that resembled growth cones. The innervationof SGs was sparse until about 27 days pc, when theybecame more consistently innervated by many slendernerve fibers.At six weeks, innervation throughout the pad appeared

superficially to approach normal density (Fig. 1F). Meiss-ner’s corpuscles were normally distributed, although theterminal nerve coils were still not as well organized as innormal corpuscles (Fig. 2A,B). SGs had an abundantinnervation that appeared to be comparable to controlglands. Merkel cells were now difficult to detect. The mostobvious defect of reinnervation was the decreased densityof epidermal nerves and their incomplete penetration into

the epidermis. The reinnervating axons were not as wellcompartmentalized as those seen in controls. More nervetrunks passed through the center of the pad, many single(unbundled) fibers passed through the pad, and the SNPinvolved a much greater portion of the papillary dermis.CGRP immunoreactivity in control foot pads was abun-

dant in the nerve fibers of large nerve trunks that pro-gressed from the base to nerve bundles that formed adense SNP. Several axons from the SNP entered theepidermis, some penetrating almost to the corneum.Meiss-ner’s corpuscles did not react to CGRP antibody. Merkelcells were lightly stained. SG innervation was sparse (Fig.3A).After crush, CGRP-ir fibers had disappeared by seven

days (Fig. 9B). Reactivity returned around 17 days withinthe large nerve trunks at the base of the pad (Fig. 3B).During the fourth week, nerve bundles throughout the padbecame reactive and the SNP was reforming at the apexand lateral aspects of the pad (Fig. 3C). Single nerves werepresent in most dermal papilla; a few thin nerves enteredthe epidermis, and others entered some SGs. Staining wasmuch brighter during the fifth week (Fig. 3D). Most SGscontained a few CGRP-ir nerves, but branching was mini-mal. The pattern of staining in the foot pad appeared to besimilar to normal by the sixth week pc (Fig. 3E). SeveralSGs were well innervated but others were not innervated.By the seventh week pc, CGRP-ir innervation throughoutthe foot pad was heavy (Fig. 3F). The SNP was extensivebut less well organized than in the control pads. Somenerves entered the epidermis and a few extended almost tothe corneum; however, they appeared to be more widelyspaced than in control samples. Some large CGRP-ir fibersended in papillary tufts where Meissner’s corpuscles arelocated, whereas these structures were not CGRP-ir incontrol samples. Innervation was absent in some SGs, butbright-staining nerve profiles, not seen in controls, werepresent in other SGs.Substance P-ir nerves in control foot pads extended from

the large nerve trunks at the base of the pad into the SNP(Fig. 4A). A few fibers penetrated the full depth of theepidermis at approximately equal intervals. SP-ir fiberswere present in papillary tufts but were not part ofMeissner’s corpuscles. The keratinocytes and corneum ofthe epidermis were also stained. After nerve crush, immu-noreactivity to SP disappeared by the seventh day (Fig.9C). Two SP-ir fibers appeared in the proximal trunks atday 18, and over the next week a few fibers extended intothe SNP (Fig. 4B). The amount of reactivity increasedslowly over time (Fig. 4C). A few axons entered theepidermis by the sixth and seventh weeks, but less fre-quently than in normal samples (Fig. 4D). The innervationof SGs by SP-ir nerves was minimal at seven weeks, butmore obvious than in control samples.Normal foot pads had a dense network of VIP-ir nerves

in SGs (Fig. 5A). SG staining was similar to staining withPGP 9.5 except that the staining pattern in individualnerve fibers was more reticular. VIP-ir fibers were absentin the epidermis, but there was occasional staining ofnerves in the apical SNP; some of these extended into thepapillary tufts. VIP immunoreactivity disappeared withinthe first week pc (Fig. 9D). It first reappeared at day 17 asbright staining in nerve trunks passing through the centerof the foot pad (Fig. 5B). By three weeks some nervesextended from the trunk into the SNP, basal areas of the

166 X. NAVARRO ET AL.

epidermis, and papillary tufts (Fig. 5C). There were fewnerves in the SGs. During the following weeks the innerva-tion of SGs increased. VIP-ir nerves in the SNP began to

decrease, but still appeared to be more numerous thanthose seen in controls (Fig. 5D). SG innervation graduallyincreased to a near normal appearance by six weeks.

Fig. 1. Laser scanning confocal micrographs (LSCM) of proteingene product 9.5 (PGP 9.5) immunofluorescence in control (A) andreinnervating (B–F) mouse foot pads following sciatic nerve crush.Images were projected from 10–15 optical sections acquired at 5 µmintervals. A: Nerves in control foot pad form a dense network.Sweatglands (SGs) are very heavily innervated. Large trunks extendthrough the central SG area to form the subepidermal nerve plexus(SNP). Many nerves arise from this plexus to innervate the epidermisandMeissner’s corpuscles in the tufts of papillary dermis.B:By 7 dayspostcrush (pc) immunofluorescence is greatly reduced. Faint stainingis only present in large nerve trunks, but not in the epidermis and SG

areas.C:At day 14 pc, PGP 9.5-ir fibers returned in large nerve trunksfrom the base of the pad to the papillary dermis. D: At day 18 pc, PGP9.5 stain is brighter. The large nerve trunks divide to form the SNP.Few single fibers enter the epidermis for a short distance. Meissner’scorpuscles are reforming in dermal papilla. E:At day 22 pc, axons arebrighter and more distinct throughout the pad. The SNP is morecomplex and numerous axons extend halfway through the epidermis.Meissner’s corpuscles are well formed. F: By day 46 pc, the pattern ofinnervation resembles the controls, but regenerated nerve fibers tonerve trunks, SNP, and SGs are less well compartmentalized, and theepidermis has shorter nerve fibers. Scale bars 5 200 µm.


Reinnervation after nerve section

After transection of the sciatic nerve, PGP 9.5-ir profileswere seen mainly in the endoneurial sheaths of formertrunks throughout the dermis with progressively de-creased intensity. At 28 days postsection (ps), the firstregenerating fibers were observed within nerve trunks atthe base of proximal pads. By day 35 a sparse SNP waspresent, and some nerves with growth cone terminationsextended into the dermal papilla (Fig. 6A). Rarely, a nervefiber entered an SG. By day 42, SGs contained manynerves, although innervation was far from normal (Fig.6B). Intra-epidermal nerve fibers were rarely observed,and those present penetrated the epidermis only a shortdistance (Fig. 6C). No corpuscular endings had re-formed.Innervation in the remainder of the pad increased onlyslightly at later times. In general, after nerve section theregenerated PGP 9.5-ir profiles were of considerably lowerdensity and thinner than in control or postcrush samples.CGRP-ir nerves were first seen at 35 days ps in small

groups at the base of the pad, but only a few nervesreached the superficial dermis. By day 42, more nerves

were present (Fig. 7A). Some SGs contained a sparse nervefiber network. There was also a thin SNP with some fibersinto the papillary dermis and possibly a few in theepidermis.Only a few SP-ir nerves had reached the base of the pad

by day 35 ps, and only one was seen approaching the apex.However, by day 42 the trunks contained several SP-irnerves; some approached SGs, and an occasional nervereached the epidermis via a thin SNP (Fig. 7B).Reinnervation by VIP-ir nerve fibers after nerve section

was the least successful. There were several VIP-ir nervesin the main nerve trunks, but only a small number hadentered the SGs by day 42 ps; a few were in the SNP, andnone were in the epidermis (Fig. 7C).

Quantitative results of SGs reinnervation

One week after sciatic nerve crush there was no func-tional response of SGs to pilocarpine stimulation. The firstreactive SGs reappeared after 18 days pc in plantar pads,indicating that functional reinnervation had started; theyincreased in number on later tests. Quantitative analysisof nerves in SGs at 7 days pc showed very low levels of PGP9.5 and an absence of VIP reactivity. The percent of volumeoccupied by PGP 9.5-ir nerves increased, from 14 days pc,to reach values that were near those in unoperated controlpads from 39 days pc. On the other hand, the levels ofVIP-ir nerves were null until 20 days pc, very low from 20to 25 days, and increased rapidly from 27 days, reachingmaximal values slightly lower than in control pads. Thecorrelation between quantitative values of functional recov-ery and of immunohistochemical reinnervation (Fig. 8)was highly significant both for PGP 9.5 and for VIPlabeling (p , 0.0001, simple regression test).

DISCUSSION

A detailed sequential observation of skin reinnervationby regenerating nerves was made using the pan-neuronalmarker PGP 9.5, seen in thick sections under confocalmicroscopy to reveal the spatial organization of nervefibers within the mouse foot pad. Labeling with antibodiesto CGRP, SP, and VIP added information about specificaxonal subpopulations. Reappearance of PGP 9.5-ir fiberspreceded by a short time the reappearance of functionalrecovery in target organs, whereas reappearance of detect-able levels of transmitter neuropeptides was delayed, asdemonstrated in the quantitative evaluation of SGs rein-nervation.The complete disappearance of neuropeptide immunore-

activity to CGRP, SP, and VIP in the foot pad nerves soonafter a lesion is consistent with the interruption of theirsupply from the neuronal body after axotomy, and therapid elimination of their content from the degeneratingdebris. PGP 9.5 immunoreactivity was also absent fromareas formerly innervated by thin fibers such as epidermisand SGs. However, faint PGP 9.5-ir stain remained inlarge nerve fibers of the dermal nerve trunks until reinner-vation began, evidenced by the appearance of brightlystained slender axons. Other authors have reported com-plete disappearance of PGP 9.5 in skin flaps within a fewdays after denervation (Karanth et al., 1990; Manek et al.,1993). However, Figure 3c of Manek et al. (1993) appearsto show minimal residual PGP 9.5 fluorescence in skinnerve trunks. Another report showed a few PGP 9.5-irfibers in denervated foot pads eight weeks after graft

Fig. 2. Higher magnification LSCM of PGP 9.5 labeling in theapical area of control (A) and 46 day pc (B) mouse foot pads. Imagesprojected from 26 optical sections acquired at 1 µm intervals. A:Myelinated fibers are present in the SNP and extend to innervateMeissner’s corpuscles in the apex of the foot pad. Many nerve endingsare present between Meissner’s corpuscles, extending into superficiallayers of the epidermis. B: The spiral pattern of Meissner’s corpusclesis present, but less well formed than in controls. Thin fibers in theepidermis often exhibit branching and follow a tortuous route. Scalebars 5 25 µm.


repair of guinea pig nerve (Santamaria et al., 1994). Wehave observed that faint PGP 9.5 labeling remains as longas 13 weeks in foot pads of the chronically denervatedmouse paw (unpublished observations). The residual PGP9.5 reactivity is likely retained in vacated endoneurial

tubes in the same location at which myelin protein PO-irfibers are detectable (Navarro et al., 1995). Its appearancewas accentuated in the thick sections used in our study.Although retention of PGP 9.5 immunoreactivity might beconfounding in short-term studies of reinnervation, its

Fig. 3. LSCM of calcitonin gene-related peptide (CGRP) immunore-activity in control (A) and reinnervating (B–F) mouse foot pads.Images projected from 11–19 optical sections acquired at 5 µmintervals. A: CGRP reactivity resides in large nerve trunks, whichbranch to form the SNP. Many fine fibers enter the epidermis. B: Atday 17 pc, CGRP-immunoreactive (ir) fibers reappear in large nervetrunks from the base of the pad to the subepidermal area.C:At day 25pc, CGRP-ir fibers are present throughout the pad. Large nerve trunks

give rise to fibers that innervate some SGs. The SNP is continuous.D:At day 32 pc, CGRP labeling has increased in the SNP and in the SGs.E:At day 39 pc, the SNP contains many CGRP-ir nerves. Many fibersenter epidermis but extend only about halfway to the corneum. SomeSGs contain bright CGRP-ir nerves. F: At day 46 pc, the SNP isextensive but not well organized. CGRP-ir nerve fibers are present inthe epidermis, but few reach the corneum. Some SGs have brightlystained nerve profiles. Scale bars 5 200 µm.


presence did not interfere with detection of the intenselylabeled, newly regenerating axons in our serial study. Infact, residual activity was helpful for locating the originalmajor nerve pathways through the target tissue. In stud-ies of human polyneuropathy, in which disease processesare protracted, PGP 9.5 has provided excellent labeling ofsurviving neural elements (Karanth et al., 1989; Levy etal., 1992; McCarthy et al., 1995; Kennedy et al., 1996).As expected, reinnervation was more successful after

nerve crush than after section. Re-entry of regeneratingaxons into the foot pad was marked by the reappearance ofbrightly stained PGP 9.5-ir axon profiles at 14–15 daysafter sciatic nerve crush and 28–35 days after nervesection. The new nerve profiles appeared first as brightlystained regenerating fibers within the empty nerve trunksheaths in the proximal part of the pad, and soon after inthe epidermis and SGs.After nerve crush, most Meissner’scorpuscles were reinnervated but the morphology of thecoiled endings was abnormal. Although many thin nervefibers approached the epidermis, they appeared to be fewerin number than in normal skin and few penetrated morethan half-way to the corneum. Recently, Stankovic et al.(1996) found a similar defect, with intraepidermal axonsoccurring mainly in the basal layer, after crush or sectionrepaired by suture of the sciatic nerve in rats. On the other

hand, we found that SGs were well reinnervated, althoughthe nerves appeared to be thinner than in control pads.Quantitation of nerve fibers within SGs revealed that theamount of PGP-ir nerve in reinnervated samples reachedlevels similar to controls by 39 days after crush injury.Immunoreactivity to VIP and CGRP reappeared at 17

days after crush and increased in parallel. SP-ir fibersarrived one day later and increased more slowly. Thenear-simultaneous reappearance of VIP, CGRP, and SP isin disagreement with observations of Karanth et al. (1990),who found earlier reinnervation by CGRP- and SP-ir thanfor VIP and NPY-ir fibers in skin flaps of the mouse.Therefore our observations do not support the putativetrophic roles attributed to those neuropeptides (Terenghiet al., 1986; Karanth et al., 1990). The difference betweenthe types of target cells in the skin of the dorsum of theback, studied by Karanth et al., and the foot pads used inour study in which many SGs are profusely innervated byVIP-ir axons, may account for these variations. Compar-atively, CGRP-ir fibers achieved higher levels of reinnerva-tion than SP- and VIP-ir fibers, the difference being moremarked after crush than after section. McLachlan et al.(1994) also found that regenerating CGRP-ir fibers pro-vided a higher degree of distal reinnervation than didSP-ir fibers in the skin of the rat tail.

Fig. 4. LSCM of substance P (SP) immunofluorescence in control(A) and reinnervating (B–C) mouse foot pads. Images projected from11–16 optical sections acquired at 5 µm intervals. A: SP-ir nerves arepresent in large nerve trunks and SNP. Single fibers penetrate to deepepidermis at about 120 µm intervals. B:At day 22 pc, SP-ir fibers first

reappear in nerve trunks; some reach the papillary dermis. C: By day32 pc, SP-ir fibers are seen in the large nerve trunks extending to theepidermal area. Some SGs have reactivity.D: By day 39 pc, more SP-irfibers reach the SNP surrounding the central SG area. Scale bars 5200 µm.


Reinnervating nerves fibers did not always express thesame immunoreactivity found in normal foot pads. Incontrol mouse foot pads, VIP-ir nerves are abundant inSGs and only occasionally reach the base of the epidermis;and CGRP-ir nerves are mainly in the SNP and epidermalfree nerve endings, with a limited number in contact withblood vessels and SGs tubules (Navarro et al., 1995). It wastherefore unusual that the mid-stages of reinnervation(25–35 days postcrush and 42–46 days postsection) exhib-ited comparatively more extensive immunoreactivity forVIP in subepidermal and epidermal fibers and for CGRPand SP among the SGs. These abnormal patterns may beexplained bymisdirectional growth of sensory and sudomo-tor fibers within the foot pads, as well as by shifts in theneuronal synthesis of neuropeptides. Following peripheralaxotomy, the expression of neuropeptides and their recep-tors is considerably changed in primary sensory neurons;it mainly involves the downregulation of excitatory neuro-peptides such as SP and CGRP, and the upregulation ofneuropeptide Y, mostly in large neurons, and of VIP andgalanin in small andmedium-sized neurons (Hokfelt et al.,1994; Noguchi et al., 1993). Axotomy also induces VIPexpression in secretomotor ganglionic sympathetic neu-rons (Klimaschewski et al., 1994). The expression of

neurotransmitters by sympathetic neurons is also influ-enced by the target tissue (Stevens and Landis, 1990).Changes in peptide expression are thought to representadaptive responses of the organism to promote neuronalsurvival and regeneration (Hokfelt et al., 1994). Theseplastic changes are long-lasting, unless axons regenerateand reestablish peripheral connections (Himes and Tessler,1989; Nielsh and Keen, 1989). The abnormal pattern ofreinnervation tended to regress to a more normal-likedistribution in the last intervals of our study. These latechanges might be due to the retraction of terminal axons,or to a new change in the synthesis and expression ofneuropeptides of axotomized neurons that reinnervatedincorrect target cells. In long-term studies of nerve regen-eration, neuropeptide expression has been shown to bemarkedly modified when regenerating axons reinnervateinappropriate targets (McMahon andGibson, 1987;McMa-hon et al., 1989), suggesting the existence of regulatoryinfluences by factors that arise in peripheral tissues.Therefore, neuropeptides can provide semi-specific label-ing of reinnervation in different target organs, althoughthey may not label the original type of nerve fibers. It isalso recognized that the presence of immunolabeled axons

Fig. 5. LSCMof vasoactive intestinal peptide (VIP) immunofluores-cence in control (A) and reinnervating (B–D) mouse foot pads. Imagesprojected from 7–17 optical sections acquired at 5 µm intervals. A:VIP-ir fibers are seen mainly in nerves to SGs. Occasionally, a smallamount of reactivity is near the apex of the pad, but does not extendinto epidermis. B: At day 17 pc, VIP-ir profiles first appear in nerve

trunks leading to the apex where single fibers disperse in the SNP. C:At day 21 pc, VIP-ir fibers are present in large nerve trunks and in theextensive SNP. Reactivity is not seen in SGs. D: At day 46 pc, VIP-irfibers are primarily in the SGs. A small amount of reactivity is presentin the apical SNP. Scale bars 5 200 µm.


in the skin does not always imply a corresponding recoveryof function (Kinnman andWiesenfeld-Hallin, 1993).Reappearance of regenerating PGP 9.5-ir axons to skin

and SGs occurred before the return of nociceptive and

sweating responses, which averaged 18 days after crushinjury in our previous studies (Navarro and Kennedy,1989; Navarro et al., 1994). After a crush lesion, functionalrecovery was evident earlier than the accumulation of

Fig. 6. LSCM of PGP 9.5 immunofluorescence in reinnervatingmouse foot pads after sciatic nerve section. A: At day 35 postsection(ps), PGP 9.5-ir profiles are present in large nerve trunks and the SNP.Image projected from 15 optical sections at 2 µm intervals. B: At day42 ps, SGs are innervated, myelinated nerves are in dermal papilla,but nerve fibers rarely enter epidermis. Image projected from 11optical sections at 2 µm intervals. C: Detail of day 42 ps, epidermisdepicts thick fibers entering dermal papilla and a few fibers in theepidermis. Image projected from 17 optical sections at 1 µm intervals.Scale bars 5 200 µm inA,B, 50 µm in C.

Fig. 7. LSCM of neuropeptide immunofluorescence in reinner-vated mouse foot pads after sciatic nerve section. Images projectedfrom 10–19 optical sections acquired at 2 µm intervals.A:At day 42 ps,CGRP-ir fibers are present in large nerve trunks and in the SNP, butnot in SGs and epidermis. B: At day 42 ps, SP-ir profiles are in thenerve trunks with a few fibers seen in the SNP. C: At day 42 ps, lightVIP immunoreactivity was detected at the large nerve trunks, withsome fibers running within the SGs and a few reaching the SNP. Scalebars 5 200 µm.


visible amounts of transmitter neuropeptides immunoreac-tivity in nerves reaching the epidermis or SG tubules.Although slightly different levels of the nerve lesion anddifferent intervals chosen for sampling may invalidatestrict comparison between histological and functional stud-ies, it appears that once nerves re-enter the foot pad,synaptic reconnection and functional restoration occurrapidly (Gutmann et al., 1944). In our study, reinnervationof Meissner’s corpuscles and epidermis occurred simulta-neously, but both had abnormal configurations. Our histo-logical findings are in agreement with Gutmann et al.(1944) who estimated that the rate of axon return to the

end organ was probably identical for myelinated (motor)and unmyelinated (pain) nerves after crush.The number of nerve profiles and the density of reinner-

vation progressed rapidly during a three-week period afterthe initial reappearance of growing axons in dermal nervetrunks; then the number and density appeared to stabilizein both experimental groups. The overall density of reinner-vation of all structures by nerves immunoreactive to eachlocalized neuropeptide was close to normal levels after acrush injury, but much worse after nerve transection.Aftera repaired section, the number and extension of PGP 9.5-iraxons reinnervating the epidermis has been shown to bereduced as compared to crush (Stankovic et al., 1996).Other studies have also shown that the number of reinner-vated corpuscular terminals is reduced after crush, andeven fewer after unrepaired nerve section (Wong andKanagasuntheram, 1971; Nurse et al., 1984; Zelena, 1984;Koshima et al., 1993). We previously found that the degreeof functional recovery of pain and sweating was also betterfollowing crush injury than after nerve section (Navarro etal., 1994). The parallel quantitation of SGs reinnervationshowed that morphological reappearance of PGP 9.5-irnerves was well correlated by recovery of the sweatingfunction, both parameters reaching levels of about 90% ofpreoperative control values by the sixth week after nervecrush. Further correlation of return of function and mor-phology after nerve lesions for other target organs willrequire simultaneous quantitative, repeatable measure-ments of both in the same experiment.

ACKNOWLEDGMENTS

This work was supported by grants from FIS (95-0033-02) and CIRIT (EE93-2-219) (Spain); from the U.S. Na-tional Institutes of Health (NS 26348, NS 31397); and fromToray Industries, Inc. (Japan). We thank Michele Illies,

Fig. 8. Number of reactive sweat glands (SGs) and percentage ofvolume occupied by PGP 9.5- and VIP-ir nerves in the SGs of foot padsafter sciatic nerve crush. For each time point, the number of reactiveSGs is the sum of those secreting in the six plantar pads, while the

percentage of nerve is the average of measures from three sets ofimages obtained through LSCM. PGP9.5, protein gene product 9.5;VIP, vasoactive intestinal peptide; ir, immunoreactive.

Fig. 9. LSCM of (A) a control foot pad processed without primaryantibody, and denervated pads at 7 days pc incubated with antibodiesagainst (B) CGRP, (C) SP, and (D) VIP. Note the absence of immuno-fluorescence throughout the dermis and epidermis. Dim fluorescenceappears in the stratum corneum. Scale bars 5 200 µm.


Sally Haugen, Tim Johnson, and Dr. Joaquim Fores fortechnical assistance.

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Immunohistochemical study of skin reinnervation by regenerative axons