EL:Ii~CTRON MICROSCOPIC STUDY OF LEPROMATOUS CHANGES IN THE IRIS 1

HISAKO HASHIZUiVIE, M.D. and EINOSUKE SHIONUMA, M.D. A isei·en L eprosar·iu7I1

Olcayama, Japan

Ultrastructural details of M. leprae, and skin and peripheral nerve lesions of lepromatous and tuberculoid types of leprosy, have been studied since the introduction of electron microscopic technics in the pathologic study of leprosy C' 8, 11 , to , 17 , 18.30 ).

In the field of ophthalmology, anatomic detai ls of various normal tissues of the eye have been studied by electron microscopy, and many problems that could not be understood clearly by light microscopic studies are now being elucidated ( 9,19,24,25,27,28,29). However, few r e-ports at the ultra-structural level are available on pathologic changes in the eye except in trachoma and sympathetic ophthalmia ( 5,6,7, H, 15, ),

and no reports are availabl e yet on ophthalmologic changes in leprosy at the electron optic level.

In the present article, we shall describe the electron microscopic details of lepromatous changes of the iris. In the study here reportecl we shall elucidate the details of lepromatous changes in smooth muscle cells that could not be seen clearly with a light microscope. In addition the problem of the origin of lepra cells in the iris stroma on which ther e have been many different opinions, will be discussed on the basis of electron microscopic features.

MATERIALS AND METHODS Five specimens of iris obtained f rom 5 lepromatous p atients in the Aisei-en Lepro­

sarium were used in this study. These specimens were secured by optical i.ridectomy or by ITidectomy before extirpation of the lens. The iris in each case was fixed immediately in 1% osmium tetroxide (pH 7.2-7.4) in phosphate buffer , and left ill a refrigerator fo l.' 3 hours. After the fixation, the specimens were dehydrated in ethyl alcohol (70 %, 90 % and 100% ) and embedded in a 7:3 mixture of n-butyl- a nd 1Ilethyl-1Il ethacrylate.

Ultrathin sections were made with the J CM-3 ultramicrotome (Japan Electro1l Optics Laboratory Co. Ltd.) with glass knives. The electron microscope used in this s tudy was the Akashi Tronscope 50 of the Leprosy Research Laboratory, Kyoto Uni ­versity. Pictures were taken at direct magnifications of 1,000 to 15,000.

ELECTRON MICROSCOPIC OBSERVATION

For the orientation of leprosy lesions in the whole structure of the iris, wide field composite pictures of the iris are more useful than num­ber s of single separate pictures. For this r eason we have made com­posite pictures of serial fi elds (from several to 30 serial pictures) in order to cover the whole area of the iris. Figures 1 and 2 r epresent such composite pictures.

Histopathologic studies of the lepromatoUF; iris by li ght micJ'oscope examination have been reported by many authors ( 3,4, 12.13,21, 22,23 ).

1 Received for publication October 28, 1964.

61

62

N

lB ~~-tn

FS

lC

M

ll1t e1"/w tiollal ./oll1'llCtl of 1, e p1'().~ ,Ij 1965

HR

~m-¥~~~~~S

o

l<'IG. 1. };Iedron mi croseopi c chara cter of th e l epromatou s iris . 'l' he figure shows a n e ntire c ross section of th e iris , in whi ch the uppe r pal·t i s endothelium (END ) ~nd th e lower pa rt pigm e nt e pithelial cell s (PEC ) . L ('promatous changes a ro more f requ ent in th e pos t e l·ior half of the iri s. L (' prosy bncilli (LB ) are found ~ s s ingl e ba cilli or groups of bacilli in pigm ent epith elial coll s, and yariou s s tru ctures, such a s small vacuol ('s, smooth · surfaced endoplasmi c r e ti cululll (SER) and llIito ehondri a ( M ) ~ re rCl·ognized in th e cyto · plasm . Chrom a tophol·es (CHR) with oval nu clei a re sho\\"n gl·oupNl togeth ~ r imlll C' diatc ly h eneath th e endoth elium , in th e anterior border la ye l· .

L epra cells (LC ) simila r to tho 0 found in le promatous skin l es ion s a r e seen he re a nd thero in th e iris strom a . In th eir cy topl asm, no t onl y th e normal cytopl asmi c compon ents a re seen, such as rough -surfaced end oplasmi c re ti cululll (RER ) , mitochondri a, a nd pigm ont granul es (PG) , lJUt a lso pathologic compon ents su ch HS leprosy bn cilli, foam y s tructures (FS) and opaque dropl e ts (OD ) . Th e figur e show s th a t the cell membran e of l('pra c('lI s is brok en (BCM) and th e cy topla smi e eOI1'1Jon ents nalll ed a!Jo,·o are extruded frolll th o ce ll.

A slend er group of ce lls shown a t th (' right of tho t ente r of the figur e is composed of smooth mu scle coli s (SMC ) , ' I' he tran sYC'rs(' s('(· tion of :l norm a l nonllly (' l in~t C' d ncn ·l' d ema rcated b y a basement m embra n e (BM) is a dj ac('nt to th e c('l!. L (' prosy bac illi f o rm

68

Chief iindillgs in the leproma/om.; iris Hrc (CI) illriltl'ution of lepra eell s in iris stroma , (b) changes in the nonmyelinated nerves of the iris, i.e., cnlargem0nt or shrinka ge of the axons, alld the presence of M. lep1"lae in ,:lXons, (c) the presence of M. lep'ra e in endothelial cell s of the capil­laries of the iris, (d) the presence of M. lep'I'(( e in smooth muscles of the sphincter pupilla e and th e dilatator pupil lae, and also in the inter ­s titium between muscle fib er . , and (e ) th e j) l'ese1lce of !II. leprae in pig­ment epithelium of th e iri s.

In the specimens used in this s tud y, \\'e were able to study the de­tails of (1) lepra cell s in iri s stroma , (2) lepromatous changes in smooth muscles in the iri s, and (3) lepromatous changes ill the p igment epithelium (Figs. 1, 2.) , \1 e were no t able to find distinct lepromatous changes in the hlood vessels and l1 erV0S in the iris stroma of these specimens.

General a ppco'r,u'll ce of lepromat ous chunges in the iris .- L epro­matous changes arc usually more abundant in the posterior part of th0 iris stroma than in the anterior part (~ . l ~. ~I) . A characteristic f eature of lepra cells in the iri s, which differ s from their appearance in skill and periph eral nerve truuks, is the presell ce in most of them of pig­ment granules ill varyin g numbers in their cytopla sm.

The iris is a tissue rich in pigments. Clump cells, cbromatophores of iris stroma, and pigment epithelial cells contain 11Umerou s pigment granules in the physiologic state. Smooth muscle cell s of th e dilatator pupillae and macrophages also contain a f ew pigment g ranules e O) .

In iris stroma lepra cells are usua]] y not packed toge ther tightl~r ,

as in lepromas of the skin. III most cases they ar e separated from each other and located in the posterior layer of the iris. 111 ad vanced cases, however , in which" iris pea rl s " Ot' "miliary lepromata '" are found clinically, many lepra cells are found tightl y packed togeth er in th0 anterior layer of the iris (2 t) . At times the cell membran es of lep ra. cells are broken, and leprosy bacilli, opaque droplets, foamy structure. , pigment granules and mitochondria are extruded f rom the cells into the extracellular environment.

Collagen fib er s are usually normal in appearan ce in the extracellu­lar media, but sometimes slight edema is found.

In advanced lesions of the stroma of the lep;'omatous iris, plasma cells and many vacuolated cell s, which r epresell t the final s tage of de­generative change in lepra cell s, are found. Also, many cytoplasmic organelles derived from hroken lep ra cells 1:I 1'e scatter ed in the extra­cellular media (Fig. 3).

groups in smooth muscle ce lls, espec i>;t lly in ju xtanu clear pa r ts, co ntainin g ma ny mit o· chondri a; a n electron·tra nspa rent zo ne (ETZ ) is observed a roun d the a rea.

L ep rosy bac illi a l'e not foun d in th e up per en dothel ium of th e iri s a nd also not i n th e' ,ascul a r endo th eli a l ce ll nround the erythrocyte (ECR ) on th e left side of the p ictur e-, Vac uoles a rc found in ce ll s r esemblin g plnsmn ce ll I'i ,' h in rou gh ·sur facecl endoplasmi c r et iculum. V al' ious corrclat ions between lep rosy bacilli, pi gment g ranul es, foamy st ru t'­t urcs a nel opa que d ropl e ts in lepn, cells Ill ay be note(l . Tn th e ex trn rell ul a r pa rt of th e iris s trom a, tra nsve rse (CF(T» nncl long itll (lin nl (CF(L» se-di ons of co ll a.gen fib ril s a re fo un d; th ey show n o patholog ic changes.

FH ;, :!, EIf'(,t-I'(Jll Jlli.'rog-r:!J11! of ;111 ultr:tliJill s(·(· tioll uf :l 1('llrulIl:!tlIlIS iris. 'nll' illHS­tl'at.ioll is :1 ('Olll}l{)sitl' on e 1 1I ~ld(' LIp of 1(J\\, - III ;q"~' lIifi~' :l tion (·Il't'troll luil' )'Og"l':l ld lS sh owing' :lll l"I'I'OIlI :t lllllS ( 'I "" I ~"'" Tit .. 111' 1"'1' I':t 1'1 i" tI,,· 1:t,"('1' (If' .. n""I I,,·li,1l1l of' II,,· il'; " :111" tI,,· lo\\'1'!' is t ill ' postl'rio)' P:ll't of tiJ,· iri s, I '('PI';I (,(·11", ('onl:lillillg' piJ,!'Ill('Ilt- gl':I IIIII ('s of \ ' ;ll'yillg" si z(' ;l lI d irJ'i'g,t11: l r di :"'\ triill i tioll, f O;IIll Y :-: trLll'tlll'l', a lHI Iv pr() ~y I ): will i , :1J' t ' fHlI ll d ill s<'\'(,I': 11 " Ia"t's .. ?- 1:i g'1I ili,,: tl ion I ,:WO X ,

SYlIll,ol,,: FS, YO:!!)IY "II' IH'III!"' : cell; CHR, eh l'nlll<llopliol'l',

33, 1 H ashiz llni C d'; S lt iorwlll a: Lepl'Ol1wto lt s Changes in 11"1:s 65

L epra cells in t7, e iris.- L epra cells in the iris resemble those of lepromatous skin and peripheral 11 er ves . rrhe chief differ ences are that lepra cells in the iris fr equentl y contain pigment granules, and have at times slender protrusion s.

Lepra cells in iris stroma have an oval nucleus. vVhen leprosy bacilli multiply in their cytoplasm and later degenerate, leaving large electron-tram;parent vacuolar structures . hehil1(J , th e nucleus of the lepra cell is pressed asi(le hy the contents of these vacuoles. Tn the cy toplasm of lepra cells various cy toplasmi c organ ell es a re seen, such as mitochondri a and smooth-snrfa ced and rough-surfa cecl endopla smic reticulum wi th Paladc's granules . . Mitochondria are not frequent, just (\ s in ordilla ry lepra cell s of th e skill. LT sually in the cytopl asm of lepra cells, varioll s-s izNl vacuol es, opaque dropl ets, pigment granules, and leprosy bacilli ar e seell (Fig. -1:). Some of the vacuoles are separated from each other , but other s have coalesced. E specially in old and de­generated lepra cell s, most of the cytoplasm is occupied with large vacuoles. Opaque d ropl ets are of vary ing size, electron density and quantity in differ ent lepra cells, hut in most cases they a re surrounded by a limiting membrane. 1 suall y vacuol es of varying size and pig­rnent granules a re embedde(l in opaque droplets, but ther e are also opaque dropl ets that have an electron density uniform with that of electron-dense small granules (0.2-0.6 mIL in diameter). Sometimes 1-3 leprosy bacilli are found in an opaque droplet (Fig. 5). There is a tendency for small opaque droplets to be round and for large ones to be of irregular shape. ']'hese opaque droplet s closely r esemble those of the skin and peripheral nerv es, "which have been described by Nishi­ura et .al. (17.18 ).

On the other hand, opaque droplets, which ar e r elated .to the pig­ment granul es in lepra cell s of the lepromatous iris stroma, r esemble normal and ahn ormal pigment formations seen by electron microscopy in epithelioid cell s of the uvea in sympath etic ophthalmia, which were described by 1kui et {fl. (5. G). On the hasis of electron microscopic find­ings he concluded that epith elioid cell s in th e uvea in sympathetic oph­thalmia might originate from chromatophores.

~rhe similarity thus noted might suggest that the lepra cells seen in iris stroma in our studies also have been derived from chromatophores. However, normal clump cells and chromatophores have many uniform­sized pigment granules, and they too are uniformly distributed through­out the cytoplasm. In contrast, the qua.ntity of pigment granul es in lepra cells is usually small, and the size of the granules varies. They are distributed unevenly in the cytoplasm, and fr equently from small numher s up to several doz.ens of pigment granules are grouped together in the cytopla sm. Sometimes they are found isolated in the cytoplasm of lepra cell s, hilt in many cases they are emhcdcled in opaqu e droplet s toge ther with leprosy hacilli. rr'he pigmont mas. os vary in size from powd er-like fine g ranules of low electron density to el ectrOll -dense largo gr anules with capsul es. Tn old and degCll erated lepra cells in which

I" ig-.:l. 1';il'el"OIl IlIi("l"og-l' :l pll "r " " 1I111'"I I,ill " " 'I io ll 01' :III ,,1' so"'I ,' 1,' P"'"II :l IOli s il'i ,;. L~I'g p fo ,,,,, .'" s ll'ud,,,·p, d,'g- l' IIPI' :l t e d pig-nl t' li t gr:lIl"iPS, opa"II (' "I'oplds , :Llld ntil" (' holldl'i,, IUl' sc ntl l' J' l' d p :dJ'nct' lIul " I' ly ill till' iri s ,tl'onln . Thl' "1'11 , 0 11 Ih e' I ('ft. ("o ll tainillg pig-nll'n!; g":lnllil' s of "nifo l'lI! (' h:ll':lrt l'l' , is ,1 11 0 ,.",,11 r hl'ollln t ophol"('. ~l'\' (' I"" ]'"11 (' 11:11' , ll'lIdun's [I"" s et' n . \I"g' lI if i",,[ioll .) ,H Of) X .

~.'"II"/OI s: FS, f" " "I .\· ' [,·II..t II 1'( ' ; PG, pig'lI" ' 1I1 g' 1' " 11111,,; OD, 0 1""1"" dl'opl " t : M , lIIilo ­('hondl'i,, ; CHR, ,· 1'1'(1111 :" " ph," ·" .

J " IG,~, 1'; 1""11'011 IIlinog.,." I'll 01':111 IIl1r:"llill s"l'l ioll (If typi .. :" )('1'1':1 ,'plls ill tht' iris strO lll:l , !."prosy I':"'i lli s ho w illg \' "rious s l:lg" s "I' ,IPg"lI ,'r:l tioli ill Ih,' <",\' lopl:lslll :In' fo und :IS si ll glc iso )"t c d I""' illi :1I1l) gr(Jl IPS of I,,,<"illi , All ,' I('d roll -tr:l lI s pn n' III' zO l1 e :lilt.! o P:lfl IJ(' "I'oplds :11'(' ollS,'rn'" :11'011 1,,1 tlll' lIl, ~) i flJ('holldri:l :11'" 1101 I1UIIIl'I'OUS :lllil rOllgh -s lIri'n (-('d :111 (1 silioolh -sll rf" ,'"" (' 11"opl:l,,"i, ' r"'i"lllulll is ,,'(,II, ~) :lgl1ifi":ltioll l!l ,:;OO X ,

S,\'Il1l,ols: Le, I(' pr:l (" , 11 ; LB, I"prosy 1"((' illll s; ETZ, I'It'('tru ll -tr:1IlSp:1I'l' llt ZU lil'; RER, rOllgh -s llr f :l,"' " ('lI dopl :IS lIli,' 1'(' li(, lIll1lll ; SER, s illoulh -s urf"""" l' lli loplaS IlJi (' 1'('t ic u11l1l1; OD, opnqu l) tll'opl,'I; M, 1IIil'I('hol1dri:l ; LM, lili lililig 111('11 " '1''' 11 ''; FS, fO:IIIlY st rudlll'l'; PG, )I ig- IlI CIl t gr:l 11 U fLo,

68 In lcl'natiollol .!ollnlOl of LCP1'OSY 1065

most of the cytoplasm is occupied by electron-t rall spar ent vacuola r structures, opaque droplets and pigmellt gnlllul C'~ are few.

Characteristics 0/ the s lll ooth 1nuscle of the iris .- '-Cile l'e a re two smooth mu scles in the iris st roma, viz., the sphincte r pupillae and the dilatator pupillae. Some smooth muscle is found also around arte ri oles. Numerous electron microscopic studies on the ultra structures of the sphincter pupillae and the dilatato r pupillae of the ll ormal human eye have been reported ('n. ~:; . ~7. ~8. ~U) . According to these studies, the two mu cles r elated to the movement of pupil lae are cOll str ucted of smooth mu sCl le cell s contain ing myo fibril. ·, rn~' ofi l a rn C' 1lt ~, mitocho1ldria , endo­plasmic r eticulum, Uolgi apparatus, and a small Humber of pigment granul es. lVl itocholl(lria of the smooth muscles of the sph incter pupillae a re located in a juxtanuclC'a r part of the cytoplasm ; they are of long­rod-shape, with dist inct cristae in side them. 1n contrast, the mitochon ­dria of the smooth muscle ce ll s of the dil atato r pupill ae are distrib nted even ly th roughout the cytoplasm, and they are of short-rod-shape, with the cristae not well developed. There are basement membranes outsid e the plasma membranes of smooth muscles. 'When smooth muscle cell s ar e sepa rated from each other a little more widely (0.2 fL ), collagen fibril s and nerve fibers are fOUl ld between the adjacent basement mem­branes (2(;. :iD). As observed by light microscopic studies, many ner vC' fibers are distributed in a definite pattem, and smooth mu scles of the iris are innervated by the vegetative nervou s system. Elect ron micro­scopy also has shown that smooth muscles of the iris are richly in­nervated by vegetative nerves (25. 2D) .

Electron, mic1'oscopic f eatures of lep1"oma t o'Us changes in the smooth muscles of th e iris .-A few descriptions based on light micro­scopy have been r eported on lepromatous changes in the sphincter pupillae and the dilatator pupillae (4. 13.21). According to these, leprosy bacilli are found in smooth muscle cells and also extracellul arly be­tween muscle cells. .

In the biopsy specimens examined in the present study, smooth muscle cells were found in the sphincter pupillae and around arterioleR, but lepromatous changes were seen only in smooth mu scles of the sphincter pupillae .

.Ill these specimens most of the sphincter pupillae cells a re grouped together form ing mu scle cell bundles, but some of them are isolated by collagen fibers and nerve fibers from the main muscle cell bundles . Each muscle cell has a basement membrane around the plasma membran e, and in their cytoplasm there are mitochondria, endoplasmic reticulum, Golgi apparatus, small vacuoles, myofilam C'nts, and pigment 0Tanules in small numbel'. N umel'OUS smooth mu cle cells contain :") . .

leprosy bacilli and foamy structures in their cytoplasm (Figs. 6 and 7). l\f ost of the mitochondria ar e of long-rod-shape; they arc located along the axis of the cell body and especially near the nucleus. In smooth mu scles affected by lep romatou s change, many mitochondria a r e found, accumulated around the foamy structures. In such cells

;~;l, 1 IIashi : IlJl/( (l' HMoI/IIlI/a: Lf'I)/'OIJIfI/()IIS ('/tall{Jts ill I /'is 69

FIG. 5. Electro)} mi<'Togl'aph of an ult nlthin ~('ctioll of n lepromatous iris , Large foamy struc'tul'PS containing man," <IPgc'll(,l'Htc'(1 h' pl'os,I' hncilli, opaquc dl'oplt'ts :\1I d pig· ment granules of varying si~c') with high eleetron·density, arc shown. Magnifkation O,800X.

Symbols: LB, leprosy hac'illu~; FS, fOHmy stl'udurc; PG, pigment granule; OD, opnquC' droplet.

I"w. Ii. 1';1"" 11'0 11 III il'l'ogr;IJdl of ;111 IIltr:l -tllill sedioll of " It ' prOIIl:ltolis iris. .\ hig'II 1I1:lg'llifi":ltioll pil'IIlI'l' of :I s llloolli 1lIII S('\P ('('11 ill tile iris. '1'11(' tr:lllsl"('rsl' :llld IUIig-illltiill:l1 Sl'd ioll s of 111"".\' I<'I" 'OSY I",,·illi ;11,,1 Illilu .. llolldri:l ;lrl' o"s('I'\ ' "d ill IIII' (,l'n ll' ;11 :11'1':1 of till' ' 11100111 11111", 1(, ('1'11, SIII'I'Olilld l'd "y "'1"' 111(' 111 1I1(' IIII,r;III('s. 'I'll" ,,\P"lroll -tr:l Il Sp;lrl' lIt zO ll e is ShO\l'lI, IJilt 110 Op'I(JlIl' drllpll'l is ('\ ' i<ll'llt 11" ;11' "'I"'("Y ";H·illi. .\ s ll'lIdl' r ('c ll ]Jro('('ss, sec ll 011 thc rig'ht side of II" , Sli looth 11111 ",1,' (',, 11 , \l'lli"I, "olll:liIlS pig-IlI( ' III' g-r;IIlII"'s, is I'l'g-nrtlril as a P:lr( of ;1 ,·hl·olll,,'oJlhor, '. 'I'll" pillo(·.doli(· l"('si('ll's ;11"(' \' isi"'" dir l'(· I1 .\· II ll d('r till' plnsmn 111('1111.1';111(' of II", SlIIool li 11111 ", 1(, "1'11 . ~ 1 ;1g-lli(il';ll ioll l!l,:iIlIl X .

:';1'111 "ols: SMC, s lllooth 11111"'1 1' ("l' II; LB, II'Jlrosy h:1('ilills; M , lIIil(ll'holldrin; PV, P;IIO ­('ytot;';, \,(,8;('1 (' ; PG, 11;glll C' llt gr'llIui c.

33, 1 T1as /li ?: IIIII(' (Co S Ii 'iOIl IlIlI(f: / ,(' jJrOIiW/O/lS C II({II !JI'S ill Iris 71

swelling allcl vacuoli7.cdion of mitoch ondria are ll ot ed, and tlw cristae mitochondrial rs havc he come indistinct.

In t-h e smooth mu sclcs with lepromatou s cha ngrs, pigmen t g r anules are absent or prese llt in small number s only. rl'hc picture is in striking contra st to that- of lep l"a cclls ill the iri s st roma, which almost always cOllb,in man y pigmen t g ranules. Pigmcnt g l'allulcs ill a f"f('ctcd smoot l] Illll scle c(' ll s do no t (Ii ff e l" from thosc in intact s lllooth muscle cell s .

As all'l'culy notcd, pi g-HI Cllt g l'anul cs illl c])I'R ccll s of thc iri s stroma ar<' of VHI'~r ill g s i7.(' 1-Ind diff(' J'(' ld (· lccLl'oH d(' ns iLy, alld ar(' di st l"ihuted ull ('vcnl y ill tll (' ir c.vto pla slll. ~Io s t- of th c pigm l' llt g rClnul es ill lepra cell s aI'(' ('Hdwddl'd ill opaq u(' droplc, ts. Oil thc ot h(' r h1-lnd , ill smooth mu sc lc c(' ll s, OJd.," 2 o r :-3 pigm(·nt grallul es of' 1I1liforlll s i7.(', with the san\{' cl('ct 1'011 d (' ll s ity, li (' iso la tcd ill thc cytopla sm. ' I'hcsc filHlin .!.;s suggest that I(' prosy ha cilli haY(' no cssclltinl r c la tiOllship to pigm ent granules in smooth lllll scl(' s.

Leprosy hacilli Cll"(' usually foun(l in tl1(' ce ntral part of tl1<' sphin c­ter pupillae c('ll s . Nome of them li (' ill so li ta r~r positioll S ill thc cyto­plasm, whil e othcl's arc agg l'egnt.ccl . ' I' h e s izc of th e haci lli Hnd th(' deg ree of hacillal'.v d('generRtion in smooth muscl(' cell s r csemhl r what is seen ill the bacilli in lepra ccll s of th e iri s stroma . \\"hell scv(, l"al bacilli a rc aggregated, th ey tend to be a rranged along the long axis of the smooth muscle ccll s . -Ill ohsolete leproma tous case~, fORlll~' strnc­tu res with f ra gmCld s of degenera ted hR cilli arc foull(l n (' ,-11' th c llllClC'us of the smooth muscle cell s . Frcqu entl~r mitochondri a an' accumulat('cl a round groups of leprosy bacilli 01' ckct ron -transpH 1' (, llt foam ." s truc­tLu'es (Fig. 8). rl'he textuJ'(' of the juxtnnucleal'portioll of thc smooth muscle cell is looser than that of othe r portion s of till' c)rt opJa snl \\'h er (' there are abn ndant myofih rils, HnclJc>prosy ha cilli alld mitochondria accumulate in this part h ecause of its lesse r res i ~ta n cc.

Opaque choplets, which arc obse rved almost cO)l stantl y ill o]'(li]lclJ'~' lepra cell s, are not fo uml in lepromatous ]('s ion s of s lll ooth mu scles of the iris . No particular changes arc founel in the llllCl eus, cndopla s­m ic r eticulum, pinocytotic vesicl es, plasma m em hran es, awl ha semen t membranes of smooth muscles infected with leprosy hacilli.

Around the smooth muscle huncHes Rnd a lso lwtween the individual smooth mu scles, where there are thick layers of ha s('mt'nt m emhran es, ther e are numerous 11 0nmyelinated Herve fibcrs and lI ervC endin g-so gvcll at the ll eUr0I1111 SCular junctioll, the]'e "'R ,' always a ha s('m cnt lll C'mbrall e, a nd ]10 dircct- contact of the pla sma mcmhran c of smooth mu scle cells awl axon memhran e 01' c(' 11 memhrall (, of th(' ll c rvc endin g­wa s f01lnel in these s l)('cinlC'lls. III the matcrials cxamin ccl ill the 11J'CS­cut study, n o pathologic chang('s wer c found in ne rvc fiber s adjaccnt to smooth mu scle fib er s with 'di s tinct lr promatous changes (~--' ig . 9).

L C}Jr'om.a fo li S r lI(f1'lg ('s 0 l 111 (' pig'/ll ('lI t (' pi tll ('1 i'lliI/ of 111 (' i'ris.- rl'h c pos terior surf'nc(' of th e iri s is C'ove J'('(j ",ith (1011]) 1{' iH)T(' rS of' pigm cnt epithclial cell s . Posj,(']'ior-layt' l' piglll l'nt (' piUw lial ccll s I'a cing thc pos terior chamber have pigm en t g ra nul es, mitochondria, endoplasmic

72 J ntc1' national Journal of L eprosy 1965

reticulum, and Golgi apparatus in their cytoplasm. Infolding of the free cell membrane facing the posterior chamber is observed in many cells. Anterior pigment layers make direct c0l1tact with the posterior pigmel1t epithelial cells by apposition of the cell membranes. The

Ilashiz nlll c (C; S hionwJla : 1,cpl'o1Jlotons Chang es in h 'is 73

llllclear portioll s of Lhe <lilataLol' pupillae a re located in this laY0 r. Cytoplasmic organ ell es of anterior pigment layer cells a re few as C0111 -

par0cl with those of the posterior pigment epithelial cells e5 ) .

Lepromatolls changes affecting the pigment epithelium of the iris, a s studied with the light microscope, have been r eported by Mitsuda (13), Shionuma el.~2.2a ), and Ribi (4). They stated that in light micro­scopic studies in general, even when leprosy bacilli have multiplied and degenerated in pigment epithelial cell s, no dist in ct foamy structure formation is found there. Some of the globi form ed in the posterior pigment epithelium of the iris are thrown out of the cell s through the broken cell membranes and later attach themselves to the po teriol' surface of the iris or float in the posterior chamber .

Characteristic features of leprosy bacilli and degeneration of mitochondria in pigment epithelial cells were observed in detail by electron microscopy. E lectron microscopy of the pigment epithelium of the lepromatous iris has also r evealed the presence of foamy struc­tures in small quantity in pigment epithelial cells, which could not be found by light microscopy (Fig. 10).

Leprosy bacilli are found in the cytoplasm as single organisms or in groups. Some groups of bacilli are also found near the nucleus. The appearance of lepr osy bacilli in pigment epithelial cells is similar to that of leprosy bacilli in ordinary lepra cells. ~s the degeneration of leprosy bacilli advances, the breadth of the electron-transparent zones around degenerated bacilli becomes greater. In a few pigment epithelial cells examined in this study, we have found large foamy structures, with limiting membranes, which contained severely degen­erated leprosy bacilli. Opaque droplets, which are usually found in or­dinary lepra cells, were not fo und in pigment epithelial cell qf the iris.

Pigment granules of the pigment epithelial cells of the iris ar e larger than those of the chromatophores, and are distributed evenly in large number in the cytoplasm. Even in the pigment epithelial cells with lepromatous changes, the number, size, morphology and distribu­tion of the pigment granules do not differ from those of the pigment granules in normal pigment epithelial cells. This findin g of the pig­ment granules is different from that of the pigment granules in lepra cells of the iris stroma. In the lepra cells of the iris stroma, the" com­pound body" of Dalton and F elix (Z), and opaque structures r esembling

FlO. 7. E lectron mic rograph of a n ultrathin section of a leprom ato us iri s. '1'he sec­tion sh ows a b lood yessel, a cell ri ch in rough-surfaced endopl asm ic reticulum, nonmyelin ­ated nerves, and a smooth mu scle ce ll. On th e extreme right, two leprosy bacill i are ob­served a mong many mitochondr ia in th e celltral a rea of a smooth muscle cell ; a few pig· men t granul es a re in clud ed in th e cell. Half of th e lon gitudi nal section of a s in gle bacillus is Sh OW11 s lightly downwa rd f rom th e cente r of the right edge. A cell rich in rough­surfaced endoplasmic r eticulum, an d w it h a clen r Golgi a ppa ra tus, is fou n d between the endotheli a l cell of a bloo d vessel and n smooth mu scle cell. Leprosy baci lli are not foun d in it. P a thologi c changes are no t fo un d in n onmyelin ntecl n er ves . Magnification 8,300X.

Symbols : ENC, endoth eli al cell of b lood vessel ; ERC, e rythrocyte; A, axon; SCH, chwann cell ; SMC, smooth muscle cell ; BM, basement membrane; LB, lep rosy haeillus;

PG, pigment granu le; RER, rough·smfa ced endoplasm ie ret iculum; N , nu ch· us.

74 Inte1'?/atiollal JO/(1'Iwl oj'Lcp1'osy 1965

FIG. 8. Elcdron mi('rogr<lph of nil ultrnthill sl'dioll of n lcprol1llitotl8 iris. This shows the foamy structure of a smooth llIuseIc cel l ill nil olJ~oletc lepromatolls iris. lIfnllY fonmy structures are ohseryed ill the neighborhood of a llUeleus, nll(l Icprosy bntilli. arc not found. Magnification 7,300X.

Rymbols: SMC, smooth mus(·le ('cll; FS, foamy structure; M, mitodlOndria; N , nncl('us .

75

1' 1(:, !l , 1':1( , .. 11'011 III inog.,." "I I of :111 III11':I1I, ill ,w('l ioll o f:l "'1'1'0111:11011 ' il'i s , TI' :l ll SVCI'S(' s ('('li o ll of:l II OlIlll ,I' (, lill :I1('d II (' I'\ 'P ill lh,' i l'is, !. l'P I'O S,\' i>:I(' i lli :11,,1 01 11l'1' p:l th o logi ('. r hallgps :l l' l' 11 01' fOil "" , ~ 1 "g lli (i<'''lio ll ~~ , -lllil X ,

1-',I' IIlI, o ls: SCH, 1-'(' 1111' :111 11 "" Ii ; A, ,,''') 11 ; N , II" .. "' " S; BM, ""lS(' II I(,llt 111 1'1111.1':111 (',

76 International J o1t1'nal of Leprosy 1965

FIG. 10. Electron micrograph of an ultrathin section of a lepromatous iris. It shows 11

large vacuole and degenerated leprosy bacilli in l.I. pigment epithelial cell of the iris. A nucleus is pressed laterally and has become flat. The size and distribution of pigmellt granules are almost normal. Magnification 7,100X.

Symbols: LB, leprosy bacillus; FS, foamy stmetul'e; N , nucleus; PEe, pigment epi· thelial cell; PG, pigment granule.

33, 1 JIashiZll lil r (C, Shionullla: Lepromatolls Changes in Tn's 77

" mela llin illclusion bodies" were fo und (Fig. 11), but in the pigment epithelial cell s such bodi es were not found.

']'he ]lUmher of mitochondria is 1l0t so large, but often they becom e enlarged, with sign s of degeneration, such as vacuolization and ob­scuring of the cri sta e mitochondrial es .

DISCUSSION

There a re several hypotheses on the origin of lepra cell s in the iri s. Phillipsoll and Ba bes stated that chromatophor es in the iris s troma could change in to lepra cells, and GOll e) r epo rted that hoth clump cell s and chromatophores phagocytize leprosy hacilli and also that hi .. tio­cytes change into lepra cell s by phagocytizing leprosy bacilli and pig­ment g'rlllluics. According to his desc riptioll pigment g ra1IUles of chronlHtophores infected with leprosy bacilli are more irregular and large r than those of normal cell s, and also some of the cytoplasmic processes of infected chromatophores are being torn off. Tn contrast, Shiolluma stated that chromatophores that cannot take up carmin e or India illk whell s taill ed vitally will 110t take up leprosy hacilli, and for this reason he concludetl that lepra cell s ill the iri s are derived from clump ce ll s that have phagocy ti7.ec1 leprosy hacilli and f rom histiocytes that have taken up leprosy bacilli as well as pigment g ran ul es (2~. 23 ).

As lloted above, electron microscopy of lepra cell s in the iris has shown that most of these cells contain pigment granules in the cyto­plasm. ~Jo]'phologically ther e seem to he two types of lepra. cell s in the iris. On e type consists of lepra cell s containing pigment granules of varying size with differing electron density, together with precursors of the pigment. rrhis type of cell has al so slender processes projecting from its body. ~rhese findings suggest that this type of lepra cell was derived from chl'Omatophores in which variou s stages of pigm ent for ­mation can he seen. The other type of lep ra cell ha s pigment granule .. of ullifonn size, which surround groups of leprosy hacilli in their cytoplasm. rrhe appearan ce of cells of this type suggests an origin from macrophages. These electron microscopic findings might sugg-est that both chromatophor es and macrophages can convert themselves into lepra cells in the iris, but final conclusion s on this probl em still need further investigation.

·With r eference to lcproma tous chang'c in smooth mu scles, it i ~ noteworthy that by elect ron microscopy Nishiurl:1 found leprosy hacilli in smooth muscl e~ arollnd blood ve~sel s of the na sal muco~;;a, and that with li!tht microscopy Hirako found leprosy hacilli ill smooth muscle cells of the erectores pili and also between muscle cells. Ultrastructural detail of lepromatous changes in smooth muscle cell s in the iris is similar to that of smooth mu scles in the na sal mucosa.

According to Ollr clillical ('xpe rience, som e lepromatous patients have pupils fix ed in thc statc of miosis, with 11 0 light r eaction of pupils. As about 70 per cent of patients with lepromatous disease ha.ve chronic iridocyclitis, the lesion is likely to he complicated by post erior synech-

7 Intel'natiOllal JOllnwl of Lepto y 1963

FIG.]1. 8Ied"on mil·rogl":\Jlh of an ultrnthin s('dion of n I('Jlroll1ntou~ iris. A "ml'lnnin inclusion hody ( Dalton a ,"1 ~'('lix )" is o"s('n' ('<1 in a so·tullp(] manophnge in th e iris stroma. Leprosy hnl'illi an' not found. (Jroups of pignH'nt granules with sud, <"lear limiting membranes al'e ra,'(, in the cells of lep"omatous lesions. Magnifil"ntio n ~6,~OOX.

ymbol : N , nudeus; PG, pigment granule; MIB, melanin inclu ion body.

J[a shi z /flll l' ([' Sh im/ /III/a; 1'('7)1'0 1ll0tO/f S Chang('s 1' /f Trig 79

iilt' , heCHU S(' tlw posh' rior half of th e iri s is more fr('quc' ldJy aJfcctt'd hy lepros)' bacilli. Altho11gh sYllC'chia e arc th e most freque ll t ca ns(' of Illiosis ill lepromatous patiellts, there arc cases of miosi s ill which there are no synechia e, the shape of pupil is round, aIld mydriatic drugs do not challge the state of mios is . .In such cases of miosis para lysis of the dilatator pupilla e is most probable.

In other lepromatous patients, however, the pupil is fixed in the state of mydriasis hy paral ys is of the spliinctel' pupill ae.

'I' he elect ron microscop ic finding of lepromatous chan ges in smooth muscles, and the illtact sta te of the ll erves of the iri s, suggest that direct damage of smooth muscle by leprosy bacilli might be one of the ca uses of smooth mu scle paral ys is in fun ct ional di sturhance of the pupil ill lep romatou s pati ents.

Although some cases of tuberculo id leprosy show continuou s mydria s is during the course of tubercu loid reaction, it is consider ed that such mydria sis is caused by nerve damage ill the parasympathetic system during the phase of r eaction, and not clue to direct damage of smooth mu scles in the iri s by leprosy bacilli.

I t is generally sa id , on the basi s of light microscopic studies, that foam~T structure is Hot found in the pigment epithelium of the iris. In our studies by electron microscopy, how ever, small quantities of foamy structures were founel around leprosy baci lli in the cytoplasm of pig­ment epithelial cells of the iris. \Ve have identifi ed these cells as pigm ent epithelial cells on the basis of their location in the iris and the appea rance of the pigment granules ill them.

SUMMARY

l1 ltrast ructural features of lepromatous chl:llIge in the iri s str oma, smooth muscles alld pigment ep ithelial cell s of the iri s arc described.

Lepra. cells arc more ahu ndan t in the posterior- half of the iris st roma than the a nte rior half, and these lep ra cells frequently contain pigment g ranul es. rrhis is in contrast with the condition of lepra ce ll s in the skin a nd peripheral nerves, which have no pjgJnent granules in the cytoplasm.

rl1he origin of lepra cells in the iris stroma is discussed on the basis of electron microscopic features of these cells. ~h chromatophores and macrophag('s seemed to change in to lepra cell s ill the iri s, but a. dec isive cOllclu sion could not he r eached evell hy e]ectron microscopy.

Lep rosy hacilli \\" e re foull(\ in smooth mu scle ce lls of the iris. ThC'l"e were also e]ectron-t ran spa rent ZOll es and foamy structures in smooth muscle cells, and, in obsolete lepromatous cases, large vacuolar struc­tures without hacilli in th e c?toplasm of smooth muscle cell s, beli eved to be the r esult of complete disin tegration of hacillary bodies. Smooth muscles of th(' iri s arc rich ly innervated hy nOllmye]inat('d nerve fibers, but no nerve changes were fou nd ill specimens examined ill the present study, in striking contra st to di stinct damage of s rn ooth muscle cell s. Lepromatous change of smooth mu scles in the iri s is of exceptional

80 I nte1'natiol'lal .T01lrnal of L C1?rosy 1!J65

illtert'sL as a Muse of di s tlll'hallcr of t he li ght l'cacLioll of the pnpil ill lepromatoll s patients.

Leprosy bacilli \\'(,ft' found also ill pi gnwllt ('piLhelial ce lls, either ill groups within th e cytoplasm, or lying free hut separated fl'om each other. Small quantities of foam y structmes were found ill pigment epithelial cell s. This finding is contrary to the general opinion that foamy structure. are neve r rncountrl'ea in pigment epith eli al cell s.

RESUMEN

Se describen los as pe~tos de los cflIllhi os leproillatosos en 1'1 e~trOlIl I1 (!I'I iri s, como Hsi en' los 11I1lscu los liso;; y las celul l1s epitl'liall's pigl1lt'ntadas.

Las celulas leprosHs son I1II1 S abundantes ell la lIIitad posterior del estrOllla del iris que I'll la lIIitad anterior, y estas celulas lepro .. as conti enen f recupntcllI cnte g rflnulos pig mentlll'ios. E sto contrasta con las condicion es de las celulas leprosl1s en ]a piel y ncrvios pel' iferi cos, Ill S ('uales no contienen granulos pig l1lenwrios en el citoplasilla.

Se discute el origen de la .. celulas lepl'osas en el estrollla del iris en base de los aspectos lIIi croscopicos electroni cos de estas celulfl s . Tflnto los cromatOforos como los lIIacr6fflgos pareccn cOl1vertirse en celulflS leprosas en el iris, PCI'O no se puede lI egar a un a conclusion decisiva flun pOl' el micl'oscopio electronico.

Se encontl'al'on bfl cilos lepl'osos en las celulas de los lIlusculos li sos en el iri ~. 'l'ambien hubo en las celulas de los IllUSC lllos li sos zonas electl'oni cas tt'!Lnsparentes y est L'U r tul'as es pulllosns, y en caRos leproma tosos ohsoletos, estl'uctllrns vn.cuolares g l'andes sin bacilos en el citopla:; 11111 de las cclulas de los lIIusculos lisos, 10 fJu e se crce es el r esul ­tnrlo de la completa desintegracion de los cuerpos bacil ares. Los mllsrulos li sos del iris esta n ricamente inervarlos pOl' fibrFl s nel'viosas no llIi elinizadas, pel:o no se encontraron call1bios nerviosos en los esp eefmenes examinados en el presente pstudio, en f uerte con­tt'aste con el danG distintivo de las celulus de los musculos lisos. En pl1cientes leproma­tosos los cam bios lepromatosos de los musculos lisos en el iris son de excepcion.al intel'es, como una causa de altel'acion de III. r eaccion a la luz de la pupila.

Los bacilos lep1'osos tam bien f ueron encontrildos en las celulas pig mental'ias epiteliales. E stos hallazgos son cOl1trarios a III. opinion general de que las estructUl'flS vacuoladas lIunca ~e (,Ilcuentran en las celulas epitelia les pig ll lCntarias.

RESUME

Sont ici deerites les caractel'i stiquf's de la fin e stl'Ucture des changements lepromateux dans Ie strollla il'idien, les muscles lisses et les cellules epitheliales pig mentaires de I'iris. Les cellules lepreuses sont plus abondantes dans la moitie posterieure du stroma iridien que dans la 1II0itie anteri eure, et ces cellules lepreuses contiennent f l'eq uemment des granules pigmentaires. Ceci est en contraste avec les cal'acteri stiques des cellules lep­reuses dans la peau et dans les neds pel'ipheriques, qui n'ont pas de granul es pigment­a ires dans Ie cytoplaslll e. L'origine des cellules lepreuses dans Ie stroma de l'iris est .cJis­('utee sur la base des cal'acteristiques r evelees au niveau de ces cellules par III. mi croscopic electronifJue. Tant les chromatophores que les ll1F1crophages sell1blent se transformer en cellules lep reuse .. dans I'iris, IllHis aucune cOll clusion defi nitive ne peut etre obtenue, meme pal' Ill. Illi r roscopie electron ique.

' Des bacilles de III. lepl'e f un'nt trollves dans les cellules des muscles lisses de l'iris. II y ava it aussi dans les cellules IllUsculail'es lisses des zones transparentes aux electrons et des structures spumeuses et de plus, dans Ie ' yieux cas lep1'oll1ateux, des gran cles tI'UC­tures vacuolisees, sans bacill es cette f ois, da ns Ie cytoplasll1e des cellules muscul airrs Iisses, et que 1'0n croit etre I'a hollti sselllcnt de la. desintegrati on co mpl ete des CO ['ps bacil ­laires. Les muscles li sses de I'iri s sont richemcnt innerves pal' des fibres nerveuses non myelinisees, mais aucune modifi cation dans les neds n'a toutefo is ete notee dans les echantillons exam ines au com's de cette etud e, ce qui est en flag rant contraste avec les

33, 1 JZashiZ/lme (C; 8hiol1l1l11a: L elJI'Ol1wtOUS Cha?lg es i?1 Tl'i~ 81

l e~iom; "aI'Hdel'i sees dl's eel lules Illuscul a il'cs lisses. Les changelll ents de type lepl'o­IIluteux au niveau des IllUseles lisse de I'il'is sont d' un interet exceptiollne\ en tant que constituant un e cause dc r eaction anol'lnal e de la pupille it la lumiel'e chez les malades leprolll atcux.

Des bacilles de la Jepl'e ont aussi ete trouve,; dans Ie,; (,l'lIull's epithelia lcs pig lllen­tail'es, soit g roupes it l 'inthieul' du rytopla sllle, soit libres mais separes les uncs des autl'es . Des structures spUllleuses ell petit nOlllbl'C ont ete tL'ouvees dans les cc!lules epithelialcs pig ll1entaircs. Ccttc obse rva ti on cst en QPposition avec I'opin ion general e­ment adllli se que des s tructures spulll euses ne sont jalllais trotlvees dans les cellules epithcli aJes p ig menta ires.

A ckno1rledg ement .- Th e authors wish to ex pl'es~ their Sll1 ce l'e thanks to Dr. 1\1. Nishiul'a for his kindness and encoura gement in this investiga tion .

ImFERENCES

1. BIRBECK, M. S. C., MERCER, E. and BAR NIC01', X. A. The structure and form ation of pigment g ranules in hUllian hair. Expel'. Cell Res. 10 (1956) 505-514.

2. DALTON, A. J., and FELlX, M:. D. Pig ment Cell Growth (Gordon, M.) New York, Arademic Press, 1953, pg. 267.

3. GON, N. On th e clinica l and histopatholog ieal flndings of the eye diseases in leprous patients. n. Histopathologica l findings. Acta. soc. ophthal, japon. 43 (]939) 2111-2142.

4. 1-IIB[, H . "Etfer',t of prOinin and other su lfones on cye leprosy. Histopathological findings. J . Kansa i Med. Sch. 12 (1960) 6H-628.

5. 1KUJ, n ., KIM UHA, K. and IWLlKT, S. S tudi es on sYI.llpathetic ophthalmia with elec­tI'on microscop c. Acta soc. ophtha\. japon. 61 (1957) 1194-1210.

6. h:UJ, H., KIM UHL\ , K . and NISl-IIO, T . Further studies on sympathetic ophthalmia with eler.tron microscope. Acta soc. ophtha\. japon. 62 (1958) 1188-1203.

7. hiAEDA, T. and CO~VlT, J. Electron microscop e study of lIIycobactel'ium lep1'lte an (l its environment in a vesicular leprous lesion. J. Bact. 82 (1962) 43-52.

8. hfLlEIM, T. and OGUIlA) 1\1. Formation of intracytoplasmic. membra ne systcm of lII yco bacteria r elated to cell division . J. Bact. 85 (1963) 150-163. .

9. IWAMOTO, T. Electron llIicroscop ir studi es on Bechet's syndrome. Report 1. Iri s tissues f rom patients of Bechet's syndrome. Ada soc. ophtha\. japon. 64 (1960) 528-540.

10. IWAM.O'J'o, '1'. Electron microsC'o pic studi es on the ceJls in the human iris stroma. Acta soc . ophtha!. japon. 65 (1961) 1296-1367.

11. 1\[ ALFA'J'TI, M. G. a nd J OKQUIERES, E. D. L . S tudy of the morpholog ical modiflca­tions of 111ycobaclel'ill1n lepme during ehel11otherapy. 1nternat. J. Leprosy 21 (1953) 323-:337.

12. MINAl\u, K. Patholog ische studi en iiber di e i\e rven in Augengebiete unter verschie­denartigen Krankheiten. 1. Mitteilung' . Pathohistologische Beohachtungen iiber die Nervcn im Augengebiete bei Lepra. II. Tei\. Uber die patho-histolog ischen Vedi.n­dcrungen dcl' Irisnerven von Leprakranken. Acta soc. ophtha\. japon. 41 (1937) 444-469.

I:'). ]\I[ ITS DII , K. On the leproll s eye. ~ihon-Igaku 65 (1910) . R ep?'in tecZ in " Papers on lep rosy by K ensuke Mitsuda ," Vo\. ] , 1935, pp. 109-129, Okayama, Japan, Chotokai }'oundation (in .J apanesp).

1-J.. ;VII'I'S(' I , Y., SIIi',UKI, A. , }"'UK\lS HlMA , S., [IIII>A R. , O(:A 'I'A, S., K UHTIJAIlA , IT ., OK,I­J\I \I /! A, H. IIlId MI URA , M. 1'; Il'l'trol; Inir'ros('oPY of tra('hoilia virus in spction . Part ) V. Ada SUl'. ophthal. japoli. 62 ( 19:18) 12·10-] 2-17.

1:1 . MI UHA, Y. Electron lIIieroscopie studi('s Oil thl' tra('holll:t g ranule. }\r>ta S fl<' . ophth:t1. japon. 62 (1958) 1226-1239.

82 TniG1'llatio llal J OIl 1'?wl of Dep1'os,!} ] 965

16. ~ I S lfl ll IlA , '!'Ii(" II AIlAI)A, 1'{ . a lld 1 ~ I AE Il A, T . 1': lp{' t l'oli 11I i(' I'os('o PY of' ult l'a-thill se('­tiO Ii S of IpPI'OIl Hltous per iphera l II (' I' VE'S . IlI tr l'lI lIt. .J. Leprosy 2S (19:'57 ) 323-:3:37 .

t7. ~ I S H.I U RA, M ., S IIl S,,"I', S . M. a lld KH ANOI.KAIl , V. H. 1 '~ I( ' c tl'u lI 11I icl'os('op ic study o r leprosy les ion . Lrp l'oHy ill Ill d in 32 (1960) 90-93.

18. N ISHIURA, M. The elect ron microscopic hasi::; of the pathology of lep ro::;y . Inter ll at. J . Lppl'osy 28 (] 960) ::157-,1.00 .

19. OKAMO'I'O, T. F; 1('r't l'on IIli c l'osco pie studi('s 0 11 the fi ll (' s t rurt llre of lI ul'lli a l hum ull eye. Acta soc. op hthal. .iapoll . 64 (J 9(0 ) 2-W2-2-10S.

20. 1:{ UDOI,P, C., G~~ORGF;, A. K a nd H I': J. lVIU'I', R . A l'ch itl'ctul'P and lI en 'e sup p ly of II lalll ­Ili a li ull s lllOoth Ill uscle tiss ll e. J . B iophys . & Bioc·helll . Cy tol. 3 (1957) 867-878.

21. S III ONU~tJI , 1<; . Patholog ica l studirs 011 ('y(' ll'p l'osy. ] . !.epm!>y of th e iris find co r­pus (· ilia I'P. Cha p. 1 r. On iri s lep l'oli l. La Lrp l'o 8 (19:H) 64 1-6-19 .

22. S HI ONU MA, K Plltholog'ica l studies Oil eye II' PI'OSY. 1. L('p l'osy of: the iri s a ll d ('OI 'pUS (' ilia I'e. Chap. J[J. On the lep rous degl'lI el'atio ll in thl' iris a lH"! att itude of the iri s pig nlent ce lls aga inst leprosy hacilli . La Lep l'o 8 (19::17) 65.1-659.

23. S l-I IONlJ MA, E. Hall dbook of Op htha lll lOlog'y ( ill Japanese) Vo lUlli e 12, Lep rous E YI' Diseases, 1953, pp. 59-68.

24. SHIOSI':, Y. E lect ron nlir l'oscopi(' s tudies on the cilifl l'Y lIlusell'. A cta soc. op htha!. japon. 6S (1961 ) 1267-1283.

25. TA NIG UCHI, Y., U~~:-: o, K., SU MI'I'A, Y. a lld X AKA11I W, K . E lect ron II I icrosco py of t he uvea l t ract. Acta SO(·. np htha!. japoli . 6S (1961.) 2302-2402.

26. 'l'lI AF::amRT, J . D. The ultl'l.ls t l'Uctul'c a lld d is posit ion of vC's i(·ul ated np l' ve p rocesses in SlII ooth muscle . • J. Cell. B ioI. 16 (1962) 361-378.

27. TOM,I'rA , 1., ]\If Al'SUO, T. a ll d K ;1'1'0, Y. E lect ron III icl'os('o py of t ill' p ig lll cnt-l'pithcli a I cells a nd d ilatato r p upill ilC' nlllscies in t he hUll ta n iris. Acta SOt. op ht ha !. japon . 6S (1961) 1177-1187.

28. TOUSIMIS, A . an d FI ~~=) K S. Ultrast m cture of the ir is : a n electron microscopic study. Ameri can J . Ophthal. 48 (1959) 397-404.

29. UEN O, K . T he' ul t rast ructure of the hUl1Ian iri s. Acta soc. ophtha l. japon. 66 (1962 ) 383-409.

30. YAMAMOTO, '1'. , NrSHI uRA, M., HAIlADA, ~ . a nd IMAr~DA , T. E lect ron Illi cr osf',op.v of ul tra.-thin section. of lepra cell s a nd MYl'o bacte1'ium lepme. In terna.t. J. Leprosy 26 (1958 ) 1-8.