Scanning electron microscopic studies of the canal of schlemm

Scanning electron microscopic studies of the canal of schlemm

F~xTtl Eye 1los. (1970) 10, 2 1 4 - 2 1 8 Scanning Electron Microscopic Studies of the Canal of Schlemm* ANDEI~S BILl. Departmv~t of Experimental O]...

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F~xTtl Eye 1los. (1970) 10, 2 1 4 - 2 1 8

Scanning Electron Microscopic Studies of the Canal of Schlemm* ANDEI~S BILl.

Departmv~t of Experimental O]>/~t/mlmology, lnslittdc
1. I n t r o d n c t i o n I n 1)rimates t h e m a i n r e s i s t a n c e to tl.te o u t t t o w of a q u e o u s h u m o r is h:wate~! in l.he t i s s u e that, is b e t w e e n t h e a n t e r i o r clmmb rw am.l t h e (.:amd of St:lde, n : l , . Tim c o m p l e x a n a t o m y of tills region h a s .mot.ivated l n a n y sttuties in w h i c h t rall-~mis.~ion e l e c t r o n m i c r o s c o p y w a s u s e d ( H o l m b e r g , 1965; V e ~ e . , 19G7 ; .Kayes, ]967 ; T r i p a t hi. I968). I n t h e s e s t u d i e s £rontM or m e r i d i o m d s e c t i o n s w e r e usc
Male a n d female v e r v e t (Cercopithecus ethiops) a n d rhesus (Macaca m u l a t t a ) monkeys, w e i g h i n g 1.8-6-0 kg each were used. The m o n k e y s were a n e s t h e t i z e d with soMium methoh e x i t a l (Brietal, Lilly), a b o u t 20 m g / k g b o d y weight, a n d anesthesia was m a i n t a i n e d with s o d i u m :pentobarbital given i n t r a v e n o u s l y . Using ,~ needle gun each eye was c a n m t l a t e d wi~h t h r e e c~nnulas. Two c a n n u l a s c o n n e c t e d th e a n t e r i o r c h a m b e r to push-pull coupled syringes c o n t a i n i n g 2-5 or 4~/o g l u t a r a l d e h y d e in m o c k aqueous h u m o r (BAr f a y , 1964). The p H was a d j u s t e d to 7-4 ~'ith 0.10 _~ N a 2 H P O 4. The t h i r d c a n n u l a c o n n e c t e d the a n t e r i o r c h a m b e r to ~ pressure t r a ns duc e r . The a n i m a l s were killed by rapid bleeding t h r o u g h ,~ e a n n u l a t e d femoral a r t e r y a nd a n overdose of p e n t o b a r b i t a l ; at t h e s a n t e time, the a n t e r i o r c h a m b e r fluid was m i x e d w i t h t h e content,s of t h e push-pull coupled syringes w i t h o u t a n y rise in eye pressure. T w e n t y m i n u t e s later th e a n t e r i o r p a r t of the eye was g e n t l y r e m o v e d , using a t e c h n i q u e t h a t p r e v e n t e d a rise in eye pressure. The p r e p a r a t i o n was d iv id ed into 4-10 sections t h a t were fixed in ice-cold 2-5 or 4~/o g l u t a r a l d e h y d c in m o c k aq u eo u s h u m o r , pI-I 7-4, for 1 hr. Some specimens t h e n were d e h y d r a t e d in g r a d e d e t h a n o l (30, 60, 95 a n d 100~/o) a n d air-dried. O t he r specimens were fixed in 1~/o osmitmt t e t r o x i d e in m o c k aqueous * T h i s i n v e s t i g a t i o n w a s s u p p o r t e d b y ,~ g r a n t ( B 7 0 - 1 4 X - O 6 C ) f r o m t ho S w e d i s h 5Iedieal I / e s e a r o h Council, and ~ grant (EY-00475) from tho l~ational E y e Institute,U.S. Public/c[ealthServico. 214

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h u m o r , p l l 7..1, for an a d d i t i o n a l 12-hr p e r i o d ; w a s h e d w i t h d i s t i l l e d w a t e r ; h a r d e u c d in el:hanol (30, 70, 95, 70, 3 0 % ) for 30 rain a t e.ach c o n c e n t r a t i o n ; a n d w a s h e d w i t h r e d i s t i l l c d w a t e r . T h e s e s p e c i n m n s were t h e n freeze-dried. S o m e p r e p a r a t i o n s werc fixed wif~h 1 0 % formalin m o c k a q u e o u s h u m o r , p H 7-I. T h e (-'~nnl of S c h l e m m w a s o p e n e d u m l e r a microscope.. w a s d o n e a t d i f f e r e n t st.ages of t h e pro(:es~iag in diflhre, n t p r e p a r a t i o n s . Iit web p r e p a r a t i o n s t h e i n c i s i o n w a s m a d e n e a r t h e antt~rior or p o s t e r i o r en([ of t h e c a n a l h ' o m t h e a n t e r i o r c h a m b e r side. T h e i n n e r wall w a s t h e n g e n t l y (ilsh:)cat.ed in s u c h a w a y thai; bo~h the, i n n e r a n d o u t e r w~tlls were e x p o s e d [Fig. l(a)], or t h e o u t e r wall waa r e m o v e d . I n dricd p r e p a r a t i o n s r a z o r c u t s were m a d e i n t o

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I"r(;. i. ta) "l'h,~ c a n a l o f S c h e l m m wits i n ci sed fl'(,m t h e a n t e r i o r c h a m b e r side. a n d t h e c h a m b e r a n g l o t i s s u e ,,vs, r,-,tate, l in silt'It n, v,':~,y at~ f:~ exl)r~::,~ t he itltler art¢l o u t e r w a l l s o f t h e c a n a l . (b) It, d r i e d p r : - p a r a i i o t m r a z o r eul.~ w e r e made; bet& i n t o t h e a n t e r i o r a n d p o s t e r i o r p a r t s o f t h e c a n a l an,[ t h e i n ~ ' r w a l l 'waz t h t ' n ~zently brt)kci~ u p .

b o t h t h e a n t e r i o r a n d p o s t e r i o r anoit:s of t h e c a n a l a n d t h e i n n e r wall w a s t h e n g e n t l y b r o k e n u p [Fig. ! (b)]. ]n s o m e wet. prepar~Ltions, a f t e r t h e c a n a l h a d b e e n o p e n e d , t h e walls of t h e c a n a l w e r e w a s h e d by a s t r e a m of fLxation fluid or eft.hanoi. I n t h r e e m o n k e y s b o t h eyes were c a n n u l a t e d a n d c o n n e c t e d to p u s h - p u l l c o u p l e d s y r i n g e s a n d p r e s s u r e trans, tucers. T h e a n t e r i o r c h a m b e r c o n t e n t s w e r e m i x e d w i t h a s u s p e n s i o n of w a s h e d b l o o d ,'.ells. Afte," 30 rain t h e m o n k e y w a s k i l l e d a n d t h e ant, crier c h a m b e r s w e r e w a s h e d l.hrough w i t h ,1°'o g l u t a r a l d e h y d e or 1 0 % f o r m a l i n . T h e e y e s w e r e t h e n d i s s e c t e d , d e h y d r a t : e d in g r a d e d e t h a n o l , a n d air--dried as d e s c r i b e d a b o v e . D u r i n g t h e 30-rain p e r i o d ~-ith blood cells in the a n t e r i o r e l a a m b e r t h e r e was a rise in eye p r e s s u r e of 3 - 8 m m H g . All f : r e p a r a t i o n s were g l u e d to sl~andard sl;ubs. T h e s e w e r e m o u n t e d a t a n a n g l e o f 45 ° to a n e v a p o r a t i o n s o u r c e a n d r o t a t e d while t h e s a m p l e s were c o a t e d w i t h a 2 0 0 - 8 0 0 A - t h i c k l a y e r of g o l d in v a c u u m . A ':Stereoscan" (Cambridge Instrtunent Company, England) scanning eleetronmicroscope was used.

3. Results T h e leng~.h o f t h e c a n a l o f S c t f l e m m i n f i x e d p r e p a r a t i o n s w a s 3 4 - 3 6 n a m (5 e y e s f r o m 5 v e r v e t m o n k e y s ) . T h e w i d t h o f t h e c a n a l , e s t i m a t e d in 5 m e r i d i o n a l s e c t i o n s f r o m e a c h o f t h e s e eyes, w a s 0 " 1 - 0 - 5 r a m , m e a n v a l u e 0-3 r a m . T h u s , i f f o l d s a n d o t h e r i r r e g u l a r i t i e s in t h e i n n e r w a l l a r e d i s r e g a r d e d , t h e s u r f a c e o f t h e i n n e r w a l l is a b o u t 1 0 - 1 1 m m s . D u r i n g a i r - d r y i n g t h e r e w a s litt.le o r n o c h a n g e i n t h e l e n g t h o f t h e c a r e d . l ) t t r i n g d i s s e c t ; i o n i t w a s n o t e d t M t t h e i n n e r and. o u t e r w a l l s o f t~he s l i t - l i k e l u m e n

216

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of t h e c a n a l were c o n n e c t e d to e a c h o t h e r b y m a n y tissue str[,.nds, w h i c h eouhl be t o r n off w i t h s o m e difficulty. The outer wall o f the canal of S c h l c m m I n m a n y places p a r t s of tim o u t e r wall were covered b y p e r f o r a t e d sept~, w h i c h were a r r a n g e d longit, u d i n a l l y . T h e a q u e o u s h u m o r is d r a i n e d from the canal of S c h l e m m b y t h e collector c h a n n e l s . T h e origins of these c h a n n e l s were often n m r e or less h i d d e n b e h i n d t h e s e p t a (i?Iatc 1). S o m e of t h e tissue s t r a n d s m e n t i o n e d al)ov(.,---whieh were b r o k e n w h e n t h e e~mal w a s opened-v.were, in fact,, connect.ions b e t w e e n t,he immr wall and s e p t a on t h e o u t e r wall. Occasionally, where t h e r e were no sept a, fla~ ellipsoid s t r u c t u r e s t h a t p r e s u m a b l y r e p r e ~ n t t h e nuclei of t.lm e n d o t h e l i a l cells couhl I)e seen l y i n g p a r a l l e l w i t h tim canM. Such stramtures also couhl be seen on t.he seI)t~ a n d tissue strands. T h e i ~ e r wall o f the canal o f S e h l e m m 13oth in w~mhed a n d unwmshcd p r e i ) a r a t i o n s one eouhl see about, 10 ~ long, often spool-like s t r u c t u r e s , whi(:h bulged i n t o the c a n a l . -ks a rule tl'e long axi,~ of these s t r u c t u r e s was p a r a l l e l w i t h t h e l u m e n of t h e c a n a l ( P l a t e 2). A t tim origin of the abovem e n t i o n e d t i s s u e s t r a n d s t h e o r i e n t a t i o n of t h e b u l g i n g st,ructnre's was often irr(~guhLr. S o m e of the b u l g i n g s t r u c t u r e s h a d v e r y long tails. A l t h t , u g h t~hc cell b,:,r~h:.rs could n o t be seen c l e a r l y it see.ms l i k e l y that, w i t h t h e s e tails, ~:aafty (.',ells w i t h b u l g i n g p~rrt.s are m o r e t h a n 70/z long. In 5 prcparatio,~s from 5 m o n k e y s w i t h th:: c a n a l opC~.ed a f t e r a i r - d r y i n g t h e a v e r a g e f r e q u e n c y of b u l g i n g ~true.ture.s was l jr,>m,~: ", T h e range. ..... tz'. was 1/180 to 1/370/z"-. E a c h area a,~sayed was a t l e a s t 18,04)0 tt'-' in size. lit is kno~na f r o m s t u d i e s l a w ) l y i n g t h e use of t r a n s m i s s i o n electron m i c r o s c o p y that, large i n t r a c e l l u l a r v a c u o l e s a n d t h e nuclei of t h e e n d o t h e l i a l cells of t h e inner wall bulge int<) t h e l u m e n of S c h l e m m ' s canal. T h e wmuoles were re.ported to communi~-ate b o t h w i t h Om c a n a l of S c h l e m m and. w i t h t h e j u x ~ a c a n a l i c u l a r tissue, a n d t h u s cons t i t u t e p a r t s of c l m n n e l s for a q u e o u s flow i n t o t,he canal of ,.~:hlemm (I[olmberg. 1965). I n t h e p r e s e n t s t u d y , b o t h in a i r - d r i e d a n d in freeze-drietl I, r e p a r a t i o n s , m o s t b u l g i n g st~ructures a p p e a r e d to c o n s i s t of a rigid part---.prob:d)ly ~ nuch.us---and a p a r t t h a t t e n d e d m collapse--probably a v a c u o l e ( P l a t e 3). A large u u m l ) e r of i n n e r wall prep'~rations were in.spectcd f r o m all sides a n d p h o t o g r a p h e d f r o m one l o n g side a t an angle of a b o u t 15 °, a n d w i t h a m a g n i f i c a t i o n of 2000. I n s u c h p h o t o g r a p h s p a r t s of the b u l g i n g s t r u c t u r e s were h i d d e n b u t one coifld see 2 of 3 openings. T h e t o t a l n u m b e r of a p p a r e n t l y p h y s i o l o g i c a l o p e n i n g s was 22.-35 per 100 blflging s t r u c t u r e s in p r e p a r a t i o n s f r o m 5 d i f f e r e n t ver~-et monke).-s. T h e m e a n v a l u e w a s 2 9 ~ . The t o t a l n u m b e r of bulgbag s t r u c t u r e s i n v e s t i g a t e d was 604. Sonic o f t h e o p e n i n g s were seen in flat a r e a s b e t w e e n t'.,,.e b u l g i n g s t r u c t m ' e s . I t is n o t clear i f t h e s e o p e n i n g s were l o c a t e d in f l a t p a r t s of t h e cells or if t h e y , in facts, were o p e n i n g s i n t o vacuoles t h a t did not bulge into the lumen. T h e d i a m e t e r of t h e o p e n i n g s in the t)ulging Stlamtures v a r i e d f r o m a b o u t 0.3-2/x. O c c a s i o n a l l y one could fred a v a c u o l e w i t h a large, p r o b a b l y a r t i f a c t u a l , o p e n i n g t o w a r d s t h e c a n a l of S c h l e m m . T h e o p e n i n g w a s c o n s i d e r e d a n a r t i f a c t i f t h e edges were i r r e g u l a r a n d w r i n k l e d as in P l a t e 4. In the e x p o s e d i n n e r wall of s u c h v a c u o l e s o n e c o u l d often see a n a l m o s e c i r c u l a r o p e n i n g w i t h r o u n d e d edges t o w a r d s t h e j u x t a c a n a l i c u l a r t i s s u e ( P l a t e 4). T h i s m o s t p r o b a b l y w a s t h e p h y s i o l o g i c a l o p e n i n g t h r o u g h w h i c h a q u e o u s h u m o r n o r m a l l y e n t e r s tlle v a c u o l e on its w a y t o w a r d s t h e c a n a l of S l c h e m m . A few i n t a c t b u l g i n g s t r u c t u r e s were f o u n d to h a v e s u c h large o p e n i n g s

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.Pi.AT~: 2. The'. it~t~c~" wttl[ ~:~t'Schh:t~xnl"~ caluxl it~ a x-t:rx-(~t~ mo,~k£~5-. 'l'h~: cat~al w~xs it~eised it1 il.~ p o s t e r i o r part. I)~.For(~ d r y i , l g . I#ulgi,lg .,4l.t'ut:t.ul.l.S ~ll:)oul. 10 t• ]or~g at'(. p a r a l h : l w i t h th,~ h t , n c n (~E th(: (:algal. S(~m(~ o f t h e cell.~ will', t~tll~in~ st.t'll(:t.tlr(...; hav~- lt.tll~ t a i l s . Its. u o ~;trall£1s Lhab |lax'~: I)e(,l~ I.)rnk(:n cs~.,~ l)e ~l.~rl lit, at'ros~-s, l'rt;c(:(i,.trc: g l u t a r a l d c h s , d o fix~IAott; e t h a n o l d e h y d r a t , i o n ; a i r - d r ) ' i , ~ g .

I'I.ATI-: tll'olilllJiy the plli'l fixatioli ;

it. ] ) f l a i l lit" t h l ' ii'llit.l" w t l l l ~ , | ~ c h l l , n i l l l ' s r(.lll-i+-~elllS il |il~.rlitilly (,<))ililJ.~t.'~[ ~.'liil'llllh~ o f I h l - cell ( ! ( i n l a i i l i l i g Ill(. illi(,]lql,~. A el | l a l i c d d c l l y d rtll i,~ll : a i r - d r y i l l g : ot~..llin

e a l l a l ~ i t l l s t i - u l . l i l i . t ~ ll~llj~ilig i l i l , i i h l , h i l l l ( , l i , %' illO~l. ,.villi Jill (~lll,iiill7 !illl'i t h ( , / , i i l l i i l . . ' ~ tli'lllJ~il,ly r~,lli-(.s(.ill~ vl~ivi:.t, i l i f i i t i ~ , v i.'it~ il~l,
I~LATE 4 . . D c i a i l o f t h e i n n e r wall oJ' S c h l e m m s c a n a l ill "~ vevvet,'m~mkey. 01,(: o f t h e b u l g i n g st,ruet u r e s h a s a larg~ art, i f a c t u a l o p e n i n g i n t o tile l u m e n o f a vacuole.~V). At. P t h e r e is a n o p e n i n g f r o m {,be u m e n o f t h e w t c u o l e t o w a r d s t h e t r a b e c u l a r side. P r o c ( : d u r e : g h i t a r a l ( l t r h y d e f i x a t i o n ; e t h a n o l d(,.hydva. t i o n ; a i r - d r y i n g ; openit~g o f t h e c a n a l .

l)L.4°r~: ;5. D e t a i l o l ' t h e illl)(q" w~dl ( ) f , ~ c h l e ) ~ m ' ~ can~ll in a vhesu.~ m o n h e y . A b u l g i n g s t , ' u e t u r e w i t h t w o ()i)(e))il,~s ( l ' ) m<).~t, l)ro},al)ly eot~tai)is ~l vtte:u<)l<". 'l'h(> s ) n a l l <)l)(+J)ing eotd(l be s e e n | h o v e .el(~arlx- a f t e r rot0.tiol'i ()|" 11~(: l)z'e|)~l'ati(,)n. Not(, tit.(,}) ~)l)~,Iiil)I~ st~t,.it t,l)|'t')~gl~ the [al'g(: ~u]")el'fi~,~itxl o|)e)xillg. ]>roeedllrt:: g l t t t a r a h | ( . i ~ y ( l e fixatic)n ; ()!),.tdu,.-.' o f the. east()|; <:).~))i~ t<'t)~oxi<[<~ f i x a t i o ~ : fveev.e-dr3"ing.

P[,A'rE 6. ])eL~til o f (,he, intl(:v w a l l o f ~ e h l o m m : ean,al in a. r h e s u s m o n k e y . T h e a n t e r i o r c h a m b e r Jlad l)(~en p e r f u s e d w i t h r e d e(:ll,~. ,Az~ e r y t h r o c . y f e (1~) w a s o n i(.s w a y otzt. o f ~ b u l g i n g s t r u c t tn'(~ inLo t h e e n r m l o f S e h l e m m at, Lhe m o m e n t , oi" fix~tLion. P r o c e d u r e : f o r m a l i n f i x ~ l t i o n ; ~t, h a n o l d e h y d r a t i o ) : ; a i r ~ d r y i n g ; o p e n i n g o f the- c a n ~ h

l>l...'lTl'." 7. {l'he ll-lllll.,cullil" nu;.~h~v(li'l~, il,,,i .~l,o;i fl'l)lll th(, ('illi|ll ,iF ,'~,hh'iiilll "ll"ll,i- lht Olllii#.l h~l(l 1.1(.'l~li i'ClliOVl'll I;t%" f~ll(.'i'~Ol io ~,v~llinT. Tli~, l lilhl,(!lilill lll,iilil.,-; I;iill hi, ,~i,l'li |lj oflho h-le.~ ill lh(' I"IO.'llll,'4 (It'l'l'l;lll, |"ili~ fil~l'il,.' ('1111 171l~ ,"i('l'll /11~1'(i.~$ l.l, II ' llttt'lliill.~ ill it!l" VOI'VOt Inollle.(.~v. WII~ ll.--:l'(.I. I"r()('~,ihil'(,: ~,~"hli~lr;llcl~hvdo, l'].~li!illll , ,l~llliilil+ll (li'hvdl'~ili~li,

~, ilill(,i. ,,t'llll "H" Ilia. iiilt~i'(.+~liill-(~l. ~llllli, IIIl~'l'l'lllll.~l li.#'illl'l, .,?ll llil'.lll'%'lliT,.,.

SCANNING

I,]I~EC'I'ItONb|IC.iC.OSCOI'Y OF ,NCH:LlgMM'S CANAL

217

t h a t t.he i n n e r wall of the vacuole cotth[ bc seen ( P l a t e 5). I n t h e inner wall t h e r e seems to l)c a n Opetkitlg t(:,wanls t h e j u x t a c a n , ' d i c t d a r tissue. To i)erl~lit, corn parisons wit lt r e s u l t s o b t a i n e d w i t h t r a n s m i s s i o n electron m i c r o s c o p y lines were d r a w n a t r a n d o m I)ut l)erpcndicttlar to the l o n g a x i s of t h e canal in p h o t o gr~q)hs ()f t h e i n n e r wall of Schlen~nt's canal. In 7 eyes f r o m 7 v e r v e t m o n k e y s t h e n u m b e r of 1)ulging st,ructures t l , a t were ]fit b y these lines v a r i e d from 3.9-9.3/100/1. lengt.h of line. 'l'ho nleatl value w a s 5.9/100/z. A l e n g t h of more t h a n 5 0 0 / , of l i n e s was inspeete, t for e;~.¢.:}~eye. When re~l cells ha¢i been tmrfused t l t r o u g h t,he a n t e r i o r c.hamber one could find red. cells l,rotru, ling Iron, the Ol,enings i J, the t,ul/.png s t r u c t u r e s . P l a t e 6 shows a r e d cell thal. was olx its w a y into tim crmal of Sch.lcmm w h e n t h e c h a m b e r angle tissue w a s tix¢.Jl. There was no tJ}.~vious difference. Imtween v e r v e t and. r h e s u s m o n k e y s . '['lit; {ti ffere, tit. f[xlit,ion itnd d r y i n g procedures p r o d u c e d simil~fr results, t I o w e v e r , aird r y i n g and .~llbsequent ,qmning o1" tho (.anal g a v e p r e p a r a t i o n s with. large ~reas of t h e i n n e r wall suitah],, for c.ah'.ulat.ion of b u l g i n g s t r u c t u r e s per u n i t area, a n d t h e n u m b e r of hulgizlg st rm,tu res w i t h open iI~,gs. Freeze-d r y i n g g a v e s m a l l e r areas with.out~ o b v i o u s :trlitS,.'t.% b u t w i t h i n these m'eas t h e r e seemed t<~ be s o m e w h a t less collapse of tim hulgint4 strut-tares t h a a in air-::[ried p r e p a r a t i o n s . T h e f a c t t h a t some collapse of bu!gin;$ s t r u c t u r e s also was seeu in freeze-dried p r e p a r a t i o n s s u g g e s t s that. s o m e collapse ha~l alremt v oectlr1"t'.d d u r i n g the dissection of the eyes or earlier. "1'/~" I m & ' c u&~ r ..:;sharer]:

In so,he pr,-.par~,.tions the inner wall of t h e c a n a l of ,~chlemm was w a s h e d so vigorou.~lv t h a t p a r t s of the wall were r e m o v e d a n d t h e u n d e r l y i n g t r a b e c u l a r m e s h w o r k w~Ls expose.'l. P l a t e 7 shows the a p p e a r a n c e of the m e s h w o r k in such. a p r e p a r a t i o n . One cat~ see c l e a r l y ~he w a y in w h i c h the tral)ecular b e a m s i n t e r c o n n e c t . T h e r e a r e rt.l'ttively large o p e n i n g s in t.he t.rabe.cular bea.ms a n d s o m e of t h e o p e n i n g s o v e r l a p in several layers. ,%ram of the i n n e r m o s t o p e n i n g s can be seen to be p a r t i a l l y c o v e r e d b y fine fibrils. ~The t:rat)ecular Imams are c o v e r e d b y t,h i n e n d o t h e l i M ceils ( H o l m b e r g , 1965). N e i t h e r the nuclei nor t h e cell borders of these cells could be seen c l e a r l y u n d e r t h e s c a n n i n g electron microscope.

4. Discussion T h e liten~t,ure a b o u t t h e canal of S c h l e m m u p t,o 1965 ha~ been r e v i e w e d i n d e t a i l by H o l m b c r g (1965). T h e s e p t a a n d t i s s u e s t r a n d s s h o w n i n P l a t e s 1 and 2 have, o f course, been observed b y m a n y i n v e s t i g a t o r s w h o e m p l o y e d l i g h t a n d t r a n s m i s s i o n electron m i c r o s c o p y to s t u d y xfieridionM or f r o n t a l sections. T h e s c a n n i n g e l e c t r o n microscope very clearly shows the general arrangement of the septa since large areas of the structures c~n be observed at one time. A compariso~ betweml Plates 1 and 7 shows that the septa are similar to the trabecular beams, with interconnections betwcen t h e sepuum and t h e o u t e r wall of t h e s a m e t y p e as f o u n d b e t w e e n t h e different t r a b e e u l a r beams. T h e f u n c t i o n of the s e p t a is n o t clear, b u t one effect of s u c h s t r u c t u r e s ( P l a t e 1) would no doubt be to prevent occlusion of the collector channels by the inner wall o f the canal if tlds is displaced against the outer wall (Robert and :Rentsch, 1968, 1969). This may be of special importance in such monkeys as the v e r v c t in which the scleral

218

A. B I L l ,

s p u r is v e r y p o o r l y d e v e l o p e d ( R o h e n a n d Ba,-a.ny, 1967). Inter~,stingly, in such m o n k e y s there are m a n y more septa in the ean,d t h , m in hu, n a n s (l[oll,fl)erg, 1965). T h e nuceli a n d v a c u o l e s t h a t bulge into the l u m e n of the, canal from 1he inrw, r wall h a v e been d e s c r i b e d m a n y t i m e s (see t l o l , n l m r g , 19(;5; Vegge, 19(;7; .l('ty,.'s, 1967). 1-Iolmberg• ( I c.:'tk)) '~" r e p o r t e d (;hat tim vaeuoh.'.s, in fa(-t , re.l)res(',nt, p a r t s of (-h mn(,ls t h r o u g h t h e cells. T h e o p e n i n g s f r o m t h e trai:)ceular st(It into the. v,~euoles ar(.' relat i v e l y e a s y to find when the ~ransmission ch,etron lui(:roscol)c is used, b u t t h e o l w n i n g s i n t o t h e canal of S c h l e m m as a rule can be found o n l y if m a n y seri:~l secti,)z~.* ar('. zmule. H o h n b e r g and Bhr~'tny (196G) eom~tcd t h e n u m b e r of vacuoles witlt a d i ' m m t e r of m o r e t h a n 0.2/x and t h e nuclei t,hav can be seen in the in)mr wall of Sehtt, m m ' s canal in m e r i d i o n a l sections. In vervet, monke.ys t h e y fotmd 35.7 "=5-2 nuclei per 100(1 / t l:ttl,l 45'5=t=6-7 v a c u o l e s per 1000/L. In the present, s t u d 3- lim..~ ~lrawn on photogral~h:~ to s i m u l a t e such sections crossed a b o u t 60 b u l g i n g s t r u c t u r e s per 10()0 t ~, which s u p p o r t s t h e conclusions t h a t the b u l g i n g s t r u c t u r e s were nuclei and vacuoles. I n a h u m a n eye K a y e s (1967) has e s t i m a t e d t h e n u m b e r of (.h,mne.ls throu~lx tim i n n c r wall of Sctflemm's c a n a l to be 300-50(I per m i l l i m e t e r of the canal. T h e | l u m b e r of o p e n i n g s seen u n d e r t h e s c a n n i n g electron microscope in vcrvt:t n~onk~,'¢s wa.~ ~tt~)ttt 350 p e r m i l l i m e t e r l e n g t h of the canal. T h e size of t h e o p e n i n g s b e t w e e n (.he c a n a l of S c h l e m , n and tim va('~uol(,s, a b o u t 0-3-2/x, was the same. as r e p o r t e d b y I t o l n , b e r g (1965). A_fter r e d cells h a v e b e e n perfu.~ed t h r o u g h into the anterir)r eh,uul)o," .'~u(::|t ('.ells c a n be seen in t h e i n t e r t r a b e c u l a r spaces, t.hr~ j u x t a e a n a ~ i c u l a r tissue, t | w va~:uoles of t h e e n d o t h e l i a l cells, a n d also in t h e openil,gs of t h e vaeuoIes ( I n o . n a i a , Bill a n d Smelscr, u n p u b l i s h e d d a t a ) . In the p r e s e n t e x p e r i m e n t s w i t h e r ) ' t l m ) c y t e s th(; red cells could be seen p r o t r u d i n g i n t o the l u m e n of S e h l e m m ' s canal from the b u l g i n g structures. I~a t h e p r e s e n t s t u d y the p r e p a r a t i o n of t h e s p e c i m e n s created n u m v art.ifact.s. S o m e of t h e s e ~ s u e h as breaks, a n d s m a l l slit.s p r o d u c e d b y the electron b e a m - - w e r e obviott% o t h e r s ~ s u e h as collapse of vacuoles d u r i n g dissection .... m a y }m s o m e w h a t m i s l e a d i n g . Still, i~ seems l i k e l y t h a t the scareling electroll m.ieroscope will be v,.:ry useflfl in f u t u r e s t u d i e s of t h e p h a r m a c o l o g y a n d p h y s i o l o g y of t h e e n d o t h e l i a l cells; t h e n u m b e r of large b u l g i n g s t r u c t u r e s a n d visible opeuings in t h e i n n e r wall of S c h l e : n m ' s c a n a l in t r e a t e d a n d c o n t r o l eyes c a n be c a l c u l a t e d q u i t e easily. ACI,2NO'WLE DGM I'~NTS I wish to t h a n k Miss Anit'~ Pcrssoa and 5Iiss Monica Thor(.n for their w:duable techr, ical assistance, hIr GSran Alsterborg and Mrs A n i t a I ( a j l a n d , AB Analyt.ica, Sollentuna, gave

expert assistance at the s c a n m n g elect.ronmicroscope. REFERENCES

B£r£ny, E. I-I. (1964). Invese. Ophthalnwl. 3, 135. :EIolmberg, z~-. S. (1965). Doc. Ophthal~ml. 15, 339. Holmberg, A. S. and B£r£ny, E. H. (1966). Invest. Opthal.mol. 5, 53. Kayes, J. (1967). Invest. Ophlh•lmol. t5, 381. t-Cohen, J. and :Bhrhny, E. H. (1967). Arch. Ophthalmol. 172, 23. Rohen, g. and Rentseh, F. J. (1968). Arch. Ophthalmol. 176, :309. I~ohen, J. and Rentsch, F. J. (1969). Arch. OphthalmoL 177, 1. Spencer, W. H., zklvarado, J. and Hayes, T. L. (1968). Invest. Ophthalrnol. V, 651. Tripathi, P~. C. (1968). Exptl Eye Res. 7, 335. Vegge, T. (1967). Zellforsch. Mikrosla~, Anal. Abt. Histochem. 77, 267.