Clinics in Dermatology (2015) 33, 46–54

☆

Mechanisms of nerve injury in leprosy David M. Scollard, MD, PhDa,⁎, Richard W. Truman, PhDb, Gigi J. Ebenezer, MBBS, MDc

aDirector, National Hansen’s Disease Programs, Baton Rouge, LouisianabLaboratory Research Branch, National Hansen’s Disease Programs at LSU, Baton Rouge, LouisianacDepartment of Neurology, The Johns Hopkins School of Medicine, Baltimore, Maryland

Abstract All patients with leprosy have some degree of nerve involvement. Perineural inflammation isthe histopathologic hallmark of leprosy, and this localization may reflect a vascular route of entry ofMycobacterium leprae into nerves. Once inside nerves, M leprae are ingested by Schwann cells,with a wide array of consequences. Axonal atrophy may occur early in this process; ultimately, affectednerves undergo segmental demyelination. Knowledge of themechanisms of nerve injury in leprosy has beengreatly limited by the minimal opportunities to study affected nerves in man. The nine-banded armadilloprovides the only animal model of the pathogenesis ofM leprae infection. New tools available for this modelenable the study and correlation of events occurring in epidermal nerve fibers, dermal nerves, and nervetrunks, including neurophysiologic parameters, bacterial load, and changes in gene transcription in bothneural and inflammatory cells. The armadillomodel is likely to enhance understanding of themechanisms ofnerve injury in leprosy and offers a means of testing proposed interventions.Published by Elsevier Inc.

Introduction

Nerve involvement occurs in all patients with leprosy.The earliest definitive clinical sign of this disease ishypoesthesia in a cutaneous lesion. In skin biopsy speci-mens, perineural inflammation is the histopathologic hall-mark of leprosy (Figure 1), and because Mycobacteriumleprae is the only bacterium that infects nerves and Schwanncells (SC), the demonstration of acid-fast bacilli within aperipheral nerve is pathognomonic (Figure 1). Theseobservations—SC infection and perineural inflammation—comprise the two major concepts around which nerve injuryin leprosy is understood and are the primary foci of researchinto the mechanisms of nerve injury in leprosy.1 A third,

☆ Supported in part by the Department of Health and Humans Services,Health Resources and Services Administration, Healthcare Systems Bureau,National Hansen’s Disease Program and National Institute of Allergy andInfectious Diseases (NIAID IAA-2646).

⁎ Corresponding author. Tel.: +1-225-756-3713; fax: +1-225-756-3819.E-mail address: dscollard@hrsa.gov (D.M. Scollard).

http://dx.doi.org/10.1016/j.clindermatol.2014.07.0080738-081X/Published by Elsevier Inc.

newer focus of research is the study of epidermal nerve fibersin this disease.

Mycobacterium leprae is an obligate intracellular organ-ism that prefers a cool growth temperature (Table),influencing the location of clinical lesions to cooler areasof the body.2 Approximately 95% of adults have nativeimmunity to M leprae3 and will not become infected evenwith substantial exposure. After infection with M leprae,nerves are apparently infected very early in the course of thisdisease, possibly before a cellular immune response (CMI)has developed.

The immunopathologic spectrum of leprosy2,4 is recapit-ulated in nerves (Figure 1): In polar tuberculoid disease (TT),bacilli are rare because the host’s CMI response to M lepraeis strong, and well-organized granulomas may replace theentire nerve. In polar lepromatous disease (LL) the host’sCMI to M leprae is ineffective and, in nerves as elsewhere,the bacterial load is extremely high and the inflammatoryresponse within the nerve is a disorganized collection ofhistiocytes with foamy cytoplasm. Between these two polarforms of leprosy is a wide borderline region, furthersubdivided into borderline tuberculoid (BT), mid-borderline

Fig. 1 Inflammation and infection of cutaneous nerves across the leprosy spectrum. The inflammatory responses in and around cutaneous nervesare shown in the upper panel; arrows highlight recognizable nerve twigs. The immunopathologic classifications of leprosy, TT to LL, are indicatedat the top of the figure (see text; mid-borderline, BB, is not shown). The TT lesion (upper left) is composed of a well-organized epithelioidgranuloma that has nearly destroyed the nerve, remnants of which are shown by S-100 staining. The granulomatous inflammatory responsebecomes less organized across the spectrum until, at the LL extreme, it is composed of disorganized aggregates of foamy histiocytes, seen heresurrounding a nerve (upper right). (TT: S-100, original magnification × 10; BT, BL, LL: hematoxylin-eosin, original magnification × 250.) Thedemonstration of acid-fast bacilli within nerves is pathognomonic of leprosy. In the lower panel, Fite-stained sections reveal thecorresponding intensity of M leprae infection in cutaneous nerves across the spectrum. M leprae are rare and difficult to demonstrate innerves of TT and BT lesions; they have been photographically enlarged in the insets. In contrast, bacilli are abundant and easily recognized inBL and LL lesions. (Fite/methylene blue, original magnification × 1000.)

47Nerve injury in leprosy

(BB), and borderline lepromatous (BL).5 Slowly progres-sive, insidious, “silent” neuropathy occurs in patients with allforms of leprosy—tuberculoid, borderline, and lepromatous.Acute neuritis develops in some patients and often alsoaccompanies the immunologic complications of leprosycalled “reactions.” Reactions are episodes of sudden,unexplained, spontaneous enhancement of an individual’simmune response to M leprae, and the immunologicmechanisms that characterize them in the skin may alsooccur in nerves.1 This review will focus on the pathogenesisof nerve injury in leprosy not related to reactions, becausereactions have been reviewed elsewhere.6,7

Early changes in nerves

Axonal atrophy, measured as reduction in axon caliber,has been described in nerves of leprosy patients.8 Thepathogenesis of this atrophy is not clear, but abnormalities ofphosphorylation of neurofilament proteins have been observedin nerves from treated and untreated patients with differenttypes of leprosy and in nerves with inflammation varying fromminimal to extensive.9 This has been investigated in a mousemodel, in which hypophosphorylation of neurofilament

proteins was observed in noninfected sciatic nerves of miceinoculated with M leprae in the hind footpads.10 Thesefindings suggest that M leprae may initiate early biochemicalchanges in the axonal compartment, preceding structuralchanges in myelinated fibers.

M leprae entry into nerves

Perineural inflammation in leprosy, a dermatopathologic“pearl,” may provide clues to the route by which M lepraeenters nerves. M leprae infection of endothelial cells is welldocumented in human leprosy11; studies in the armadillomodel of leprosy (Table) indicate that these organisms collectin epineurial lymphatics and from there are able to infect theblood vessels of the epineurium.12 Inflammation developsaround foci of infection, and classical studies in leprosydetermined that this inflammation ascends proximally fromcutaneous lesions to subcutaneous nerves to nerve trunks.13,14

The endothelial location offers a vascular route of entry forM leprae through the impermeable perineural sheath and intothe endoneurial compartment, possibly carried within macro-phages.15 Such a mechanism of entry into nerves does notpostulate any role for specific immunologic activities but is

Table Challenges and possibilities in experimental investigation of nerve injury in leprosy

M leprae Tissue culture Mouse Armadillo

Culture ofM leprae

Cannot be cultivated onartificial media

M leprae survive withincells; cannot assess growth

Limited growth in footpads Disseminated infectiondevelops

Multiplication ofM leprae

Doubling time = 13 days;Optimal growth at 32oC

Most cells in culture die ordivide in b13 days

Mouse experiments requiremonths or years to complete

Armadillo experimentsrequire months or yearsto complete

Determinationof viability ofM leprae

Viability cannot bedetermined by growthon agar, etc.

Radiorespirometry; fluorescent methods for live/dead bacilli; RNA-based methodsbeing developed

Killing ofM leprae Killed by freeze-thaw M leprae die rapidly attemperature N35oC

Growth in footpads(cooler site)

Good growth (core bodytemp = 32oC)

Study of effects ofviable organisms

Viable bacilli must beobtained fresh frominfected tissues (mouseor armadillo)

Standard cell cultureconditions (37oC) do notsimulate infection withviable bacilli

M leprae viability high;limited proliferation ofbacilli in footpads

M leprae viability high;infection progresses andbecomes disseminated

Genomics Genome has beensequenced

Genome known for cellsused in tissue culture

Genome has beensequenced

Genome has beensequenced

Mutations Mutations associatedwith drug resistancehave been identified

Immortalized cells aregenetically abnormal;may not replicate somenormal functions

Gene knockoutmice available

Molecular probes arebeing developed forcytokines, etc.

ModelingM leprae–nerveinteraction

n/a Schwann cells and SC/axonco-culture modelsare available

M leprae does not infectnerves in mice; knockoutmice enable modeling ofsome aspects

M leprae does infectnerves in armadillos;recapitulates many featuresof human infection

Suitability fortranslationalstudies

n/a Minimal Footpad infection mayinitiate biochemicalchanges in regional nerves

Neurophysiologic studiespossible; can study effectsof drug interventions onsensory fibers in skin punchbiopsy specimens

48 D.M. Scollard et al.

probably mediated by adherins or similar nonimmunologicmechanisms involving endothelial cells.

Schwann cells

Once inside the fascicle, M leprae is ingested by SC. Theextraordinary spectacle of acid-fast bacilli within SC hascaptured the imagination of investigators for decades.Although direct study of affected human nerves is difficult,SC can be studied in tissue culture, and so they have offered aready model to study M leprae infection in vitro (Table),which has generated a large literature of its own.

Several molecules have been identified that are respon-sible for M leprae’s adherence to and ingestion by SC.16,17

Once inside SC, M leprae has a viability profile similar tothat in macrophages (the more common host cell).18 Bothmyelinating and nonmyelinated Schwann cells may beinfected, but M leprae is a very well adapted, minimallytoxic pathogen, capable of inhabiting various cells withoutmarked injury or dysfunction. The general morphology ofSchwann cells is not altered by infection in vitro, and theirbasic ability to interact with axons and produce myelin isnot impaired.18

Some SC functions may be different in lepromatous than intuberculoid patients. The foamy appearance of infected SC inlepromatous nerves is due to lipid droplets, microorganellesrecruited into M leprae–containing phagosomes by M lepraethrough Toll-like receptor-6–dependent signaling.19 Inlepromatous patients, these lipid droplets may facilitatepersistence of M leprae in SC. Schwann cells infectedin vitro are also able to present antigen and can serve astargets for primed cytotoxic T cells.20,21 If this occurs in humandisease (which has not been confirmed), it would apply totuberculoid patients who develop CMI toM leprae but wouldprobably have little or no application in lepromatous patientswho do not produce M leprae–specific T cells.

Ultimately, individual neurons in leprosy undergosegmental demyelination.22,23 Demyelination may be in-duced by a variety of insults, including high levels of someproinflammatory cytokines.24–26 The major toxic effectormolecule known to kill M leprae is nitric oxide (NO), andNO has been demonstrated in the inflammatory infiltrates ofnerves in leprosy lesions.27 Nitrotyrosine, an end product ofthe metabolism of NO, has also been observed in nerves inBL lesions, and this molecule has been associated with lipidperoxidation of myelin leading to demyelination of nerves inother diseases.28,29 M leprae also bind and activate thetyrosine kinase receptor ErbB2, which induces the Erk1/2

49Nerve injury in leprosy

signal transduction pathways. In experimental systems thiscan result in demyelination and dedifferentiation of theterminally differentiated Schwann cell.30–32 No evidence isyet available to indicate if this sequence of events occurs inhuman leprosy.

Schwann cells are clearly at the center of the pathogenesisof nerve injury in leprosy; however, it is not clear how muchof this is due to the fact that some SC are infected and howmuch is due to the toxic inflammatory milieu within andaround these nerves.

Perineural inflammation

The extent to which the various observations in vitro areactually occurring in vivo is not clear, however, and suchin vitro studies do not replicate the immune and inflamma-tory milieu of the infected peripheral nerve. To validate andextend such findings, mechanisms must be examined in thehuman patient or tested in an experimental animal model. Forpractical and ethical reasons, peripheral nerves are rarelybiopsied in humans. When diagnostic biopsies are done, thesural or radial cutaneous nerves are selected because biopsyof either of these sensory nerves will not cause motorimpairment; however, these nerves may not be significantfoci of infection, and inferences about mechanisms fromstudies of such biopsies must be made cautiously.

Dermal macrophages within inflammatory infiltrates arethe major host cells for M leprae. This chronic, persistentinflammation in cutaneous leprosy lesions may lead todestruction of dermal appendages such as sebaceous andsweat glands, leading to dryness of affected skin and todestruction of hair follicles, causing madarosis, for example.Cutaneous nerves may be injured by the same inflammatoryprocesses. The well-organized granulomatous responses intuberculoid disease may function aggressively to destroyadjacent structures, but even in lepromatous patients, withineffective CMI, chronic inflammation will ultimatelydestroy surrounding tissue, including nerves.

Edema regularly accompanies cutaneous leprosy lesions,and it has been invoked as a potential mechanism of injury toperipheral nerves. Endoneurial edema can cause peripheralnerve injury by various mechanisms and has been exploredin several models.33 The immunoinflammatory milieu ofnerves in leprosy, especially during reactions, could lead toedema and transient pressure increases causing intermittentischemia, possibly comparable to that seen in models ofischemia-reperfusion injury.34 This has not yet beendemonstrated in biopsy specimens or in animal models ofleprosy neuropathy. No measurements of endoneurialpressure have been done in leprosy to document increasedpressure or edema, although surgeons who operate onpatients with acute lepromatous neuritis describe theprocedures as “decompression.”35 Notably, available datado not provide support for the value of this surgery,36

probably due to problems in study design. In any case, edemaseems an unlikely mechanism for the chronic, painless,“silent” nerve injury that is common in leprosy patients,including those who do not have reactions.

Evidence from human nerve biopsies

Very limited molecular data are available regardingimmunologic or neural mediators in leprosy-affected humannerves because nerves are seldom biopsied. The levels oftumor necrosis factor (TNF) in skin and nerves of leprosylesions were similar in one study,37 suggesting that theimmunologic phenomena studied extensively in leprosy skinlesions also apply to nerves. A larger study confirmed theseobservations and also found substantial amounts of trans-forming growth factor β (TGF-β) and inducible nitric oxidesynthase (iNOS) in nerve fibers by immunohistochemistry.38

Other elegant immunohistochemical studies have re-vealed high levels of matrix metalloproteinases (MMP) 2and MMP-9 within both inflamed and noninflamed nervesfrom leprosy patients. These MMPs appeared to be producedby SC and intraneural macrophages, and the highest levels ofMMPs were observed in nerves with endoneurial inflamma-tion.39,40 Such MMPs may play a role in demyelination41

and contribute to the neuroinflammatory response in manydiseases of the central nervous system.42 Other studies haveexamined the mRNA and protein levels of Ninjurin-1, anadhesion molecule involved in response to injury andregeneration in peripheral nerves. Both mRNA upregulationand elevated levels of the protein were observed in nervebiopsy specimens from leprosy patients with neuritis.43

Studies of human nerve biopsies are difficult for manyreasons, and the duration of infection and nerve involvementbefore study cannot be clearly defined. To obtain suchinformation, an animal model is required.

Armadillo model

The nine-banded armadillo (Dasypus novemcinctus) isthe only other natural host for M leprae44 (Table). Naïvearmadillos can be experimentally infected and offer the onlymodel of leprosy in an immunologically intact mammal.Infection of nerves was recognized when the susceptibility ofthis animal to M leprae was first described,45,46 anddissections of peripheral nerves in infected animals demon-strated that the infection of nerves in armadillos recapitulatesmany features of the human host.11,47

Armadillos do not reliably respond to thermal, light, ortactile nociceptive stimulants, but measurement of nerveconduction can be used effectively to assess function of theirmotor nerves. Demyelinating events result in a decreasednerve conduction velocity (NCV) measured in m/s, whereasaxonal loss and muscular atrophy lead to a decrease in

Fig. 2 Gene expression profiling of posterior tibial nerves from naïve, M leprae–infected untreated and treated armadillos. Alteration inexpression of genes was observed armadillos in comparisons of posterior tibial nerves from (1) naïve, uninfected, normal animals; (2) infecteduntreated animals; and (3) infected, treated animals that had received 12 months of rifampin. Genes analyzed were those associated withmaintenance of neurons, such as PGP9.5 (UCHL1), PMP 22, β-tubulin, and neurofilament (NCAM); nerve growth factors such as NGF-β andDLK-1; and inflammation (tumor necrosis factor α [TNF-α] and interferon γ [IFN-γ]). Relative expression was computed by using theΔΔCtmethod and data were normalized using GAP3DH. Results represent mean ± SD from duplicate experiments on five animals in each group.

50 D.M. Scollard et al.

the compound motor action potential (CMAP) measured inmV.48 Conduction deficits have been observed in theposterior tibial nerves of 75% of all experimentally infectedarmadillos, with onset occurring as early as 90 dayspostinfection. Similar to observations on humans, depressedcompound motor action potential amplitude (b0.9 mV) is themost common presentation, but abnormal nerve conductionvelocity (b40m/s) also can be observed.49,50 Most armadillosprogress from normal conduction to a total conduction blockby the latest stages of their experimentally induced infectionswith M leprae. Onset of conduction abnormality generallycoincides with evolution of a detectable immune response toM leprae and is a significant predictor of other nonspecificsymptoms of clinical neuropathy such as plantar ulcers andnail avulsion or hypertrophy.

Molecular studies of armadillo nerves

Nerve segments obtained at necropsy from armadillosenable extensive assessment of gene transcripts inM leprae–infected and noninfected nerves at a defined duration ofinfection. Cross-species hybridization using human genechips has revealed a total of 4313 differentially regulatedgenes (a greater than fourfold change) in the nerve of late-stage (N2 years) infected armadillos. This gene expressionprofile includes genes pertaining to antigen processing andpresentation, apoptosis, cell adhesion, cell migration, cellproliferation, cellular trafficking, chemotaxis, degeneration,growth factors, and inflammation, as well as neuronaldegeneration and regeneration activities in infected armadillo

nerves.49 In addition, the recently available whole genomesequence has enabled us to design armadillo-specificquantitative reverse transcriptase–polymerase chain reaction(RT-PCR) assays. Genes associated with inflammation(interferon γ [IFN-γ], TNF-α) and constitutive neuropro-teins (protein gene product 9.5 [PGP9.5], β-tubulin,neurofilament) were found to be upregulated, and growthfactors (DLK-1, nerve growth factor β [NGF-β]) requiredfor regeneration in the peripheral nervous system51 weredownregulated in affected nerves (Figure 2). Further studiesof armadillo nerves are likely to elucidate much about themechanisms of nerve injury in leprosy and enable potentialnew interventions to be assessed before clinical trials.

Epidermal nerve fiber and Schwann cell density

Tactile sensitivity is mediated by thickly myelinated Aβfibers of the dermis, whereas thermal sensitivity is mediatedby thin myelinated Aδ fibers and unmyelinated C type fibersthat end in epidermis as free nerve endings.52 Some evidencesuggests that the C fibers in the epidermis are the earliest toundergo degeneration in leprosy, consistent with clinicalobservations indicating that the assessment of thermalsensation is probably the primary component in establishingearly diagnosis.53,54

Immunostaining of punch skin biopsy specimens forPGP9.5, a neuronal pan-axonal marker, has been used byseveral investigators to visualize the intraepidermal nervefibers and dermal nerves and antibodies to NGF receptor p75to identify Schwann cells. Enumeration of these cells andfibers can identify small nerve fiber impairment that is not

Fig. 3 Epidermal nerve fibers and Schwann cells in armadillo skin. Skin sections from naïve animals, immunostained with anti-PGP9.5, aneuronal marker, reveal dense epidermal innervation of the ear lobe (A) and abdomen (B) compared with the distal leg (C). Confocal imaging of askin section from the distal leg (D) demonstrates dermal axons (red; anti-PGP9.5) and Schwann cells (green; anti–nerve growth factor receptor,p75) counter-stained with DRAQ5 for nuclear staining (blue). The deep dermal neurovascular bundles with nerve bundles (red arrow) andSchwann cells (green arrow) exhibit co-localization of staining (yellow) demonstrating nerves ensheathed by Schwann cells. (Scale bar: A, B, andC = 50 μm, D = 20 μm).

51Nerve injury in leprosy

detected by nerve conduction tests. For example, a decreasein epidermal density in the distal leg has been demonstratedin sensory neuropathies associated with diabetes, HIV, andidiopathic small fiber sensory neuropathies.55–58 Althoughleprosy neuritis has been well described clinically andhistologically, very little morphologic work has been done todetect early damage to sensory nerves.

In naïve armadillos, quantitation of epidermal fibers inskin biopsies of ears, abdomen, and a distal leg has showna length-dependent innervation similar to humans59

(Figure 3). The infected animals showed a lower meanepidermal nerve fiber density (ENFD) compared with naïveanimals, suggesting early small fiber degeneration. Doublestaining of cutaneous axons and Schwann cells in naïvearmadillos also mimicked the human cutaneous nervenetwork pattern (Figure 3). Preliminary studies of SCdermal cutaneous nerves in infected armadillos showed atrend toward increasing density (unpublished) and thusprovide indirect evidence that during early infection SCmay proliferate while harboring M leprae. These studiesindicate the feasibility of studying small fibers and earlyinfection in the armadillo using this technique and thepossibility of using it as a novel tool to test new drugs andtherapeutic interventions. (See Figs. 2 and 4).

Effects of treatment on nerve injury in thearmadillo model

The effects of treatment on nerve injury in leprosy canalso be studied in the armadillo. In one study, nerves wereobtained from armadillos that were infected for 12 monthsfollowed by treatment for another 12 months, and fromcontrol animals infected for 24 months but not treated (fiveanimals per group). The treated group received 10 mg/kgrifampin orally once monthly for 12 months. Nerve trunksfrom treated and untreated animals were divided into distaland proximal portions and processed for simultaneous DNAand RNA extraction. The total number of leprosy bacilli ineach nerve was enumerated using an M leprae–specificquantitative PCR assay targeting the RLEP gene andnormalized per centimeter of nerve. Although each of thetreated animals showed clinical improvement in skin lesionsand ulcers as a result of the antimicrobial therapy,examination of their posterior tibial nerves showed continuedpresence of M leprae (Figure 4).60

The number of viable bacilli was calculated using aquantitative RT-PCR assay targeting 16S rRNA.61 Theresults indicated that the organisms had been effectively

Fig. 4 Comparison of M leprae-infected armadillo posterior tibialnerves among treated and untreated animals. Bars indicate the meanand standard deviation of the number ofM leprae per centimeter of theposterior tibial nerves obtained at sacrifice 24 months after infection.Treated animals had received once-monthly oral rifampin (10 mg/kg)for 12 months. Nerve trunks were divided into distal and proximalportions and processed for simultaneous DNA and RNA extraction.Bacilli were enumerated using anM leprae–specific quantitative PCRassay targeting the RLEP gene and normalized per centimeter ofnerve. Untreated armadillos had higher overall numbers of M lepraein nerves, but treated animals also retained large numbers of dead Mleprae in their nerves even after 1 year of treatment. Although noviable bacilli were detected after 1 year of rifampin treatment, up to10E + 04 dead bacilli/cm nerve were still present in the nerves oftreated armadillos. Bacterial counts were significantly higher (p b .05)in segments of the distal half compared with the proximal halfof posterior tibial nerve from untreated animals with active infection(p = .0023) as well as after treatment (p = .01).

52 D.M. Scollard et al.

killed by rifampin therapy; however, bacterial countsaveraging 104-5 bacilli per cm of nerve were still observedeven after the conclusion of a full year of treatment.

This experimental finding parallels the very slow declineof deadM leprae in skin biopsy specimens of human leprosylesions, which may demonstrate the presence of dead bacillifor years after they have been killed by antimycobacterialtherapy.62 The continued presence of deadM leprae providesa continuing source of antigenic stimulation and may be amajor factor in the immunopathogenesis of long-term nerveinjury in leprosy.63

Conclusions

In summary (the natural sequence of these events isnot certain):

• Biochemical changes such as hypophosphoryation ofaxonal neurofilaments may occur soon after infectionwith M leprae.

• M leprae appears to enter the innermost compartment ofnerves via the blood supply and endothelium.

• Several different molecules on Schwann cells bindM leprae and facilitate its ingestion.

• Epidermal nerve fibers are damaged and destroyed; someSC are infected and some proliferate.

• M leprae live and proliferate within SC.• Stimulated by M leprae, SC produce several proteins thatmay contribute to demyelination.

• Infected nerves are physically at the center of chronicinflammation and immunologic activity that may persistfor many years.

• Some immune and inflammatory mediators are toxic tonerves and promote demyelination.

• Segmental demyelination occurs, regeneration followsin some fascicles, and these processes may coexistfor years

• After killing ofM leprae with rifampin, a large number ofdead bacterial cells remain within the nerve and maycontinue to elicit immunologic responses and acute andchronic neuritis.

Identifying the mechanisms of nerve injury in leprosy isparticularly challenging, because the major peripheral nervesaffected in humans can seldom be biopsied and studied. Thesingular difficulties posed by this pathogen and by theunusual animal models available have been reviewed. Inspite of these difficulties, extraordinary progress has beenmade in the last decade, in large part due to new toolsresulting from the sequencing of the M leprae genome andthat of the only animal model of leprosy neuropathy, thenine-banded armadillo.

References

1. Scollard DM. The biology of nerve injury in leprosy. Lepr Rev.2008;79:242-253.

2. Scollard DM, Adams LB, Gillis TP, et al. The continuing challenges ofleprosy. Clin Microbiol Rev. 2006;19:338-381.

3. Lazaro FP, Werneck RI, Mackert CC, et al. A major gene controlsleprosy susceptibility in a hyperendemic isolated population from northof Brazil. J Infect Dis. 2010;201:1598-1605.

4. Scollard DM. Association of Mycobacterium leprae with humanendothelial cells in vitro. Lab Invest. 2000;80:663-669.

5. Ridley DS, Jopling WH. Classification of leprosy according toimmunity—a five-group system. Int J Lepr Other Mycobact Dis.1966;34:255-273.

6. Walker SL, Lockwood DN. Leprosy type 1 (reversal) reactions andtheir management. Lepr Rev. 2008;79:372-386.

7. Scollard, submitted for publication.8. Shetty VP, Antia NH, Jacobs JM. The pathology of early leprous

neuropathy. J Neurol Sci. 1988;88:115-131.9. SaveMP, ShettyVP, ShettyKT, et al. Alterations in neurofilament protein

(s) in human leprous nerves: morphology, immunohistochemistry andWestern immunoblot correlative study. Neuropathol Appl Neurobiol.2004;30:635-650.

10. Save MP, Shetty VP, Shetty KT. Hypophosphorylation of NF-H andNF-M subunits of neurofilaments and the associated decrease in KSPXKkinase activity in the sciatic nerves of swiss white mice inoculated in thefoot pad with mycobacterium leprae. Lepr Rev. 2009;80:388-401.

53Nerve injury in leprosy

11. Fite G. The vascular lesions of leprosy. Int J Lepr. 1941;9:193-202.12. Scollard DM, McCormick G, Allen J. Localization of Mycobacterium

leprae to endothelial cells of epineural and perineural blood vessels andlymphatics. Am J Pathol. 1999;154:1611-1620.

13. Dehio K. On the Lepra Anesthetica and the pathogenetic relation of itsdisease-appearances. Proceedings of the International ScientificLeprosy Conference in Berlin, Oct. 1897. Translated and reprinted.Lepr India. 1952;24:78-83.

14. Gerlach W. Investigations on anaesthetic skin patches. Virchow Arch.1891;125:126-145.

15. Scollard DM. Endothelial cells and the pathogenesis of lepromatousneuritis:insights from the armadillo model. Microbes Infect. 2000;2:1835-1843.

16. Rambukkana A, Yamada H, Zanazzi G, et al. Role of alpha 2-dystroglycan as a Schwann cell receptor for Mycobacterium leprae.Science. 1998;282:2076-2079.

17. Teles RM, Krutzik SR, Ochoa MT, et al. Interleukin-4 regulates theexpression of CD209 and subsequent uptake of Mycobacteriumleprae by Schwann cells in human leprosy. Infect Immun. 2010;78:4634-4643.

18. Hagge D, Robinson SO, Scollard D, et al. A new model for studying theeffects of Mycobacterium leprae on Schwann cell and neuroninteractions. J Infect Dis. 2002;186:1283-1296.

19. Mattos KA, Oliveira VG, D'Avila H, et al. TLR6-driven lipid dropletsin Mycobacterium leprae–infected Schwann cells: immunoinflamma-tory platforms associated with bacterial persistence. J Immunol.2011;187:2548-2558.

20. Ford A, Britton WJ, Armati PJ. Schwann cells are able to presentexogenous mycobacterial hsp70 to antigen-specific T lymphocytes.J Neuroimmunol. 1993;43:151-159.

21. Steinhoff U, Kaufmann SH. Specific lysis by CD8+ T cells of Schwanncells expressing Mycobacterium leprae antigens. Eur J Immunol.1988;18:969-972.

22. Swift TR. Peripheral nerve involvement in leprosy: quantitativehistologic aspects. Acta Neuropathol (Berl). 1974;29:1-8.

23. Jacobs JM, Shetty VP, Antia NH. Teased fibre studies in leprousneuropathy. J Neurol Sci. 1987;79:301-313.

24. Constantin G, Piccio L, Bussini S, et al. Induction of adhesionmolecules on human schwann cells by proinflammatory cytokines, animmunofluorescence study. J Neurol Sci. 1999;170:124-130.

25. Fujioka T, Kolson DL, Rostami AM. Chemokines and peripheral nervedemyelination. J Neurovirol. 1999;5:27-31.

26. di Penta A, Moreno B, Reix S, et al. Oxidative stress andproinflammatory cytokines contribute to demyelination and axonaldamage in a cerebellar culture model of neuroinflammation. PLoS One.2013;8:e54722.

27. Ledeen RW, Chakraborty G. Cytokines, signal transduction, andinflammatory demyelination: review and hypothesis. Neurochem Res.1998;23:277-289.

28. Schon T, Hernandez-Pando R, Baquera-Heredia J, et al. Nitrotyrosinelocalization to dermal nerves in borderline leprosy. Br J Dermatol.2004;150:570-574.

29. Antunes SL, Chimelli LM, Rabello ET, et al. An immunohistochemical,clinical and electroneuromyographic correlative study of the neuralmarkers in the neuritic form of leprosy. Braz J Med Biol Res. 2006;39:1071-1081.

30. RambukkanaA.Mycobacterium leprae-induced demyelination: amodelfor early nerve degeneration. Curr Opin Immunol. 2004;16:511-518.

31. Tapinos N, Rambukkana A. Insights into regulation of human Schwanncell proliferation by Erk1/2 via a MEK-independent and p56Lck-dependent pathway from leprosy bacilli. Proc Natl Acad Sci U S A.2005;102:9188-9193.

32. Rambukkana A. Usage of signaling in neurodegeneration andregeneration of peripheral nerves by leprosy bacteria. Prog Neurobiol.2009;91:102-107.

33. McManis P, Low PA, Lagerlund TD. Microenvironment of nerve:blood flow and ischemia. In: Dyck PJ, Thomas PK, Lambert EH, eds.

Peripheral Neuropathy. 3rd ed. Philadelphia: WB Saunders; 1993.p. 453-473.

34. Iida H, Schmeichel AM, Wang Y, et al. Orchestration of theinflammatory response in ischemia-reperfusion injury. J PeripherNerv Syst. 2007;12:131-138.

35. Van Veen NH, Schreuders TA, Theuvenet WJ, et al. Decompressivesurgery for treating nerve damage in leprosy. Cochrane Database SystRev. 2012;12:CD006983.

36. Van Veen NH, Schreuders TA, Theuvenet WJ, et al. Decompressivesurgery for treating nerve damage in leprosy. A Cochrane review. LeprRev. 2009;80:3-12.

37. Khanolkar-Young S, Rayment N, Brickell PM, et al. Tumour necrosisfactor-alpha (TNF-alpha) synthesis is associated with the skin andperipheral nerve pathology of leprosy reversal reactions. Clin ExpImmunol. 1995;99:196-202.

38. Lockwood DN, Suneetha L, Sagili KD, et al. Cytokine and proteinmarkers of leprosy reactions in skin and nerves: baseline results for theNorth Indian INFIR cohort. PLoS Negl Trop Dis. 2011;5:e1327.

39. Teles RM, Antunes SL, Jardim MR, et al. Expression of metallopro-teinases (MMP-2, MMP-9, and TACE) and TNF-alpha in the nerves ofleprosy patients. J Peripher Nerv Syst. 2007;12:195-204.

40. Oliveira AL, Antunes SL, Teles RM, et al. Schwann cells producingmatrix metalloproteinases under Mycobacterium leprae stimulationmay play a role in the outcome of leprous neuropathy. J NeuropatholExp Neurol. 2010;69:27-39.

41. Kieseier BC, Seifert T, Giovannoni G, et al. Matrix metalloproteinasesin inflammatory demyelination: targets for treatment. Neurology.1999;53:20-25.

42. Rosenberg GA. Matrix metalloproteinases in neuroinflammation. Glia.2002;39:279-291.

43. Cardoso CC, Martinez AN, Guimaraes PE, et al. Ninjurin 1 asp110alasingle nucleotide polymorphism is associated with protection in leprosynerve damage. J Neuroimmunol. 2007;190:131-138.

44. Truman RW, Singh P, Sharma R, et al. Probable zoonotic leprosy in thesouthern United States. N Engl J Med. 2011;364:1626-1633.

45. Walsh GP, Storrs EE, Burchfield HP, et al. Leprosy-like diseaseoccurring naturally in armadillos. J Reticuloendothel Soc. 1975;18:347-351.

46. Balamayooran G, Pena M, Sharma R, Truman RW. The armadillo asan animal model and reservoir host for M. leprae. Clin Dermatol.2015;33:108-115.

47. Scollard DM, Lathrop GW, Truman RW. Infection of distal peripheralnerves by M. leprae in infected armadillos; an experimental model ofnerve involvement in leprosy. Int J Lepr Other Mycobact Dis. 1996;64:146-151.

48. Franssen H. Electrophysiology in demyelinating polyneuropathies.Expert Rev Neurother. 2008;8:417-431.

49. Sharma R, Lahiri R, Scollard DM, et al. The armadillo: a model for theneuropathy of leprosy and potentially other neurodegenerative diseases.Dis Model Mech. 2013;6:19-24.

50. Truman RW, Ebenezer GJ, Pena M, et al. The armadillo as a model forperipheral neuropathy in leprosy. Institute of Laboratory AnimalReseach (ILAR). 2014;54:304-314.

51. Hammarlund M, Nix P, Hauth L, et al. Axon regeneration requires aconserved MAP kinase pathway. Science. 2009;323:802-806.

52. Ebenezer GJ, Hauer P, Gibbons C, et al. Assessment of epidermal nervefibers: a new diagnostic and predictive tool for peripheral neuropathies.J Neuropathol Exp Neurol. 2007;66:1059-1073.

53. van Brakel WH, Nicholls PG, Das L, et al. The INFIR CohortStudy: investigating prediction, detection and pathogenesis ofneuropathy and reactions in leprosy. Methods and baseline resultsof a cohort of multibacillary leprosy patients in north India. LeprRev. 2005;76:14-34.

54. van Brakel WH, Nicholls PG, Wilder-Smith EP, et al. Early diagnosisof neuropathy in leprosy—comparing diagnostic tests in a largeprospective study (the INFIR cohort study). PLoS Negl Trop Dis.2008;2:e212.

54 D.M. Scollard et al.

55. Holland NR, Stocks A, Hauer P, et al. Intraepidermal nerve fiberdensity in patients with painful sensory neuropathy. Neurology.1997;48:708-711.

56. Polydefkis M, Hauer P, Sheth S, et al. The time course ofepidermal nerve fibre regeneration: studies in normal controls andin people with diabetes, with and without neuropathy. Brain.2004;127:1606-1615.

57. Goransson LG, Brun JG, Harboe E, et al. Intraepidermal nerve fiberdensities in chronic inflammatory autoimmune diseases. Arch Neurol.2006;63:1410-1413.

58. Ebenezer GJ, O'Donnell R, Hauer P, et al. Impaired neurovascularrepair in subjects with diabetes following experimental intracutaneousaxotomy. Brain. 2011;134:1853-1863.

59. Ebenezer, G. unpublished observations.60. Truman, RW. unpublished observations.61. Truman RW, Andrews PK, Robbins NY, et al. Enumeration of My-

cobacterium leprae using real-time PCR.PLoSNegl TropDis. 2008;2:e328.62. Scollard D, Stryjewska BM. Treatment and prevention of leprosy. 2011.

[UpToDate [serial on the Internet]].63. Scollard, DM. unpublished observations.