Expression of capsaicin receptor immunoreactivity in human peripheral nervous system and in painful neuropathies

RESEARCH REPORT

Expression of capsaicin receptor immunoreactivityin human peripheral nervous system and in

painful neuropathies

Giuseppe Lauria1, Michela Morbin2, Raffaella Lombardi1, Raffaella Capobianco2,Francesca Camozzi1, Davide Pareyson3, Mauro Manconi4, and Pierangelo Geppetti5

1Neuromuscular Diseases; 2Neuropathology; and 3Biochemistry and Genetics Units, National Neurological Institute‘Carlo Besta’; 4Department of Neurology, S. Raffaele University Hospital and Scientific Institute, Milan; and

5Department of Clinical Pharmacology, University of Florence, Florence, Italy

Abstract We describe the expression of the capsaicin receptor (TRPV1) in humanperipheral nervous system (PNS) and its changes in sural nerve and skin nerve fibersof patients with painful neuropathy. Dorsal root ganglion (DRG), root, and spinal cordautopsy specimens from subjects without PNS diseases were immunoassayed with anti-TRPV1 antibodies. Bright-field and confocal microscope studies using anti-TRPV1, proteingene product 9.5 (PGP 9.5), and unique-b-tubulin (TuJ1) antibodies were performed inskin biopsies from 15 healthy subjects and 10 painful neuropathies. The density of intra-epidermal nerve fiber (IENF) labeled by each antibody was quantified. Sural nerve biopsiesfrom three patients with painful, one patient with nonpainful diabetic neuropathy, andtwo patients with multifocal motor neuropathy used as controls were immunoassayedwith anti-TRPV1 antibodies and investigated by immunoelectron microscopy. TRPV1strongly labeled laminae I and II of dorsal horns, most small-size and some medium-sizeDRG neurons, and small-diameter axons of dorsal roots. In sural nerve, TRPV1 was ex-pressed within the cytoplasm of most unmyelinated and some small myelinated axons,in the muscular lamina of epineural vessels, and in the endothelium of endoneurial ves-sels. The density of IENF labeled by TRPV1, PGP 9.5, and TuJ1 did not differ. TRPV1 colo-calized with TuJ1 in all IENF and dermal nerve bundles. Painful neuropathies showeda diffuse loss of TRPV1-positive axons both in the sural nerve and in the skin. Our find-ings demonstrated that TRPV1 is normally expressed throughout the nociceptive path-way of PNS and that TRPV1-positive peripheral nerve fibers degenerate in painfulneuropathies.

Key words: capsaicin receptor, dorsal root ganglia, immunohistochemistry, painfulneuropathy, skin biopsy, spinal cord, sural nerve biopsy, TRPV1, vanilloid type 1

IntroductionCapsaicin receptor (TRPV1) was the first cloned

member of the transient receptor potential (TRP)family of vanilloid proteins (Caterina et al., 1997).TRPV1 is a ligand-gated, nonselective cation channelactivated, besides capsaicin, by heat in the noxious

Address correspondence to: Dr. Giuseppe Lauria, NeuromuscularDiseases Unit, National Neurological Institute ‘Carlo Besta’, ViaCeloria, 11, 20133 Milan, Italy. Tel: þ39-02-2394-2378; Fax: þ39-02-7063-3874; E-mail: [email protected]

Journal of the Peripheral Nervous System 11:262–271 (2006)

ª 2006 Peripheral Nerve Society 262 Blackwell Publishing

range, tissue acidification, and endogenous lipid sig-naling molecules, such as the endocannabinoid anand-amide and lipoxygenase products (Di Marzo et al.,2002). TRPV1 is able to integrate the simultaneousexposure to these ligands, which have synergisticeffects and modulate, together with the phosphoryla-tion state of the channel, its temperature threshold(Vellani et al., 2001).

TRP receptors have a general role in thermosen-sation, on which pain-related behavior of animalslargely depend (Benham et al., 2003). TRPV1 wasshown to be essential for selective modalities of painsensation and for thermal hyperalgesia induced bytissue inflammation (Davis et al., 2000). Due to itsresponsiveness to capsaicin, which is peculiar amongTRP family members, TRPV1 was suggested to havea role also in the pathogenesis of neuropathic paininduced by nerve injury (Hudson et al., 2001; Fukuokaet al., 2002; Rashid et al., 2003), though this remainsa controversial hypothesis (Szallasi and Blumberg,1999). The nociceptors, a subgroup of dorsal rootganglion (DRG) neurons, and their peripheral pro-cesses, the unmyelinated C fibers, are characterizedby their sensitivity to capsaicin that is responsible forcell excitation and pain perception (Szolcsanyi, 1993).Paradoxically, capsaicin was used as an analgesicagent in humans (The Capsaicin Study Group, 1991)because it induces a long-term desensitization ofnociceptor terminals after prolonged exposure. Thisphenomenon is based on the destruction of sensoryaxons and, eventually, of DRG nociceptors (Jancsoet al., 1977). The expression of TRPV1 was widelystudied in human DRG and intestinal mucosal layers(Cortright et al., 2001; Yiangou et al., 2001; Smithet al., 2002; Chan et al., 2003), but it was neverinvestigated in detail throughout the peripheral ner-vous system (PNS). Skin biopsy is the ideal tool toinvestigate the expression of TRPV1 in small-diame-ter sensory nerve fibers. In fact, intraepidermal nervefibers (IENF) are somatic unmyelinated axons arisingfrom small-size DRG neurons. They can be reliablyimmunostained and quantified using cytoplasmaticand cytoskeletal markers, and their density isreduced in patients with painful neuropathy (Lauriaet al., 2004). Recently, immunoreactivity of TRPV1was assessed in different cutaneous structures(Stander et al., 2004). The aims of this study were (1)to examine the normal distribution of TRPV1 immu-noreactivity in spinal cord, DRG, roots, sural nerve,and skin nerve fibers; (2) to compare the distributionof TRPV1 immunoreactivity in skin nerve fibers withthat of specific cytoskeletal markers; and (3) to inves-tigate the changes of TRPV1-positive axon density insural nerve and skin nerve fibers from patients withpainful neuropathy.

Materials and MethodsNormal TRPV1 immunoreactivity was investi-

gated by immunohistochemistry in spinal cord, DRG,root, and skin specimens from subjects with no signsand symptoms of PNS involvement. Sural nerve biop-sies from two patients with multifocal motor neuropa-thy and normal sensory nerve conduction were usedas normal controls. TRPV1 expression was investi-gated in sural nerve biopsies from one patient withsevere and one patient with milder diabetic neuropa-thy who complained of similar degree of neuropathicpain. Sural nerve biopsy from one patient with non-painful diabetic neuropathy was also immunoassayedwith anti-TRPV1 antibodies. Skin biopsies from 15healthy controls and 10 patients with painful neuropa-thy of different etiology were immunostained withanti-TRPV1, anti-protein gene product 9.5 (PGP 9.5),and anti-unique-b-tubulin (TuJ1) antibodies usingeither bright-field immunohistochemistry or indirectimmunofluorescence with confocal microscopy forcolocalization studies. In each tissue, negative con-trols included replacement of primary antibodies withpreimmune serum. Specificity of TRPV1 immunos-taining was confirmed by preincubation of primaryantibody with cognate peptide antigen (mg of anti-body:mg of peptide ¼ 1:150 or 1:300).

DRG, roots, and spinal cord

Autopsy specimens from five patients with noclinical and pathological signs of PNS involvementwere examined. One patient had a cerebellar medullo-blastoma (male, 38 years; patient 1), one had a meta-static B-cell lymphoma (male, 63 years; patient 2),one had lung and brain bacterial abscesses (female,49 years; patient 3), one had a degenerative dementia(male, 68 years; patient 4), and one had a malignantthymoma associated with myasthenia gravis (female,49 years; patient 5). DRG and spinal cord specimensfrom cervical, dorsal, lumbar, and sacral levels werecollected from routine autopsies performed 18–48 hpostmortem. Tissue was fixed in formalin, dehydratedin graded alcohol, and embedded in paraffin. Sectionswere stained with hematoxylin–eosin (H&E), andNissl and Woelcke stain for myelin. DRGs were col-lected from patients 1, 2, and 3, whereas spinal cordspecimens were collected from patients 1, 3, 4, and5. Paraffin-embedded sections were immunostainedwith anti-TRPV1 antibodies (1:1,500; GlaxoSmithKlineR&D). Sections of 5- to 8-mm thickness were col-lected on gelatinized slides, deparaffinized in xylene,and rehydrated in graded alcohol. Slides were sub-merged in 3% H2O2 and pretreated with 20% goatserum and 1% Triton X-100 in Tris–HCl, pH 7.4, for1 h. Anti-TPVR1 antibodies were diluted 1:3,000 in

Lauria et al. Journal of the Peripheral Nervous System 11:262–271 (2006)

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0.5% Tris–HCl, pH 7.4, with 0.5% Triton X-100. Im-munoreaction product was demonstrated using theEnVision Plus/Horseradish Peroxidase system forrabbit immunoglobulins (DakoCytomation) and 3-30-diaminobenzidine (DakoCytomation). In DRG sections,alkaline phosphatase was also used to visualize thereaction in order to distinguish TRPV1 from lipofuscinstaining.

Sural nerve biopsy

Archive whole sural nerve biopsies obtained fordiagnostic purposes were examined. Normal biopsiesfrom two patients with multifocal motor neuropathywere used as controls and compared with thoseobtained from three patients with painful and onepatient with nonpainful diabetic neuropathy. One seg-ment of sural nerve was fixed in formalin, dehydratedin graded alcohol, embedded in paraffin for routinestaining (H&E), and immunoassayed with anti-TRPV1antibodies. Another segment was fixed in 2.5%electron microscopy (EM)-grade glutaraldehyde (FlukaChemie, AG) in 0.05 M phosphate-buffered saline(PBS) at pH 7.4, cut into small blocks, post-fixed in1% osmium tetroxide (Electron Microscopy Sciences,EMS) in 0.05 M PBS, dehydrated in graded acetone,and embedded in epoxy resin (Spurr; EMS) for ultra-structural examination. Immunoelectron microscopy(I-EM) was performed on sural nerves from controlsand patients with painful neuropathies. Briefly, 80-nmsections placed on 200-mesh formvar-carbon-coatednickel grids (EMS) were etched in 1% sodium period-ate for 60 min. Endogenous peroxidase was blocked,and sections were deosmicated with 3% hydrogenperoxide in methanol for 10 min. Antigen expressionwas enhanced with 0.5% Triton X-100 for 10 min.Residual aldehyde groups were quenched with 0.05M glycine (Sigma) in PBS, pH 4, for 5 min. Primaryanti-TPVR1 antibodies diluted (1:200) in Aurion-bovineserum albumin (BSA) incubation buffer (Aurion) wereapplied on grid overnight at 4�C. After rinsing inAurion-BSA, grids were incubated for 3 h at roomtemperature with goat anti-rabbit antibody conjugatedwith 10 nm gold (GAR-10) antibody (Aurion) 1:30diluted in PBS. Sections were fixed in 1% glutaralde-hyde, postfixed in 1% OsO4, counterstained with ura-nyl acetate and lead citrate, and viewed under anelectron microscope (Zeiss EM109).

Skin biopsy

Archive skin biopsies obtained from the proximalregion of the thigh (20 cm below the iliac spine; Pth)and the distal region of the leg (10 cm above the lat-eral malleolus, within sural nerve territory; Dl) in 15healthy subjects and 10 patients with painful neuropa-thy were examined. Neuropathy was associated with

diabetes in six patients and with hepatitis C virus inone patient, whereas it was idiopathic in three pa-tients. Specimens were immunoassayed for IENFquantification with polyclonal anti-PGP 9.5 (1:1,000;Biogenesis Ltd.), monoclonal anti-TuJ1 (1:2,000; Berk-ley Antibody Company), and polyclonal anti-humanTRPV1 (1:1,500; GlaxoSmithKline R&D) antibodiesunder bright-field microscopy using a free-floatingprotocol as previously described (Lauria et al., 2004).Two blinded observers quantified in three sectionsfrom each biopsy the linear density of IENF (IENF/mm) labeled by each marker. The total number of indi-vidual IENF crossing the dermal-epidermal junctionwas counted under a light microscope at 40�, withthe assistance of a microscope-mounted video cam-era. Secondary branchings of IENF were excludedfrom the quantification. The length of the epidermiswas measured using a computerized system (Micro-science, Inc.), and the linear density of IENF (IENF/mm) obtained. Densities were compared using thetwo-tailed Student’s t test. Interobserver agreementwas assessed by the Spearman’s correlation coeffi-cient.

Double staining studies with confocal microscopywere performed using a previously described protocol(Lauria et al., 2004) on three further sections fromboth controls and patients with painful neuropathies,using the following combinations: anti-PGP 9.5 andanti-TuJ1 antibodies, and anti-TRPV1 and anti-TuJ1 an-tibodies. Samples were viewed using appropriate fil-ters with a MicroRadiance 2000 Confocal ImagingSystem (Bio-Rad) and a Nikon 600 fluorescencemicroscope. Each image was collected in successiveframes of 0.5–1 mm apart at 40� magnification andwas integrated by a computerized system (Laser-Sharp 2000; Bio-Rad).

ResultsSpinal cord and roots

At each level, laminae I and II of dorsal hornswere strongly immunostained by TRPV1, whereasventral horns, corticospinal tracts, and posterior tractswere negative. TRPV1 labeled several axons of dorsalroots (Fig. 1). Sections immunoassayed with anti-TRPV1 antibodies preincubated with the cognatepeptide antigen were negative.

Dorsal root ganglia

TRPV1 stained the cytoplasm of most small-sizeand some medium-size neurons, sparing the nuclei. Alllarge-size cells were negative (Fig. 2). TRPV1 labelingcould be easily distinguished from that of lipofuscin,which is commonly found in DRG neurons, by alkaline


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phosphatase chromogen staining. Sections immuno-assayed with anti-TRPV1 antibodies preincubated withthe cognate peptide antigen were negative.

Sural nerve

Biopsies from patients with multifocal motorneuropathy were unremarkable. In these, TRPV1

immunostained most unmyelinated axons, fewersmall myelinated fibers, the muscular lamina of mostepineural vessels, and the endothelium of some en-doneurial vessels. Conversely, all large myelinated fi-bers were negative (Figs. 3A and 3B). I-EM confirmedthat TRPV1 was expressed within the cytoplasm ofmost unmyelinated axons and in some small myelin-ated fibers (Fig. 4). Patients with painful diabetic neu-ropathy showed a diffuse loss of myelinated andunmyelinated axons and thickening of endoneurialvessels. The loss of TRPV1-positive axons was pro-portional to the overall loss of nerve fibers. TRPV1immunoreactivity was not enhanced in the sparing fi-bers (Figs. 3C and 3D). Similar findings wereobserved in sural nerve biopsy from the patient withnonpainful diabetic neuropathy (data not shown). Sec-tions immunoassayed with anti-TRPV1 antibodies pre-incubated with the cognate peptide antigen werenegative.

Skin nerve fibers

In healthy subjects, dermal nerve bundles andIENF showed a strong immunoreactivity to TRPV1(Fig. 5). The density of TRPV1-positive IENF at thePth (22.5 � 4.9 SD/mm) and the Dl (14.3 � 3.0 SD/mm) did not differ from that obtained with PGP 9.5(Pth 22.6 � 4.8 SD/mm � Dl 14.4 � 3.0 SD/mm) andTuJ1 (Pth 22.5 � 4.9 SD/mm � Dl 14.3 � 2.9 SD/mm)immunostaining. Double staining confocal microscope

Figure 1. (A) TRPV1 immunostaining in human spinal cord at the lumbar level. Note the intense immunoreactivity in thedorsal horns (arrows), whereas ventral horns, corticospinal tracts, posterior tracts, and ventral roots are negative (bar ¼ 50mm). (B) High magnification image showing TRPV1 immunoreactivity in the laminae I and II (bar ¼ 50 mm). (C) High magnifi-cation image showing TRPV1 immunoreactivity in most axons of dorsal root (bar ¼ 10 mm). (D) High magnification imageshowing the absence of TRPV1 immunoreactivity in ventral root (bar ¼ 10 mm).

Figure 2. TRPV1 immunoreactivity in human dorsal rootganglion. The cytoplasm of most small-size and somemedium-size neurons is intensely labeled, whereas nucleiare spared. All large-size neurons are negative (bar ¼ 250mm). Inset shows one small-size TRPV1-positive neuronand two large-size negative neurons (bar ¼ 50 mm).


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studies confirmed that TRPV1 diffusely colocalizedwith the cytoskeletal marker TuJ1, which colocalizedwith the cytoplasmatic marker PGP 9.5 (Figs. 6A to6C). This provided an indirect clue that TRPV1 is ex-pressed in all skin nerve fibers immunostained withPGP 9.5, which is currently considered the most reli-able marker for IENF.

The density of IENF labeled by TRPV1 was signifi-cantly lower (p , 0.01) in painful neuropathies than incontrols at both the Pth (10.3 � 3.5 SD/mm) and theDl (3.4 � 3.3 SD/mm). Densities did not differ fromthose obtained with PGP 9.5 (Pth 10.3 � 3.5 SD/mm� Dl 3.5 � 3.1 SD/mm) and TuJ1 (Pth 10.1 � 4.4 SD/mm � Dl 3.2 � 4.1 SD/mm) labeling. One patient withsevere hepatitis C-associated neuropathy showeda complete loss of skin fibers with either PGP 9.5,TuJ1, or TRPV1 labeling. These findings were

confirmed by double staining studies with confocalmicroscopy (Figs. 6D to 6I). Sections immunoassayedwith anti-TRPV1 antibodies preincubated with thecognate peptide antigen were negative.

DiscussionCapsaicin, the pungent component of hot chili

peppers, is traditionally used to identify the subtypesof neurons and nerve fibers involved in hyperalgesia(Szallasi and Blumberg, 1999). Its effect is associatedwith the activation of small-diameter afferent fibersand specialized DRG neurons able to respond to abroad number of noxious stimuli (Szolcsanyi, 1993).Capsaicin also mediates some actions of the endo-cannabinoid CB1 anandamide that shares the same

Figure 3. TRPV1 immunostaining in paraffin-embedded sural nerve biopsy. (A) In control nerve, TRPV1 intensely labeledepineural vessels (arrows) and endoneurial fibers (bar ¼ 1 mm). (B) Higher magnification of the same nerve (bar ¼ 25 mm).Note the intense immunoreactivity of unmyelinated fibers, whereas large myelinated fibers (arrows) are negative. (C)Patient with painful diabetic neuropathy shows a moderate loss of TRPV1-positive axons (bar ¼ 20 mm). (D) Patient withpainful diabetic neuropathy shows a severe loss of TRPV1-positive axons (bar ¼ 20 mm).


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intracellular binding site on TRPV1 (Di Marzo et al.,2002; Szoke et al., 2002).

Several inflammatory conditions are able toenhance the expression of TRPV1 and even increasethe density of TRPV1-positive axons (Carlton andCoggeshall, 2001; Yiangou et al., 2001; Trevisaniet al., 2002; Chan et al., 2003; Gopinath et al., 2005),suggesting that this receptor is involved in the patho-genesis of specific types of hyperalgesia and mightbe a therapeutic target. Conversely, the role of TRPV1in the pathogenesis of neuropathic pain induced bynerve degeneration, such as in peripheral neuropa-thies, remains controversial. In experimental models

of neuropathic pain induced by sciatic nerve lesion,the expression of TRPV1 in DRG neurons eitherincreased (Hudson et al., 2001; Fukuoka et al., 2002;Rashid et al., 2003) or did not change (Szallasi andBlumberg, 1999; Macdonald et al., 2001). Axotomyresulted in the downregulation of mRNATRPV1 in DRGneurons (Michael and Priestley, 1999) and, in knock-out mice, TRPV1 did not contribute to nerve injury-induced hyperalgesia (Caterina et al., 2000). Overall,these findings might imply that TRPV1 is not primarilyinvolved in the pathogenesis of pain associated withperipheral neuropathies. Nevertheless, pretreatmentwith TRPV1 antiserum reduced thermal allodynia and

Figure 4. Immunoelectron microscopy with anti-TRPV1 antibodies in control sural nerve biopsy. (A) TRPV1 expression inunmyelinated fibers is revealed by gold particles. (B) TRPV1-negative unmyelinated fibers. (C) TRPV1-positive small myelin-ated fiber. (D) TRPV1 immunoreactivity is absent in large myelinated fiber.


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hyperalgesia in diabetic mice (Kamei et al., 2001) andTRPV1 antagonists reversed inflammatory and neuro-pathic pain (Walker et al., 2003).

Our study focused on the normal distribution ofTRPV1 in human PNS and on its changes in peripheralnerve fibers in patients with painful neuropathy. Wefound that TRPV1 is expressed throughout the noci-ceptive pathway, from the superficial laminae ofdorsal horns to skin unmyelinated axons. This isconsistent with previous reports in rat, mouse(Szallasi et al., 1995; Guo et al., 1999; Stenholm et al.,2002; Hwang and Valtschanoff, 2003), and humantrigeminal and DRG neurons (Caterina et al., 1997;Hayes et al., 2000; Cortright et al., 2001). In suralnerve, TRPV1 strongly labeled also epineural and en-doneurial vessels.

In hairy skin, TRPV1 proved to be an excellentmarker for IENF, which can be definitely considerednociceptor terminals. TRPV1 colocalized with the cyto-skeletal marker TuJ1 that is expressed by all the ax-ons stained by PGP 9.5, the most reliable marker forcutaneous nerve fibers (Griffin et al., 2001). Recently,Stander and colleagues (2004) reported that onlyoccasional IENF were weakly immunoreactive toTRPV1 in human skin, whereas most dermal myelin-ated nerve fibers were strongly stained. These find-ings are partly in contrast with our results and mightbe explained by the different specificity of the anti-bodies used.

In the second part of the study, we investigatedthe expression of TRPV1 in sural nerve and skin nervefibers in painful neuropathies. Intriguingly, we foundthat in patients with painful neuropathy, the density ofTRPV1-positive IENF was significantly lower than thatin healthy subjects but, like in healthy subjects, it didnot differ from that obtained using PGP 9.5 and TuJ1labeling. These observations were strengthened by

colocalization studies with confocal microscopy. Con-sistently, sural nerve biopsies showed a loss ofTRPV1-positive axons that was proportional to theoverall degeneration of nerve fibers. A similar loss ofTRPV1-positive axons was observed in one patientwith nonpainful diabetic neuropathy. The lack ofTRPV1 immunoreactivity might be caused by thedecreased expression of the protein.

Recently, an increased density of TRPV1-positiveIENF was described in one patient with post-herpeticneuralgia (PHN) compared with the skin in the normalcontralateral dermatome in which only rare fibersstained by TRPV1 were found (Petersen et al., 2002).This finding raised the hypothesis that TRPV1 is upre-gulated in regenerating axons of affected skin andis involved in the pathogenesis of neuropathic pain.However, the evidence that in the PHN area, most ax-ons were exclusively stained by TRPV1, but not byPGP 9.5, would imply that regenerating nerve fibersdid not express the ubiquitin carboxyl-terminal hydro-lase, a cytosolic enzyme widely diffuse in the PNS(Wilkinson et al., 1989), thus making this observationnot conclusive. Overall, these findings are in contrastwith previous data showing skin reinnervation aftertruncal diabetic neuropathy (Lauria et al., 1998) andthe results of this study.

The lower density of TRPV1-positive IENF wefound in painful neuropathies might reflect the lack ofregenerating axons in the skin of these patients.However, it may more likely indicate that TRPV1 andepidermal axons are not primarily involved in thepathogenesis of neuropathic pain associated withperipheral neuropathies. IENF density is a reliable toolfor diagnosing painful small-fiber neuropathy in pa-tients with no clinical and electrophysiological abnor-malities (Lauria et al., 2005). Nevertheless, clinicalstudies did not reveal a correlation between the den-sity of IENF and the degree of pain in peripheral neu-ropathies (Polydefkis et al., 2002; Pan et al., 2003).This finding suggests that skin biopsy can be used toassess small-fiber degeneration rather than small-fiberdysfunction. In fact, patients with persisting painfulsymptoms can show a complete denervation of theskin (Holland et al., 1998; Herrmann et al., 1999), asoccurred in one of our patients. We observed a dif-fuse loss of TRPV1-positive axons also in sural nervebiopsy from one patient with nonpainful diabeticneuropathy. These observations make TRPV1 a non-specific marker of unmyelinated fiber degeneration.

What structures are primarily involved in the path-ogenesis of neuropathic pain associated with periph-eral neuropathies remains poorly understood. Ourfindings suggest that the involvement of sensorynerve endings is unlikely. Neuropathic pain may bemaintained by changes of excitability in proximal

Figure 5. Skin biopsy taken in a healthy subject 10 cmabove the lateral malleolus. Bright-field immunohistochem-istry with anti-TRPV1 antibodies. Note the strong immuno-reactivity to TRPV1 of intraepidermal nerve fiber (arrows)and dermal nerve bundles (arrowheads). Bar ¼ 10 mm.


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nerve fibers and DRG neurons or by remodeling ofcentral nervous system pain pathways. TRPV1 ex-pression changes in DRG neurons, demonstrated inan experimental model of painful diabetic neuropathy(Hong and Wiley, 2005), might occur also in humanpainful neuropathies. Conversely, an upregulation ofTRPV1 expression in sural and skin nerve fibers isunlikely to occur. We showed that the density ofTRPV1-positive axons decreased parallel to the overallloss of fibers, irrespective of the degree of pain, bothin sural nerve and in skin biopsies. In particular, onepatient with severe neuropathic pain due to hepatitisC-associated neuropathy showed a complete skindenervation. This is in keeping with the hypothesisthat degeneration of skin nerve fibers is not the

primary cause of neuropathic pain in peripheral neu-ropathies but rather represents the earliest evidenceof the dying back process occurring in sensory ax-onopathies. Nevertheless, nerve fibers in the deeperlayers of the skin and resident cells might play a rolein neuropathic pain. It was recently demonstratedthat human keratinocytes express TRPV3, a warm-sensitive member of the TRP family that is activatedat innocuous temperatures, shows increased responseat noxious temperatures (Peier et al., 2002), and isable to activate cutaneous sensory nociceptors (Cookand McCleskey 2002; Southall et al., 2003). Theimmunoreactivity to TRPV3, and to the homologousTRPV4, in epidermal keratinocytes was increased inpatients with breast pain and tenderness (Gopinath

Figure 6. Skin biopsies taken 10 cm above the lateral malleolus in a healthy subject (A–C) and in patients with painful dia-betic neuropathy (D–F), and in patients with hepatitis C virus (HCV)-related neuropathy (G–I). Double staining confocalmicroscope studies with anti-TuJ1 and anti-TRPV1 antibodies (�40 objective). A, D, and G show TuJ1 immunostaining; B,E, and H show TRPV1 immunoreactivity; C, F, and I are merge images. TRPV1 colocalizes with TuJ1 in all intraepidermalnerve fiber (arrows) and dermal nerve bundle (arrowheads) of both healthy subject and patient with painful diabetic neuro-pathy, who showed a marked decrease of skin innervation density. Note the complete loss of both TuJ1 and TRPV1-positiveaxons in the skin of the patient with painful HCV-related neuropathy.


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et al., 2005). This suggests that the whole skin, andnot only the axons innervating it, may represent a poly-modal nociceptor that undergoes functional changesin painful conditions. Whether it has a role in neuro-pathic pain associated with peripheral neuropathiesremains to be addressed. Finally, different receptorsmight be involved. It has been recently demonstratedthat TRPV4, a receptor mediating mechanical hyper-algesia, plays a crucial role in taxol-associated painfulneuropathy and might represent a target for a novelclass of analgesics (Alessandri-Haber et al., 2004).

AcknowledgementsWe acknowledge Dr. John B. Davis, Neurology

CEDD, GlaxoSmithKline R&D, UK, for providing anti-TRPV1 antibodies.

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Expression of capsaicin receptor immunoreactivity in human peripheral nervous system and in painful neuropathies