INTERNATIONAL JOURNAL OF LEPROSY^ Volume 61, Number I

Printed in the

Animal Model to Study the Mechanism of NerveDamage in Leprosy—A Preliminary Report'

Vanaja P. Shetty2

Since 1881 over 30 animal species havebeen tried for the experimental induction ofleprosy, and today we arc using a few strainsof rats ( 6) and mice ( 17 ), nude mice ( 8 ), threespecies of armadillos (' 9 ), the Korean chip-munk (9 ), and four nonhuman primates ('")in this successful list. Nerve lesions are doc-umented in all of the animal models withminor variations related to route of infec-tion and with susceptibility/resistance fac-tors playing a role ( 7 ). A major limitationhas been that none of the animal modelsreally depict the spectral disease of humanleprosy, and the slow progressive nervedamage seen in these models is represen-tative, at best, of borderline disease. Thus,the search is still on ( 4 ) for a better animalmodel to use in studying nerve damage dur-ing the normal course of the leprosy infec-tion and during the various reactional states.

Our present study aims at developing ananimal model for studying the kinetics ofnerve damage induced by the tuberculoidand lepromatous types of granulomatousresponses and also the effect of selective de-letion of macrophages on the granuloma-tous response and nerve damage in a time-bound manner. For obvious reasons, suchstudies cannot be undertaken on naturallyoccurring lesions.

In a preliminary study we have used in-traneural injection of Mycobacterium lep-rae, a technique followed by Wisniewski andBloom ( 18 ) to bypass the blood-nerve bar-rier. Using this technique Mshana, et al. (U)and Cowley, et al. (3 ) have induced granu-lomatous responses within the sciatic nervesof rabbits and guinea pigs presensitized tomycobacterial antigens.

Unlike these researchers, we choose touse viable M. leprae in unsensitized, normal

' Received for publication on 3 June 1992; acceptedfor publication in revised form on 17 November 1992.

= Dr. (Mrs.) V. P. Shetty, Senior Research Officer,The Foundation for Medical Research, 84-A, R. G.Thadani Marg, Worli, Bombay 400018, India.

and immunomodulated mice since there isevidence suggesting that the molecular levelresponse to viable M. leprae may be differ-ent from that of nonviable Al. leprae, par-ticularly in the neural environment ( 13 ). Theresults obtained were encouraging and arethe subject of this report.

MATERIALS AND METHODSA total of 20 right and left sciatic nerve

biopsies obtained from ten mice were stud-ied in two sets of experiments using a pro-tocol as shown in The Table. Normal,immunosuppressed (T200 x 5R) andsilica-treated, Swiss white female mice wereinjected intraneurally with 10-20 x 10' vi-able M. leprae suspended in normal saline.

In experiment 1, the M. leprae used forintraneural injection were obtained from afresh nodule biopsy of an untreated lepro-matous patient. In experiment 2, M. lepraewere obtained from a frozen armadillo liver.The tissues were homogenized using thetechnique of Shepard ( 17 ). The bacterial sus-pension thus obtained was used for intra-neural injection within 24 hr.

Immunosuppression of mice. Three-to-four-week-old, Swiss white female mice werethymectomized and irradiated with fivegraded doses of 200 rads each x irradiationat biweekly intervals (T200 x 5R) ( 2). Thesemice were used for intraneural injection 2weeks after the last dose of irradiation.

Treatment of mice with silica for macro-phage inactivation. Adult, Swiss white fe-male mice were given intraperitoneal injec-tions of 1 ml of 20% silica quartz dust (size80% of particles, 1-5 Am; Sigma ChemicalCo., St. Louis, Missouri, U.S.A.) suspensionin 0.5% buffered saline solution (BSS), 1 dayprior to intraneural injection of M. lepraeand/or a crush injury to the sciatic nerve.The silica dose was repeated two times perweek thereafter until biopsy. Treatment withsilica quartz dust selectively inhibits mac-rophage function in situ ( 1 ). Thus, in thepresent study, normal mice with a crush

70

6 1 , 1^Shetty: Animal Model of Nerve Damage^71

THE TABLE. Number of mouse sciatic nerves studied using different experimental pro-tocols."

Experi-^Source ofment^ill. leprae

I^Human nodule

2^Armadillo liver

(1)N + ML

(4)

^

(3)^N + Si +(2)^N + Si + crush +

^

TR + ML ML^ML

(5)N + Si +^(6)

crush^N + crush Total

2^1010

Total 20

N = Normal mice; TR = thymectomy and irradiation (T200 x 5R); Si = silica-treated mice; ML = M.leprae.

injury to the sciatic nerve (standard modelfor Wallerian degeneration) and mice treat-ed with silica (+Si) followed by a crush in-jury to the sciatic nerve were used as con-trols to assess the effect of M. leprae in thenerve with or without a crush injury in sil-ica-treated mice (protocol as in The Table).

Intraneural injection procedure. Micewere anesthetized with an intraperitonealinjection of 1% pentabarbitone. Using a dis-secting microscope, both the right and leftsciatic nerves were exposed asceptically fromunder the biceps femoris, lifted from thesurrounding tissue, and 0.5 Al of M. lepraesuspension containing 10-20 x 10 6 viableAl. leprae were injected into the endoneurialspace using a 30-gauge needle and a Ham-ilton syringe. A cyclomixer was used to getan even bacterial suspension.

For inducing a crush injury, a fine watch-maker's forcep was used. In group 4 (TheTable) the nerve was first crushed, then im-mediately injected with the Al. leprae sus-pension, and the wound was sutured.

Biopsy. The sciatic nerve biopsies fromall of the mice were obtained using penta-barbitone anesthesia 2 weeks after the in-traneural injection of M. leprae and/or acrush injury. The nerves were fixed in 2.5%glutaraldehyde, postfixed in osmium, de-hydrated, and embedded in araldite. One-Am-thick, semithin sections were stainedwith toludine blue and studied under thelight microscope. Subsequent ultrathin sec-tions stained with uranyl acetate and leadcitrate were studied using a JEOL 1005transmission electron microscope.

RESULTSNormal mice with intraneural M. leprae.

One-Am-thick resin sections stained withtoludine blue revealed patchy areas of fiber

loss. There were cuffs of lymphocytes aroundthe dilated blood vessels, and a few aggre-gates of epithelioid cells and giant cells werealso seen (Fig. 1A).

Scanning the sections under the electronmicroscope confirmed the light-microscop-ic findings (Fig. 1B). A carpet of epithelioidcells and a few giant cells were seen in theendoneurium. Occasional macrophageswere seen with bacilli, single or in smallclusters. No bacilli were traced to Schwanncells. Myelin debris in various stages of dis-integration was seen mostly in the macro-phages. Schwann cells appeared active andincreased in number. A few regenerating fi-bers were also seen. The lesions seen in thesenerves compared well with the human tu-berculoid type of lesions as per the Ridley-Jopling classification ( 15 ).

Immunosuppressed (T200 x 5R) mice withintraneural M. leprae. There was a morediffuse and moderate loss of nerve fibers.There was only macrophage infiltrate inthese nerves. Occasional lymphocytes wereseen, but the epithelioid type of macro-phages or giant cells were not seen. The restof the changes seen were similar to normalmice with intraneural M. leprae. Here, again,Al. leprae were seen only in the macro-phages.

Silica-treated mice with a crush injury.There was no macrophage infiltrate in thesenerves. Fairly intact myelin rings were seenaround collapsed and degenerated axonswithin the Schwann tubes. No activation orproliferation of Schwann cells nor any re-generation or remyelinating activity was seenin these nerves (Fig. 2A and 2B).

In contrast, nerves with only a crush in-jury (group 6) revealed macrophage infil-trate, phagocytosis, and a breakdown of my-elin by the macrophages. There was

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FIG. 1. A. Part of the sciatic nerve from a normalmouse injected intrancurally with viable .1/. leprae sus-pension 2 weeks prior to biopsy showing patchy areas

of fiber loss. Note the presence of lymphocytic infiltrate

around one of the blood vessels (v). Several pale, nu-

cleated epithelioid cells and one giant cell are also seen(arrow). A few thinly myclinated regenerative fibers

are seen (arrowhead) on an edematous background.This lesion is comparable to an early human tuber-

culoid type of nerve lesion (semithin transverse sectionstained with toludine blue x 750). B. Electron photo-

micrograph of a part of the Figure IA nerve showing

one multinucleated giant cell (G) surrounded by a few

epithelioid macrophages in the endoneurial area. A fewphagosomes (P) with bacterial debris are also seen within

these cells. My = myclinated fiber (uranyl acetate plus

lead citrate stain x 6000.)

Schwannian proliferation and regenerationof fibers, suggesting that in silica-treatedmice the normal process of degeneration andregeneration following a crush injury wasretarded in the absence of macrophages.

Silica-treated mice with/without a crushinjury and M. leprae. When Al. leprae wereinjected along with a crush injury in thesilica-treated mice (group 4), the lesion ob-tained was very similar to that seen with a

FIG. 2. A. Part of the sciatic nerve with a crush

injury in a silica-treated mouse from a biopsy obtained2 weeks after a crush injury. No normal-looking my-elinated fibers, macrophages, or activation of Schwann

cells arc seen. Regeneration and remyelinating fibersare also not seen in this nerve. Note the presence of

large numbers of darkly stained myelin rings (arrow)

(semithin transverse section stained with toludine bluex 750). B. Electron photomicrograph of a part of the

crushed nerve from the silica-treated mouse in Figure2A. Note the degenerate and collapsed axons (a) andfairly intact myelin rings lying within the Schwann cells

(S). Some membraneous inclusions are also seen in the

Schwann cell cytoplasm. Note the absence of macro-

phages and regenerative activity in this nerve (uranyl

acetate plus lead citrate stain x 5000).

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crush alone, although the extent of the mac-rophage infiltrate was less. Myelin degra-dation was not as effective as in the micewithout silica but regenerative and remye-linative activity was very good (Fig. 3). Theepithelioid type of macrophages and lym-phocytes were not seen in these nerves, andbacilli were seen only in the macrophages.

When M. leprae were injected intraneu-rally without crushing the nerve in silica-

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61, 1^Shetty: Animal Model of Nerve Damage^ 73

FIG. 3. Part of the sciatic nerve with a crush injuryplus intraneural .1/. leprae in a silica-treated mouse.There are uniformly distributed, small, thinly myelin-ated fibers suggesting regeneration. Most of the darklystained structures (arrow) are the myelin debris, somewithin macrophages and some within Schwann cells.Note the presence of large numbers of bacilli withinsome of the macrophages (arrowhead) (semithin trans-verse section stained with toludine blue x 750).

treated mice (group 3), the lesion obtainedwas similar to that of TR200 x 5R micewith M. leprae. Several macrophages car-rying large numbers of Al. leprae were seenwithin the endoneurium (Fig. 4A and 4B).

DISCUSSIONThis study is a preliminary attempt to

establish an experimental leprosy modelprimarily to get tuberculoid and leproma-tous types of granulomatous responses inthe nerve. Our results show that it is pos-sible to observe a spectrum of immune re-sponses by immunomodulating the animalin relation to the nerve damage. There wasno significant difference observed in the twogroups of experiments carried out using hu-man- and armadillo-derived M. leprae. Thekinetics of the cells migrating into the lesionremain to be explored.

The observation that M. leprae were notseen within the Schwann cells even in im-munosuppressed mice further raised our cu-riosity to extend our observations to silica-treated mice in order to immobilize themacrophages. However, our results in thisexperimental group proved more interest-ing.

In Swiss white mice on silica treatmentfollowing a crush injury there was more orless total retardation of the regenerative ac-

FIG. 4. A. Part of the sciatic nerve injected intra-neurally with viable M. leprae in a silica-treated mousefrom a biopsy obtained 2 weeks after intraneural in-jection of .1/. leprae. Some of the myelinated fibersshow extensive folding of the myelin (arrow), and thereare several thinly myelinated regenerating fibers. Mac-rophages are also seen, many of them with bacilli (ar-rowhead), but no lymphocytic infiltrate (semithintransverse section stained with toludine blue x 750).B. Electron photomicrograph of the same Figure 4Anerve showing three macrophages (M) carrying largenumbers of bacilli and myelin fragments (arrow) lyingin separate compartments. No bacilli arc seen in theadjacent Schwann cells (S). One of the Schwann cellsis seen with a large myelin ring within its cytoplasm(uranyl acetate plus lead citrate stain x 4000).

tivity of the nerve fibers at 2 weeks. Thiswas unlike what has been reported by Mul-ler and Minwegan ( 14 ) in a rat model. At 4-5 weeks these authors reported a macro-phage infiltrate plus nerve regeneration butpoor myelin clearance. This difference maybe due to the dose of silica used, the timespan of study, or the animal species itself.

In our experiment, the addition of AI. lep-rae seemed to negate the silica effect, to in-

74^

International Journal of Leprosy^ 1993

FIG. 5. Electron photomicrograph of part of thesciatic nerve injected intraneurally with two doses ofM. leprae (10-20 x 10' and 30 x 10") at a 2-weekinterval. This mouse was immunosuppressed (T200 x5R) as well as silica treated. Biopsy was obtained 5weeks after first dose of M. leprae. Note the presenceof bacilli-laden macrophages (M) showing foamy cy-toplasmic changes. Myelinated nerve fibers (My) areseen on one side. This lesion is comparable to a lep-romatous-type nerve lesion (uranyl acetate plus leadcitrate stain x4000).

vite a (sub)population of macrophages intothe lesions, and to activate the normal courseof de-/regenerative activity of the nerve.What was intriguing was the observation ofthe lack of lymphocytes and epithelioid typeof response in these lesions. This observa-tion suggests a role for a specific group of(silica sensitive ?) macrophages in bringingabout a tuberculoid type of granulomatousresponse.

We failed to see M. leprae in the mor-phologically identifiable Schwann cells evenin silica-treated mice. It is difficult to ex-plain why in situ Schwann cells did not takeup any bacteria even though they were vi-able. In one of our earlier studies, a pieceof highly positive lepromatous nerve wasgrafted onto the sciatic nerve of an immu-nosuppressed mouse. Although there wasgood axon innervation into the grafted hu-man nerve, no bacilli were seen in the hostSchwann cells even after 6 months of graft-ing ( 17 ). However, in the in vitro system,both in organized dorsal root ganglion cul-tures and in monolayer cultures of Schwanncells of mice and humans (12, 16 , ,) Schwanncells do phagocytose Al. leprae. In a renewedeffort, we injected a second higher dose ofM. leprae (30 x 10 6) into the sciatic nerve

of one of the immunosuppressed, silica-treated mice and studied the nerve 5 weeksafter the first dose of M. leprae. Even in thisnerve, although there were carpets of mac-rophages with Al. leprae (Fig. 5) lying nextto denervated Schwann cells, none were lo-cated within the Schwann cells. This is con-trary to what we see in human leprousnerves.

In a study of the cell population in Wal-lerian degeneration, Gibson ( 5 ) found no ev-idence for the once quite widely held viewthat Schwann cells transform into macro-phages. Hence, it is unlikely that the mac-rophages seen in these nerves were trans-formed Schwann cells. To find the answerto what prevents M. leprae from enteringthe Schwann cells of mice in situ would bean interesting area for further study.

SUMMARYIntraneural injection of 10-20 x 10 6 vi-

able Mycobacterium leprae into the sciaticnerve of normal, unsensitized, Swiss whitemice gives rise to a tuberculoid type of gran-ulomatous response in 2 weeks. The samedose of viable Al. leprae when injected intothe sciatic nerves of unsensitized immu-nosuppressed mice (T200 x 5R) elicited amacrophage response. When macrophageswere systemically immobilized using an in-traperitoneal injection of silica quartz dustin normal mice, the lesion produced was ofthe lepromatous type, suggesting a role forthe macrophage in the induction of the tu-berculoid type of granulomatous response.In all of these in situ experiments, Al. lepraefailed to enter the Schwann cells.

RESUMENLa inyecciOn intraneural de 10-20 millones de ba-

cilos de la lepra viables en el nervio sciatic° de ratonesSwiss-white normales, origina en dos semanas una res-puesta de tipo granulomatosa. La misma dosis de My-cobacterium leprae viables inyectada en los nerviossciaticos de ratones inmunosuprimidos (T200 x 5R)indujo un respuesta macroffigica. Cuando los macr6-fagos se inmobilizaron sistêmicamente inyectando in-traperitonealmente polvo de silicato de cuarzo en ra-tones normales, la lesiOn producida fue del tipolepromatoso sugiriendo un papel de los macrOfagos enla inducciOn de la respuesta granulomatosa de tipotuberculoide. En todos estos experimentos in situ nun-ca se observ6 que el M. leprae penetrara en las alulasde Schwann.

6 1 , 1^Shetty: Animal Model of Nerve Damage^ 75

RESUMEL'injection intra-neurale de 10-20 x 10' organismes

de Mycobacterium leprae viables dans Ic nerf sciatiquede souris blanches Swiss normales et non sensibiliseesdonne lieu dans les deux semaines A une reponse gra-nulomateuse de type tuberculoide. La meme dose deAl. leprae viables inject& dans Ic nerf sciatique desouris non sensibilisées mats ayant suhi une immu-nosuppression (T200 x 5R) a produit une reponsemacrophagique. Quand les macrophages ont Olesystematiquement immobilises par ('injection intra-peritoneale de poussiere de quartz de silice chez lasouris normale, la lesion produitc êtait du type lepro-mateux, cc qui suggere quc le macrophage a un roledans la production de la reponse granulomateuse detype tuberculoide. Dans aucune de ces experimenta-tions in situ, A1. leprae n'est entre dans les cellules deSchwann.

Acknowledgment. I am grateful to Dr. N. H. Antia,Trustee and Director of the Foundation for MedicalResearch, for supporting this work; to Drs. TannazBirdi and Nerges Mistry for helpful suggestions, andto Miss Suchitra Khade for technical assistance.

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