an immunohistochemical, clinical and ... · an immunohistochemical, clinical and ... 1 laboratório...

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Braz J Med Biol Res 39(8) 2006

Nerve fiber markers in neuritic leprosyBrazilian Journal of Medical and Biological Research (2006) 39: 1071-1081ISSN 0100-879X

An immunohistochemical, clinical andelectroneuromyographic correlativestudy of the neural markers in theneuritic form of leprosy

1Laboratório de Hanseníase, 2Departamento de Ultra-estrutura e Biologia Celular,Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brasil3Departamento de Patologia, Universidade Federal do Rio de Janeiro,Rio de Janeiro, RJ, Brasil

S.L.G. Antunes1,L.M. Chimelli3, E.T. Rabello1,

V.C. Valentim1,S. Corte-Real2, E.N. Sarno1

and M.R. Jardim1

Abstract

The nerve biopsies of 11 patients with pure neuritic leprosy weresubmitted to routine diagnostic procedures and immunoperoxidasestaining with antibodies against axonal (neurofilament, nerve growthfactor receptor (NGFr), and protein gene product (PGP) 9.5) andSchwann cell (myelin basic protein, S-100 protein, and NGFr) mark-ers. Two pairs of non-adjacent histological cross-sections of theperipheral nerve were removed for quantification. All the fascicles ofthe nerve were examined with a 10X-ocular and 40X-objective lens.The immunohistochemistry results were compared to the results ofsemithin section analysis and clinical and electroneuromyographicdata. Neurofilament staining was reduced in 100% of the neuriticbiopsies. NGFr positivity was also reduced in 81.8%, PGP staining in100% of the affected nerves, S100 positivity in 90.9%, and myelinbasic protein immunoreactivity in 90.9%. Hypoesthesia was associ-ated with decreased NGFr (81.8%) and PGP staining (90.9%). Re-duced potential amplitudes (electroneuromyographic data) were foundto be associated with reduced PGP 9.5 (63.6%) and nerve fiberneurofilament staining (45.4%) by immunohistochemistry and withloss of myelinated fibers (100%) by semithin section analysis. On theother hand, the small fibers (immunoreactive dots) seen amid inflam-matory cells continued to be present even after 40% of the largermyelinated fibers had disappeared. The present study shows an in-depth view of the destructive effects of leprosy upon the expression ofneural markers and the integrity of nerve fiber. The association ofthese structural changes with the clinical and electroneuromyographicmanifestations of leprosy peripheral neuropathy was also discussed.

CorrespondenceS.L.G. Antunes

Laboratório de Hanseníase

Fundação Oswaldo Cruz

Av. Brasil, 4365

21045-900 Rio de Janeiro, RJ

Brasil

E-mail: [email protected]

Research supported by FAPERJ

and POM funds of the FIOCRUZ.

Received November 28, 2005

Accepted April 20, 2006

Key words• Neurofilament protein• Nerve growth factor

receptor• S-100 protein• Myelin basic protein• Gene product 9.5• Neuritic leprosy• Peripheral neuropathy

Introduction

Leprosy is primarily a peripheral nervedisease despite prominent involvement ofthe skin in a variety of cutaneous lesions and

damage to the upper respiratory tract, eyes,and viscera (1). Pure neuritic leprosy, how-ever, is a form of the disease in which, exceptfor hypoesthesia, no cutaneous manifesta-tions are found. In this respect, the risk of

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developing a disability due to leprosy isdirectly related to the presence of Mycobac-terium leprae in the nerves and to the nervedamage detected on the occasion of the firstpatient visit (2). On average, neuritic leprosyaccounts for about 10% of all leprosy casesand is particularly difficult to diagnose ifacid-fast bacilli are not found in the skinsmears or histological sections of the nerves(2-5). In this regard, PCR has proven to bevery useful in confirming a diagnosis of pureneuritic leprosy (6).

The peripheral neuropathy occurring inleprosy is an inflammatory disease of thenerves. The inflammatory infiltrate of theaffected nerves may be composed of either amature epithelioid granuloma or of acid-fastbacilli-loaded macrophages exhibiting foamychanges (7,8). The pathogenesis of leprosyneuropathy is far from being fully under-stood, especially with respect to the mechan-isms involved in nerve fiber lesions. In addi-tion, axonal loss accompanied by Walleriandegeneration is observed at the end stage ofleprosy neuropathy (8,9), and T-cell cyto-toxic activity on Schwann cells (Sc) hasbeen demonstrated in vitro (10). Axonal de-generation following Sc death, demyelina-tion, and the environmental changes inducedby this specific inflammatory process couldbe the pathogenic mechanism responsiblefor nerve function impairment. Demyelina-tion, best detected by the teasing of axonalsegments, is also found in leprosy (11). In2002, for example, Rambukkana et al. (12)reported that M. leprae had a strong demy-elinating effect on infected Sc neuron cul-tures and Rag-1 knockout mice.

The inflammatory process undeniablyplays a crucial role in the destruction ofnerve fibers. However, the molecular mechan-isms leading to axonal degeneration and/ordemyelination in leprosy have rarely beenexplored in the literature. Thus, in the pres-ent study, we investigated the expression ofaxonal and Sc markers in nerve biopsiestaken from 11 pure neuritic leprosy patients.

An attempt was made to characterize thespecific pattern of change in leprosy neuro-pathy and to describe the modifications inmolecular expression within the leprosy-af-fected nerves. Immunoperoxidase stainingof the axonal neurofilament protein (13) andprotein gene product (PGP) 9.5 (14) wascarried out. Sc S-100, nerve growth factorreceptor (NGFr) (15,16), and myelin basicprotein (17) were also employed for immu-nostaining. The results of immunohistochem-istry were then correlated with the patients’clinical, electroneurophysiologic, and histo-logical data.

The purpose of this study was to searchfor correlations between nerve fiber status inpure neuritic leprosy and the clinical andelectroneuromyographic presentation of thedisease.

Material and Methods

Patient selection and routine biopsyprocedures

The nerve biopsies of 11 patients withpure neuritic leprosy were submitted to rou-tine histopathological analysis. Biopsies werecollected in the Leprosy Outpatient Clinic ofthe Department of Tropical Medicine, Os-waldo Cruz Foundation, Rio de Janeiro, RJ,Brazil. The biopsies were fixed in Carson’sfixative solution (Millonig buffered forma-lin) (18), embedded in paraffin and 4-µm-thick sections were stained with hematoxy-lin-eosin, Gomori’s trichrome, and Wadestaining.

A diagnosis of neuritic leprosy was con-firmed on the basis of criteria elaborated inour laboratory (6). Briefly, the diagnosis ofneuritic leprosy can be based on the follow-ing parameters 1) defined diagnosis: patientshave a clinical presentation consistent withleprosy, with histopathological examinationsdemonstrating an inflammatory infiltrate withacid-fast bacilli, and M. leprae-specific DNAdetected by PCR and serum anti-phenolic

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Nerve fiber markers in neuritic leprosy

glycolipid-1 (PGL-1) levels, and 2) prob-able diagnosis: patients have a clinical pres-entation consistent with leprosy, with thepresence of epithelioid granulomatous neu-ritis, mononuclear cell endoneuritis and fi-brosis. In this case, while a diagnosis ofleprosy is not defined by a golden test, thereis a high probability (over 99%) of it beingconfirmed if the case is from Brazil, whereleprosy is a highly prevalent cause of periph-eral neuropathy.

Serum PGL-1 determination

Detection of serum anti-PGL-1 antibody(19) and of M. leprae DNA by PCR (20) wascarried out in order to provide an additionalparameter for a leprosy diagnosis. NT-P-BSA was used as the semi-synthetic ana-logue of the PGL-1 antigen. ELISA wasperformed essentially as described (19) ex-cept for the following modifications: serumdilution was 1:500, the conjugate dilutionwas 1:10,000, and the substrate used was0.04% tetramethylbenzine and 0.04% ureaperoxide in 0.1 M sodium acetate citric acidbuffer, pH 4.0. The cut-off value used forpositivity was the optical density (OD) at450 nm = 0.2. In order to control for plate-to-plate and day-to-day variation, a positivereference serum was included in triplicateon each plate. The color reactions of theentire plate were stopped with 50 µL 2 NH2SO4 when the OD at 492 nm from a posi-tive control serum reached a value of 0.6.The ODs were measured in a spectropho-tometer using a 492-nm filter. All sera weretested in duplicate and the ELISA results arereported as mean absorbance of the dupli-cates. The final OD value of each serumsample was calculated by subtracting theOD value of wells coated with BSA from theOD value of the test wells coated with DBSA.

Immunohistochemistry

Immunoperoxidase staining with anti-

bodies against neural markers of axons (neu-rofilament (NF), NGFr, and PGP 9.5) and Sc(myelin basic protein, S-100 protein, andNGFr) was performed on paraffin-embed-ded sections. Two biopsies with normal his-tological appearance taken from patients withsymptoms of neuropathy were used as con-trols. Only pure neuritic leprosy patientswere selected for this study since the aboveprocedures are only ethically warranted whenit is necessary to confirm a leprosy diagnosisin patients without cutaneous lesions.

Some of the routine paraffin-embeddedsections were stained with the avidin-biotincomplex and submitted to immunoperoxidasestaining with streptavidin-biotin-peroxidase(Dako A/S, Glostrup, Denmark). Primary anti-bodies were mouse anti-NGFr (Dako Corp.,Carpinteria, CA, USA, 1/50), rabbit anti-PGP9.5 (UltraClone, Cambridge, England, 1/400),rabbit anti-S100 (Dako Corp., 1/100), mouseanti-NF200 protein (Sigma, St. Louis, MO,USA, 1/400), and rabbit anti-myelin basicprotein (Dako Corp., 1/200) diluted in Tris-buffered saline, pH 7.4 (Sigma). Immuno-staining of the sections was developed in asolution containing diaminobenzidine (Sigma)and hydrogen peroxide. The sections wereplaced on slides, counterstained with Meyer’shematoxylin, dehydrated, and mounted withEntellan medium (Merck, Darmstadt, Ger-many).

Control sections with non-immune se-rum from the same animal of the secondantibody (Dako Corp.) and another slidewith only droppings of diaminobenzidine(Sigma) were utilized as specificity and en-dogenous peroxidase controls, respectively.Paraffin-embedded sections of skin, whichare S-100-, NF-, NGFr, PGP 9.5-positive,were also used in order to control processingof the tissues (internal controls). The immu-nostaining results were correlated with thesemithin section analyses and clinical andelectroneuromyographic data. Immunoreac-tivity was scored from 0 = no staining to 4 =maximal staining.

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Semithin section processing

A small fragment of the nerve was pro-cessed to obtain semithin sections whichwere fixed with 2.5% glutaraldehyde, rinsedwith 0.1 M sodium cacodylate, postfixedwith 2% osmium tetroxide, dehydrated withacetone, embedded in epon (21), and sec-tioned with a Leica (Leica Microsystems,Wetzlar, Germany) ultramicrotome. The 0.5-to 1-µm-thick sections were stained withToluidine blue, examined with a Nikon E400Eclipse light microscope (Kawasaki, Tana-gawa, Japan) and documented with a CoolSnap digital camera (Image Processing So-lutions, North Reading, MA, USA). Theimages were then saved in a tagged-imagefile format and printed on glossy paper withan HP deskjet 640 printer (Loveland, CO,USA). Fiber loss and staining intensity wereestimated semi-quantitatively using a scoreranging from 0 = total loss to 4 = normalfiber density (see Table 1).

The changes detected in the present studyreflected differences between the biopsiesfrom leprosy patients and from controls. Nofollow-up assessment was conducted.

Results

The average age (± SD) of the neuriticleprosy patients (10 males and 1 female)studied was 42.9 ± 18.09 years. None of thepatients had previously received any specif-ic anti-leprosy drugs. Sensory impairmentand paresthesia were found in all 11 patients(100%), while nerve enlargement was de-tected in 8 (72.7%), nerve pain in 3 (27.2%),and paresis in 10 (90.9%). The amplitude ofthe axonal potential was reduced in 7 of thebiopsied nerves (63.6%), conduction velocitywas reduced in 10 (90.9%), and disturbancesin potential conduction and amplitude weredetected in 7 (63.6%). In addition, 10 pa-tients (90.9%) had multiplex mononeuropa-thy and one had polyneuropathy. Table 1lists the neurological alterations found in the

biopsied nerves together with the results ofthe electroneuromyography test.

Histological examination of the nervesshowed variations in the spread of the in-flammatory infiltrate, which was related tothe number of nerve fibers still present in thenerve fascicles. The intensity and distribu-tion of the immunoreactive signals variedaccording to the number of fibers and theproportion of the endoneurium occupied byinflammatory cells.

Immunohistochemical staining of smalland large myelinated fibers (see Figures 1and 2, and Table 2) and of small immunore-active clustered dots (Figures 1B, 2A and2D) surrounded by the cytoplasm of nucle-ated cells was observed. These dots corre-sponded to a cross-section of the remainingsmall fibers (some of them presumably non-myelinated), which were preserved despiteoccupation of the endoneurium by the in-flammatory infiltrate.

A description of the staining with eachmarker follows: 1) NF200 protein producedthe strongest axoplasm staining in nerve fi-bers. Neuritic biopsies had a lower scorewith respect to both large and small fibers in100% of the biopsies (Figure 1A). 2) S-100positivity of both large (myelinated) andsmall fibers was reduced in 90.9% of thebiopsies. Sometimes irregular and exhibit-ing a granular pattern in small fibers (Figure1B), the S-100-positive pattern appeared asan immunoreactive cytoplasmic rim on theouter surface of myelinated fibers (Figure1C) and in the cytoplasm of non-myelinatingSc (Figure 1B). S100-positive Sc were foundin the endoneurial compartment of fasciclesinfiltrated by inflammatory cells where nostaining of the axonal components was ob-served. Despite some apparent similarities,S100 and myelin basic protein (MBP) stain-ing of myelinated fibers showed distinct pat-terns: anti-MBP did not stain the cytoplasmof Sc surrounding myelinated fibers but onlytheir myelin sheath (Figure 1D). On the otherhand, anti-S100 was found to stain the

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abaxonal or adaxonal rim of the Sc cyto-plasm (Figure 1D). 3) Fiber MBP immu-noreactivity was decreased in 90.9% of thebiopsies (Figure 2C). The final score de-pended on the number of endoneurial fiberspresent. Therefore, staining was restricted tothe myelinated fibers not affected by theinflammatory invasion (Figure 1D). MBPnegativity (Figure 2C) concomitant with PGP9.5 (Figure 2B) and NGFr positivity (Figure2D) indicated that in 40% of the biopsiessmall fibers remained in the endoneurialcompartment despite the absence of m fi-bers. 4) The NGFr expressed by both axonsand their surrounding non-myelinating Scshowed reduced staining in 81.8% of thebiopsies compared to control nerves. NGFr-positive Sc were seen amid inflammatorycells whose staining pattern was similar tothat obtained with S100 staining (compareFigures 1B and 2A). It was not possible todistinguish small NGFr-positive axons fromNGFr-positive Sc since both structures emit-ted positive signals on the non-myelinatedfiber profiles that were in very close proxim-ity to each other (Figure 2A and D). 5) PGP9.5 staining was similar to NF200 immu-noreactivity and was reduced in 100% of theleprosy biopsies (compare Figures 1A and

2B). Both small and large PGP-positive fi-bers could also be seen (Figure 2B).

The nerve biopsies of the controls dis-played a normal histological appearance anda maximal staining grade. In addition, alter-ations such as a reduced number of myeli-nated fibers and axonal degeneration, regen-eration (sprouting), and remyelination werecompletely absent (score = 0) on semithinsections of control biopsies (see Table 2).

Semithin section analysis showed a re-duced number of myelinated fibers in 100%of the lepromatous biopsies (Figure 3A-D).A higher number of this type of fiber wasseen when the infiltrate was lepromatous(Figure 3A and D). Axonal degenerationwas present in 10% of the biopsies, axonalregeneration in 30%, and axonal remyelina-tion in 20% even though these three mor-phological alterations were inconspicuous.In 6 biopsies (54.5%) the endoneurial space,completely devoid of myelinated fibers, wasoccupied either by lepromatous infiltrateswith acid-fast bacilli-loaded vacuolated mac-rophages (Figure 3C) or by mononuclearcells (Figure 3B), which in some cases formedtuberculoid granulomas (Table 2).

Hypoesthesia was associated with a de-crease in NGFr and PGP staining in 81.8 and

Table 1. Clinical and electroneuromyographic characteristics of the leprosy patients studied.

Patient Nerve Sensory Paresthesia Motor Neurophysiology NerveNo. pain impairment impairment biopsy

Classification Number of nerve Conduction impairments of nerve biopsy

1 - + + - Combined Multiplex mononeuropathy Present Right sural2 + + + + Demyelinating Multiplex mononeuropathy Absent Right ulnar3 - + + - Combined Multiplex mononeuropathy Absent Right ulnar4 - + + - Combined Multiplex mononeuropathy Absent Left ulnar5 - + + - Demyelinating Multiplex mononeuropathy Absent Right ulnar6 - + + + No classification Polyneuropathy Absent Left sural7 - + + - Combined Multiplex mononeuropathy Absent Left ulnar8 - + + + Combined Multiplex mononeuropathy Absent Left ulnar9 + + + + Demyelinating Multiplex mononeuropathy Absent Left ulnar

10 - + + + Combined Multiplex mononeuropathy Absent Right sural11 + + + + Combined Multiplex mononeuropathy Absent Left ulnar

Combined = concomitant presence of reduced neuropotential amplitude and reduced conduction velocity.

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Figure 1. Immunohistochemicalstaining of neurofilament (NF), S-100 protein and comparison of S-100 protein and myelin basic pro-tein (MBP) stainings of myelinatedfibers. A, A few NF-positive nervefibers in the endoneurial compart-ment (end) of a nerve fascicle froma neuritic leprosy patient. Immu-noreactive axoplasm of large my-elinated fibers (arrows) as well assmaller axons (arrowheads), notseen by routine staining, can beobserved. Inflammatory cells areseen in this field. B, S-100-immu-noreactive Schwann cells (Sc) inthe endoneurial compartment (end)of a nerve fascicle of a neuritic lep-rosy patient. The S-100 immunore-activity corresponds to the Sc ofnonmyelinated fibers, or, possibly,denervated Sc. The cytoplasmicstaining of these cells presumablycorresponds to the cell processesby which numerous axons arewrapped (arrows). There is a strik-ing loss of myelinated fibers in thisfascicle. C, S-100 immunoreactiv-ity is quite similar to MBP staining but with some distinctive features of its own. The myelin sheath is weakly stained with S-100 antibody along with astained outer rim corresponding to Sc abaxonal cytoplasm (arrows). D, The myelin sheath was more intensely stained by MBP antibody (arrows), andno rims of Sc cytoplasm were present. end = endoneurium. Avidin-biotin immunoperoxidase and scale bar = 24 µm for all panels.

Figure 2. Immunohistochemicalstaining of nerve growth factor re-ceptor (NGFr), protein gene prod-uct 9.5 (PGP 9.5) and myelin basicprotein (MBP). A, NGFr-immunore-active Sc in the endoneurial com-partment (end) of a nerve fasciclefrom a neuritic leprosy patient(cross-section). NGFr immunoreac-tivity (arrows) corresponds to theSc of nonmyelinated fibers or, pre-sumably, to denervated Sc (it is im-possible to distinguish one from theother). The cytoplasmic heteroge-neous NGFr staining of these cellsat times overlaps with S-100 immu-noreactivity. Some mononuclearcells (arrowheads) are also seen inthe endoneurium. B, PGP 9.5-posi-tive myelinated nerve fibers (ar-rows) in the endoneurial compart-ment (end) and small positiveaxons (thin arrows) can also beseen. The staining of this neuronalenzyme is not as strong as neu-rofilament staining (Figure 1A). C,Absence of MBP immunoreactivityin the endoneurial compartment(end) of a cross-section of a nerve fascicle taken from a neuritic leprosy patient. D, The same biopsy showed the presence of NGFr-positivenonmyelinated fibers (arrows) in the endoneurium (end). Avidin-biotin immunoperoxidase and scale bar = 24 µm for all panels.

end

end

end

end

A B

C D

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Table 2. Laboratory, histopathological and immunohistochemical data of the leprosy patients studied.

Patient PCR PGL-1 Histopathology Examination of semithin sectionsa Immunoreactivity of nerve fibersb

No.Presence of myel fib Ax deg Ax reg Ax rem NF200 NGFr PGP S100 MBP

1 + + MNI, AFB 4+ 0 0 0 0 0 2 1 3 0

2 + + LI, Fib, AFB 6+ 0 0 0 0 0 0 1 0 03 + - Fib 1 1 2 1 ND 0 0 0 1

4 - - TI, Fib 0 0 2 0 0 0 0 3 15 ND ND TI, Fib 1 0 0 0 1 2 1 3 ND

6 - + LI, TI, Fib, AFB 3+ 1 0 0 0 0 0 0 0 07 - - MNI, Fib ND ND ND ND 2 4 2 2 4

8 - - MNI 3 0 1 1 1 0 1 3 39 - ND TI 0 0 0 0 0 0 0 0 0

10 + - MNI, Fib 0 0 0 0 0 4 3 4 011 - - TI, Fib 0 0 0 0 2 3 3 0 0

AFB = presence of acid-fast bacilli; Ax deg = axonal degeneration; Ax reg = axonal regeneration; Ax rem = axonal remyelination; Fib = fibrosis; LI= lepromatous infiltrate; MBP = myelin basic protein; MNI = mononuclear infiltrate; ND = not done; NF200 = neurofilament 200 protein; NGFr =nerve growth factor receptor; PCR = polymerase chain reaction for Mycobacterium leprae DNA; PGL-1 = serum antibodies to phenolic glycolipid-1, PGP = protein gene product; Presence of myel fib = presence of myelinated fibers; TI = tuberculoid infiltrate.

AFB score: 3+ = 1 to 10 bacilli per x1000 field, 4+ = 10 to 1000 bacilli per x1000 field, 6+ = more than 1000 bacilli per x1000 field. aScore onsemithin sections: presence of myelinated fibers (0 = total loss/4 = normal); presence of axonal degeneration, axonal regeneration, and axonalremyelination (0 = absent/4 = maximal presence). bScore of alteration of immunoreactivity (0 = no staining/4 = maximal staining).

Figure 3. Histopathological exami-nation of semithin sections of in-flammatory infiltrate and loss of my-elinated fibers. A, Nerve fasciclewith a reduced number of myeli-nated fibers (arrows) and a few in-flammatory cells surrounding thick-ened microvessels (arrowhead) inthe endoneurial compartment (end).B, Nerve fascicle with completeabsence of myelinated fibers andfew macrophages surroundingthickened endoneurial microves-sels (arrows) in the endoneurialcompartment (end). C, Endoneu-rium (end) of a nerve fascicle witha dense inflammatory infiltratecomposed of vacuolated macro-phages (arrowheads) and lympho-cytes surrounding a prominent,branched microvessel (arrow).Note the absence of myelinated fi-bers. D, Nerve fascicle with a re-duced number of myelinated fibersand enlarged subperineurial space(sps). Atrophic axons can also beseen (arrows). Scale bar = 40 µmfor all panels.

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90.9% of the nerve biopsies, respectively.After associating the clinical and electro-neuromyographic alterations with the im-munohistochemistry results, semithin sec-tions of 100% of the biopsies that showeddecreased potential amplitudes also exhib-ited myelinated fiber loss (Table 3). Anotherassociation was seen between reduced po-tential amplitudes and a lower number ofNF200-stained (45.4%) and PGP 9.5-stained(63.6%) fibers. In 100% of the biopsies, theloss of myelinated fibers observed on semi-thin sections was associated with decreasedMBP immunostaining. Moreover, in 40.0%of the biopsies, small fibers, represented byimmunoreactive dots, were seen amid theremaining inflammatory cells even after thelarger myelinated fibers had disappeared.

Discussion

Reflecting the characteristic nerve dam-age and nerve function impairment occur-ring in this disease, the expression of neuralmarkers on the leprosy-affected nerves wasconsiderably modified. The number of my-elinated fibers with NF200 immunoreactiv-ity was decreased due to the loss of myeli-nated fibers observed on semithin sections.However, there was no noticeable decreasein the staining intensity of individual fibers.NF200 staining was also detected on thesmall fibers amid inflammatory cells. Sev-eral hypotheses have been raised to explainthe progressive axonal degeneration charac-

teristic of leprosy, and NGF deprivation iscertainly one of them. According to Anand(22), the absence of this target-derived cy-tokine could be implicated in the pathogen-esis of axonal degeneration. Kumar andSengupta (23) also found basement mem-brane multilayering of endoneurial microves-sels in addition to endothelial hypertrophy,possibly resulting in microvessel obstruc-tion and local ischemia of the nerve.

Sc S-100 protein staining of myelinatedfibers was also reduced due to the aforemen-tioned fiber loss. The finding of S100-posi-tive and PGP-negative nerve fibers in theendoneurial compartment could be attrib-uted to the presence of denervated Sc result-ing from progressive axonal degenerationand concomitant Sc survival. This observa-tion supported the detection by Shetty andAntia (24) of axon-free Sc in the advancedleprosy neuritic process during which thecollagen fibers are surrounded by Sc cyto-plasmic protrusions (collagen pockets).These surviving cells might also function asguides in axonal regeneration, which, how-ever, does not seem to be entirely effectivein leprosy neuropathy (25). The survival ofdenervated Sc is mediated by the productionof autocrine cytokines supporting resistanceof Sc to death signals and influencing theneuritic process (1). Oliveira et al. (26) havedemonstrated the presence of apoptotic Sc inthe dermal nerve branches of cutaneous lep-rosy lesions in addition to demonstratingthat synthetic M. leprae lipids could activate

Table 3. Association of patients’ clinical symptoms with immunohistochemistry findings.

Association of clinical symptoms with immunohistochemistry findings Percentage of biopsies

Reduced potential amplitudes and loss of myelinated fibers 100Reduced potential amplitudes and reduced NF200 45.4Reduced potential amplitudes and reduced PGP staining 63.6Hypoesthesia and reduced NGFr staining of small fibers 81.8Hypoesthesia and reduced PGP staining 90.9Loss of myelinated fibers in semithin sections and reduced MBP staining 100

MBP = myelin basic protein; NF200 = neurofilament 200 protein; NGFr = nerve growth factor receptor; PGP =protein gene product.

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apoptosis in the transformed ST88-14 Scline through Toll-like receptor 2 molecules.The secreted survival factors might possiblyneutralize the Toll-like receptor 2 molecules-mediated death signal.

NGFr staining of neuritic nerves wasreduced compared to control and was re-stricted to the Sc and/or axons of small fi-bers. It was impossible, however, to distin-guish axonal from Sc immunoreactivity inthe small fiber profiles since light micros-copy does not permit this type of distinction.The staining of MBP was also decreasedbecause of the reduced number of myeli-nated fibers. The MBP immunopositivity ofsome endoneurial cells not accompanied byaxonal marker staining (data not shown)might represent the myelin debris of degen-erated axons.

Axonal degeneration could also be a con-sequence of demyelination, which might beinduced by the presence of M. leprae. Thecause of demyelination in leprosy is contro-versial with two divergent hypotheses hav-ing been raised in the literature. One expla-nation refers to M. leprae-induced demyeli-nation and the other to immune-mediateddemyelination. In regard to the first, Ram-bukkana et al. (12) have demonstrated adirect demyelinating effect of M. leprae onSc neuron co-cultures, in agreement with thehypothesis that M. leprae has a direct effecton nerve fibers. Vardhini et al. (27) havepointed out the molecular mimicry enactedby mycobacterial proteins (ferredoxin-NADP-reductase and a conserved mycobac-terial membrane protein) that could mimicP0, a protein that aids in compacting myelinthrough homotypical interactions. Molecu-lar mimicry could disrupt tidy myelin layers,with the consequent occurrence of demyeli-nation. It is known that myelin-axon interac-tions are crucial for the maintenance of axontrophism and function inasmuch as axonaldegeneration is usually associated with de-myelination. Regarding the second explana-tion proposed by Spierings et al. (10), an

immunopathogenetic demyelination mech-anism in which cytotoxic T-cells could beresponsible for killing antigen-presenting Scis the causative factor of demyelination.

In the present study, PGP 9.5 stainingwas predominantly found on small fibers inthe form of clusters of small positive dotsattached to Sc in the transverse histologicalsections of the fascicles. The presence ofendoneurial MBP negativity together with PGPand neurofilament-positive clustered dots inthe cytoplasm of Sc on adjacent sectionsstrongly suggested that these dots were immu-noreactive, non-myelinated axons.

It is interesting to note that, despite theassumption found in the literature that lep-rosy predominantly affects small nerve fi-bers (12), these fibers were accompanied bythe complete disappearance of myelinatedfibers and MBP immunoreactivity and couldstill be seen among the inflammatory cellsafter staining of the PGP and NF200 axonalmarkers. As a result, small fibers (includingnon-myelinated ones) might actually be lesssensitive to the environmental endoneurialdisturbances taking place during the courseof neuritic leprosy. It is recommended thatan in-depth morphological assessment of thestatus of small fibers be conducted usingelectron microscopy.

Sensory impairment (thermal, algic, andtactile hypoesthesia) associated with smallfiber disturbances in the endoneurium sug-gested that either the number of fibers hadbeen reduced or that at the time of the biopsythese fibers, normally involved in nocicep-tive sensation, were undergoing functionalalterations. Antunes et al. (28) also raisedthis last hypothesis when they found no sig-nificant quantitative difference between thenumber of fibers in biopsies taken during orafter reactional episodes. It is worth notingthat the lack of immunoreactivity of a neuralmarker in the endoneurium does not neces-sarily mean the absence of fibers. It mightreflect a qualitative modification within thenerve fiber, which may also be accompanied

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by a functional disturbance. Again, the quan-tification and morphological status of smallnon-myelinated fibers are more adequatelyassessed by electron microscopy studies.

The reduced potential amplitudes foundin electroneuromyography were associatedwith the axonal loss shown by NF, PGP 9.5,and the corresponding semithin section anal-ysis. Moreover, in the electroneuromyo-graphic study, the presence of remyelinatingaxons on the semithin sections was accom-panied by an electroneurographic demyeli-nation pattern in only two cases. This mayhave occurred because, as demonstrated byJacobs et al. (11), the morphological signs ofactive demyelination in the nerves affectedby leprosy via the teasing technique did notseem to be as dramatic as that which com-monly occurs in both chronic inflammatorydemyelinating neuropathy and genetic my-

elination disorders. Furthermore, in leprosyneuropathy, the onset of axonal degenera-tion due to the neuritic process may be mask-ing the appearance of a fully developed de-myelinating process.

Therefore, leprosy is the cause of highlydestructive peripheral neuritis as evidencedby the reduced immunohistochemical ex-pression of neural markers and loss of my-elinated fibers on the semithin sections. Thesefindings were associated with the clinicaland electroneuromyographical manifesta-tions of peripheral neuropathy shown byleprosy patients.

Acknowledgments

We thank Judy Grevan for reviewing theEnglish and editing the text.

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