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Ultrastructure of lymphatic vessels in the vascularized rabbit cornea
Exptl .Eye Res. (1970) 10, 207- 213 Ultrastrueture of Lymphatic
V e s s e l s in t h e V a s c u l a r i z e d Cornea
Rabbit
H . I~A]~P.Y COLLZN
The Victorian College of Optometry, University of Alelbourne, Melbourne, Victoria, Australia (Received 6 .~Iarch 1970, London) A carbon atmIm1~,3ion was injected into rabbit cornem~ vascularized as a result of silver rdtnzto application. Dot h light and electron microscopic techniques were used to study the c.,rrbon-filled corneal lylnph vessels. The corneal lymphatics have a structure similar to that of lymphatic vessels ira other sites. The endobhclium is thin, there are no pericytes and no basement membrane and anchoring filamenk~ are present. Hence, they am true corneal lymphatic ve.~uels.
1. Introduction I n p r e v i o u s p u b l i c a t i o n s (Collin, 1966a, b, c) it h a s b e e n show~l t h a t in a s s o c i a t i o n w i t h t h e g r o w t h of b l o o d vessels i n t o t h e m a m m a l i a n c o r n e a , t h e r e is f o r m e d i n t h e c o r n e a a s e t of c a n a l s w h i c h is q u i t e d i s t i n c t f r o m t h e n e w l y - g r o w n b l o o d vessels. T h e s e c a n a l s c a r r y p a r t i c u l a t e m a t e r i a l s u c h as c a r b o n i n t o t h e c o n j m m t i v a l l y m p h a t i c s y s t e m (Aoki, 1951; K a t a y a m a , 1957; Collin, 1 9 6 6 a ) . , n d p r o v i d e a n a v a i l a b l e r o u t e for i m m c d i a t e r e m o v a l of l a r g e m olectfles s u c h as J:~i-lab elled a l t m m i n f r o m a v a s c u l a r i z e d c o r n e a to t h e l y m p h n o d e s of t h e n e c k (Collin, 1970). As a r e s u l t of s i l v e r n i t r a t e s t a i n i n g of t-hese c a n a l s in w h o l e m o m l t p r e p a r a t i o n s , t h e y a p p e a r t o be l i n e d b y e n d o t h e l / u m in w h i c h t h e cell o u t l i n e s are s i n u o u s a n d s h o w a n i r r e g u l a r l y d e n t a t e p a t t e r n (Collin, 19'36c). A t . t e m p t s to d e m o n s t r a t e an e n d o t h e l i a l l i n i n g to t h e c o r n e a l l y m p h a t i c vessels u s i n g l i g h t m i c r o s c o p y a n d paraffin sections, h a v e b e e n unsuccesst'u] (Aoki, 1951; K a t a y a m a , 1957; Collin, 1966c). :It w a s t h e p u r p o s e of t h i s s t u d y to d e m o i m t r a t e , u s i n g b o t h l i g h t a n d e l e c t r o n m i c r o s c o p y , t h a t t h e c o r n e a l l y m p h vessels possess a c e l l u l a r e n d o t h e l i a l I i n i n g a n d to d e s c r i b e t h e ultr:,,stoact.ural c h a r a c t e r i s t i c s of t h e s e c o r n e a l l y m p h a t i c vessels. I n o r d e r to i d e n t i f y t h e c o r n e a l l y m p h a t i c vessels t h e y w e r e l a b e l l e d w i t h a n e l e c t r o n o p a q u e rr, a r k e r , viz. c a r b o n . O n l y vessels c o n t a i n i n g c a r b o n w e r e c o n s i d e r e d i u t h i s i m : e s t i g a t i o n a n d no vessels c o n t a i n i n g c a r b o n b u t r e c o g n i z a b l e as b l o o d vessels w e r e c o n s i d e r e d . I n fact, c a r b o n w a s n e v e r f o u n d w i t h i n t h e c o r n e a l b l o o d vessels.
2. Method The left ~ye of each of four a d u l t albino rab b its was a n a e s t h e t i z e d w i t h 0.4~/o Novesine (Wander) aLd silver n i ~ a t e stick was applied to t h e c e n t r e of t h e cornea for approxim a t e l y 2 see. Am area 4 to 5 m m in d i a m e t e r was d a m a g e d . I n t h e following d a y s ~he corneas b e c a m e h a z y a n d o e d e m a t o u s a n d t he r e was a p m ' u l e n t conjunctivitis. Blood vessels grew i n t o t h e corneas. After periods of 7 weeks (two animals) a n d 12 weeks (two animals) t h e r a b b i t s were a n a e s t h e t i z e d ~with i n t r a v e n o u s N e m b u t a l (Abbott) a n d a n in~ection of a c a r b o n suspension (1% in sterile isotonic saline of 100 rag/eros--from G u e n t h e r Wag n er, l-Iannover,
207
208
tt[. B. COLLIN
G e r m a n y . N u m b e r CI1/1431a) was m a d e into t.he c e n t r e of e a c h c o r n e a using a 30 g u a g e needle. I n one a n i m a l of each p a i r t,he c a r b o n was s.een to e n t e r ~t n e t w o r k of corneal c a m d s an,! t h e n c e flow i n t o the c o n j u n c t i w d l y m p h . l t i e vessels. T h e con~ea was flooded w i t h ice-cold buffered g l u t a r a l d e h y d e fixative consisting of 2 . 5 ~ g l u t a r a l d e h y d e in 0.067 ~t s o d i u m e a e o d y l a t e buffer at p H 7-4. Tim a n i m a l was t h e n killed with a n overdose of N e m b u t a l a n d t h e corneal tissue w.ls r e m o v e d a n d pla(:e:l in t h e fixative for 1 hr. The tissues were w a s h e d and f u r t h e r disscctc,1 i n t o om'~ll piee,:s in buffer of the s a m e m o l a r i t y and t h e n post-fixed in o s m i u m tet.roxide in 0.067 .~l s o d i m n eaeodylal.e ata p H 7-4. ( S h e p p a r d , p e r s o n a l communic.,~iion.) T h e tissues were d e h y d r a t e d in a c e t o n e and embe,.hled in Aral,lite. Urar~)'i steel:de (0"50/0 in 750'0 a c e t o n e ) bli,(rk s t a i n was followed 153, lead c i t r a t e (l~ev:mMs. 19~;3) atilt u r a n y l a c e t a t e ( 1°'/o aqueous). T h i n sections were cut (m ~tn l', l( 13 l.:ltr()t~:me [ using, glass. k n i v e s a n d e x a m i n e d on ::n A E I E,M t3B (Figs I nnd 3) or a S i e m e n s E l m i s k o p 101 ole,'tr,,n mieroscol)e. T h i c k sections were s~ained w i t h llichards,.),x's .~,tain (lli,:har~[:.;on..lar,.'£-tt an,I F i n k e , 1960) a m i x t u r e of A z u r e I I ~ 3 I e t h y h . m e blue :l u,l light mi~:ro;gr:lphu were t a k e n w i t h a Leica ,35 m m c a m e r a a t t a c h e d t.o a Zei.ss T r i n o c u l a r mierosvo]:~.
3. R e s u l t s
Seven weeks All o f t h e s e vessels a r e in tile l)eril)heral are.'~ o!:-the c o r n e a in -.he re,,a,ion 0.5 t(: 1"0 m m D o r a t h e l i , n b u s . As a r e s u l t all o b s e r v e d vessels w e r e q u i t e tart,., (Pl,~t,.s t and 2). T h e e n d o t h e l i a l cells are e l o n g a t e d a n d t h i n ( I " [ , t e 3) w ~ r y i n g f r o m a r o u n d 04!71"8/x ~md u p to 4.5 l* a.t tim n u c l e u s w h i c h is m o s t l y ilatt:e.ne,:t. The. (:ytopla.-.r,,~ .'nay b~: le~s d e n s e l y s t a i n i n g that), n e a r b y b l o o d v a s c u l a r e n d a t heiial c)'*<~plasm. "l'h(- ~mcl,,.n.s Ires v e r y e x t e n s i v e l i g h t s t a i n i ~ g n u c l e o p l ' t s m , nulp.t.r(sus nuc!erar l~(sres a m ! fr,:~tu.:mt! / o n e o r t, wo p r o m i n e n t - n u c l e o l i ( P l a t e 1). B o t h lumi,~al a n d a l b m n i n a l e n d o d l e l i a i projec~cions a r e present, b u t n e i t h e r a r e l i u m e r o u s , t:im: t)-m!,hat, ic. all(:ht)ri;2g fi.lalllt:tlt.-~ a r e p r e s e n t in s o m e a r e a s o f t h e a d v e n t i t i . d eel I surfatc(~s a n d are. quit,.., well ,:i,~v,dopt.d. T h e l m n e n o f t h e vessel m a y c o n t a i n c a r b o n am{ l i t t l e (..I~: ( P l a t e 1) ,)r oc,:~,.~ionMiy it m a y be l o o s e l y p a c k e d w i t h cells t h e m a j o r i t . y o f w h i c h r e s e m b l ~ lynq)l:ov3"tr.'; (Plate 2). T h e c y t o p l a s m o f t h e l y m p h a t i c e n d o t h e l i a l celbs cont~,ins b o t h r o u n d (0.2 1'-) a~ut e l o n g a t e d (0~8/a,) m i t o c h o n ( l r i a w h i c h a r e d a r k l y s t . a i n i n g w i t h w e ! l - d e f i a e d c r i s t a e and t h e y d o n o t a p p e a r to be r e s t r i c t e d ix) t h e r e g i o n n e a r t h e n u c l e u s . T t m r e is s o m e l o n g t u b u l a r r o u g h s u r f a c e d e n d o l ) l a s m i c r e t . i c u l m n a n d s o m e r i b o s o m e s o c c t t r r i n g s i n g l y or i n c l u s t e r s , b u t n e i t l l e r o f t h e s e is v e r y plcnt.ifu!. C)'totsla.smic fibrils a r e p r e s e n t a n d in s o m e a r e a s pinocygot-ic vesicles, a p p r o x i m a t e l y 67 n m in d i a m e t e r , a r e e x t r e m e l y n u m e r o u s ( P l a t e 3). C o a t e d v e s i c l e s a r e a l s o f:',ir]y e o m n t o n . The i n t e r c e l l u l a r j u n c t i o n s a r e m o s t l y c l o s e d a l t h o u g I t a f e w o p e n j u n c t i o n s a r e presenl;. I ) e s m o s o m e s a r e r a r e l y found and w h e n p r e s e n t a r e not, w e l l - d e v e l o p e d . A b a s e m e n t m e m b r a n e and pericytes are not present.
Twelve wee]as F o l l o w i n g carbon injection l y m p h a t i c vessels were f o u n d to be present in the cornea to a distance of 6 m m from the limbus. The ly~nphatic v e s s e l s are slightly larger than ~he blood vessels of the same section, the l y m p h vessels often measuring as m u c h as 55/~ b y 10 ix. T h e wall is c o m p o s e d solely of a single layer of endothelial cells w h i c h , n a y be e x t r e m e l y a t t e n u a t e d and
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PL,~°re; 7. In~ierdigitath~g cell jur~ction (J) hi which the endot belial c~'lls (E) a,'e ~loscl3" apposcd. Vesicles arc.presellt izt t i l e c y t o p l a s m . C, Carbon : (;o, collagen ; L, lume~L ( x -10,000) I)L,¢,.'1'1.2, ~_. P a r t , o f t h e e n d o t h e l i a l w a l l o f ;~ e o t ' e l e a I l y m p h a t i c vessel, q?hero are tlltl}lel'otls vtxsioles (V) alld all albunlina[ endoLhelial projeetiotl (~'k't~l_~) o o r l t ~ t i n i t | g mitochtmdri~ (31). A base:tllent nlelllbra|10 ]s no~ visible. C o , C : o l h t g c n ; L , l u J L / e n . ( -.~. 2 2 . . 5 0 0 )
10 l'L.~'J'r: (.). E n d o t h e l i a l w a l l of a c o r n e a l 13"J:lpnatie. N ' u m e r o u s ,'esicles tLIlcl c y t o t ~ l a s m i c .fit~ril.-." at,:: l)t.csent, l , y t ~ q ) h a t i e a n c h o r i n z /ilazt:cnts (2%.F) e x t e n d f r o n l t h e albttn~i~lal endotAlelial sux'/'ace to ~:he e 0 1 1 a g ~ t i b r e s (Co). 1,, _[~ttm<:t~. (x.10,tXJ0) ]?r,.,,Tr: I0. l : ] n d o t h c l i a | ~vall o f corJ~eal l y m p h ; x t i c , b e t w e e n 2 a n d 3 m m fvon~ t h e l i m b u s a h o w i n g a n c h o / ' i n g f i l a m e n t s (.-kJF) a~¢l t h r e e eot~+ed v e s i c l e s (G'~,'). Co, C o l l a g e n ;I5, l u m e n . ( x 40,000)
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regions as thin as d00 to 500/~ arc f r e q u e n t l y seen (:Plates 5 a n d 6). T h e cell tlfickness is u s u a l l y :rather c o n s t m l t e x c e p t a t t h e area of t h e n u c l e u s w h e n t h e thic.kness is m o s t l y in t h e r a n g e 1.5-2-3 tL w i t h a s i m i l a r t h i c k n e s s tbr t h e c y t o p l a s m n e a r t h e n u c l e u s or in regions w h i c h a p p e a r to be c u t m o r e obliqucly. T h e o t h e r e l e m e n t s w h i c h n o r m a l l y c o m p r i s e a b[ood vessel wall are n o t p r e s e n t ; advcnti~ial ceils w h e t h e r of a n m s c l e cell type, or p e r i c y t e s are n e v e r observed. On occasions w h e n c y t o p l a s m i c p o r t i o n s of cells were i b u n d o u t s i d e a n d a d j a c e n t to t h e e n d o t h e l i a l ('.ells, careful e x a m i n a t i o n r e v e a l e d t h a t t h e s e were p a r t s of fibroblasts, k e r a t o c y t c s or k e r a t o b l a s t s . A c o n t i n u o t m b a s e m e n t m e m b r a n e is n e v e r seen a r o m l d the Iyml)lJatic e n d o t h e l i u m . H o w e v e r , on rare occasions a s m a l l q u a n t i t y of e l e c t r o n (lense m a t e r i a l r e s e m b l i n g a s e g m e n t of b a s e m e n t m e m b r a n e ~.nd m e a s u r i n g a b o u t 2 i~ in l e n g t h was observed. T h e nuclei are. m o s t l y oval in sl,al)e a n d in g e n e r a l are flatter t h a n in blood v a s c u l a r endot.heli~un. T h e r e is e x t e n s i v e l i g h t staining, c e n t r a l m m l e o p l a s m a n d d e n s e peripheral c h r o m a t i n w h i c h is s o m e t i m e s m o r e d a r l d y s t a i n i n g t h a n in b l o o d v a s c u l a r en(lobhelial nuclei. A s l i g h t space is s o m e t i m e s p r e s e n t a r o u n d t h e n u c l e u s b e t w e e n t h e miclcar nmmlJra, nc an(t t h e c y t o p l a s m a n d this m a y be d u e to s h r i n k a g e or artifact. N u m e r o u s / m c l e a r pores are p r e s e n t a n d in these regions close c o n t a c t is always m a i n t a i n e d b e t w e e n t.he n u c l e u s a n d t h e c y t o p l a s m . O c c a s i o n a l nucleoli are seen, C'e[1 jul~(:tions v a r y front close a p p o s i t i o n of cells witl La set)aration of a r o u n d 100 ]k ( P l a t e 7) t h r o u g h t h e s l i g h t l y open jmmbions to w i d e l y s e p a r a t e d cells or gaps of up to 2.5/* b e t w e e n endotllelial cells. W e l l - d e v e l o p e d d e s m o s o m e s are n o t o b s e r v e d , b u t a n a r r o w i n g of the int.er,,'ellular space a n d a s l i g h t increase in t h e e l e c t r o n d e n s i t y of t h e tissue in t h e region were seen on s e v e r a l occasions. T h e s e r e p r e s e n t s o m e f o r m of ad]icsion device or t i g h t juuctio_u. C y t o p l a s m i c projeceions e x t e n d i n g i n t o t h e l u m e n of t h e l y m p h a t i c vessel in t h e region of the cell j u n c t i o n s are s o m e t i m c s p r e s e n t . The), c o n s i s t of t h i n " f m g e r l i k e " exgclJsions usually a b o u t 600 ~_ in w i d t h a n d v a r y i n l e n g t h up to 0-5/~ or more. T h e c y t o p l a s m is d e v o i d of organelles. A b l u m i n a l endotheli~d p r o j e c t i o n s are present. T h e s e are larger b r o a d - b a s e d e x t e n sions of t h e c:.'toplasm wil;h d i m e n s i o n s of 0.4-0-7/x in w i d t h a n d a b o u t 2.5 t x in l e n g t h . T h e s e e x t e n s i o n s c o n t a i n q u i t e n u m e r o u s r i b o s o m e s a n d m i t o c h o n d r i a are s o m e t i m e s p r e s e n t ( P l a t e 8). V e r y n u m e r o u s l y m p h a t i c a n c h o r i n g filaments, a p p r o x i m a t e l y 60 ~ hi d i a m e t e r are present~ on t h e a d v e n t i t i a l side of t h e e n d 0 t h e l i a l cells. T h e y e x t e n d f r o m t h e cell m e m b r a n e to t h e n e a r b y collagen fibres. T h e y a p p e a r to be c o n t i n u o u s w i t h b o t h of these s t r u c t u r e s b u t a c t u a l a t t a c h m e n t s to t h e cells or t h e c o l l a g e n c o u l d n o t be i d e n t i fied ( P l a t e s 9 a n d 10.) Similar fibrils are also seen w i t h i n t h e c y t o p l a s m of t h e e n d o t h e l i a l cells. T h e s e are fh~er a n d are s o m e t i m e s o r i e n t a t e d into b u n d l e s . W i t h i n t h e c y t o p l a s m t h e m i t o c h o n d r i a are m a i n l y c o n f i n e d to tile areas n e a r t h e nucleus. T h e s h a p e of t h e mitoch.ondria v a r i e s f r o m rom~d or o v a l w i t h a d i a m e t e r of a b o u t 0.25/x to q u i t e e l o n g a t e d s h a p e s m e a s u r i n g u p to 1-1 ~L in l e n g t h . S o m e m i t o chondria are v e r y e l e c t r o n d e n s e w i t h w e l l - d e v e l o p e d cristae. O t h e r s h a v e c e n t r a l u n s t a i n e d or p a l e areas a n d less p r o m i n e n t cristae. S o m e of t h e s e give t h e i m p r e s s i o n t h a t t h e y are h o l l o w or d i s r u p t e d . E l e m e n t s of e n d o p l a s m i c r e t i c u l u m are o n l y o c c a s i o n a l l y o b s e r v e d a n d c o n s i s t o f a few s h o r t strips or t u b u l a r e l e m e n t s w h i c h are p a r t l y r o u g h a n d p a r t l y s m o o t h , arid m o s t l y s i t u a t e d n e a r t h e nuclmts. S a m e seganents of r o u g h - s u r f a c e d e n d o p l a s m i e D
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retic~dum h a v e e n l a r g e d c i s t e r n a e a n d n m n e r o u s a t t a c h e d ribosomes. T h e n u m b e r of ribosomes varies g r e a t l y . I n some areas t h e y are quite sparse, while in o t h e r s t h e y are v e r y n u m e r o u s . T h e y m a y occur in s m a l l clusters or e v e n l y d i s t r i b u t e d over an area of cell cytopla.sm. Various t y p e s of vesicles are f o u n d w i t h i n t h e cell c y t o p l a s m . (1) Pinocy-totie vesicles are p r e s e n t in quite large n u m b e r s a n d are s o m e t i m e s very n u m e r o u s ( P l a t e s S, 9 a n d 10). T h e y are l~ypically r o u n d or oval and a b o u t ,100 :~. in d i a m e t e r w i t h occasional la.rger vesicles of a b o u t I000 .~. Of these vesicle.~ (ealle,.t " p l a s m a l e m m a l vesicles" b y B r a n s a n d P a l a d e , 19138) most. lie free wii-|,in the cytoplasm, while some open on t.o either t h e l u m i n ' d or the mlve,,titial surface of the en,.tot h e l i m n (called caveolae intra¢:ellutares b y various author.% e.g.t."l¢~r,.'y, 1966). (2) A few " c o a t e d " vesicles are present. :lJhese are more t)r~m~inent in t.hat th,,v a p p e a r i~ h a v e a dense lining a n d a c o a t of fine radiMly placed bri,,t.les. T h e y arc, ,,i s i m i l a r d i m e n s i o n s t.o the p i n o c y t 0 t i c vesicle.~ (Plate. l [1). (3) Occasionally, a mult.ivesic~dar b(xty is .-.'cc.~ and l n a v lm apl)roximat~';iy 0--t. }~ in d i a m e t e r . 4. Discussion
I d e n t i f i c a t i o n of the terminal ves~cl.~ de~,,(:ril)ed in t.hi-~ l)ap..-r .~ true 15",nl;h~tic. vessels is dependent, u p o n t h e uhr~t.ructur:~] characic.,i.~tAc.~ of th," ~.',:~-~,.}s e.~;,i th,~ir d i f f e r e n t i a t i o n from the new grown blcK~d vcs~-x.~Isof the coru,:.a. TI~e ii,,~ ~tructure ,..,f blood vessels has been reviewed on n u m e r o u s ¢.x'..c~L~io~s {l.'~w~'t:':.t.," 1959. 1')'-:;3; F t o r e y , i961, 1966, 1968; P a l a d e , 196I; It[o~m a.t~d .i,'eetz~;r, 19~11: I.-~hika;va. 1~33; P_,ru.us a n d I)alade, 1968) but. !he lit.<-n~ture o:) lyml)h-'-)ti,.: ~ t r u c t u r c i~. far Jlmr~-~limit(~t and will l)e s u m m a r i z e d here. T h e l y m p h a t i c c a p i l l a r y has a wide Imn,m (Yama.~..,.i.-:.|li, 196!; t;'ra., alld Wei:~.~.: 1 9 6 l ; L e a k a n d Burke, 1966, t968) which is t-.v:~ t.<~ Iive t.im~-:s ~.l,at: of bt(~:,,t capillaries (Casley-Smit}.b 196-t) a l t h o u g h the vc~s~:ls l e a d to exi.~t in a s~.,.mi-e,:dlal,:,~,,i s t a t e ( F r a l e y a n d Vi'eiss, J961.) Tb.e endol]ael.ial cells are t h i n and attenuate,:t (Suzuki, 1956; 'faniag{zhi. 1961; I w a m o t o a n d Smelser, 1965) and flat.t.ened (lx;ak and Burke, l~,'ff;(;) thimt~r ttmn m u s c l e blood capillaries (C,~.sley-Smit:h a n d .Florey, 1961) ......all,hough (.h~l,::y-S~nit.t.~ (1964) claims t h a t l y m p h a t i c e n d o t h e l i u m is ummlly rath,:.r ~hick~.'r---:md h a v e ~ m i n i m u m t h i c k n e s s of 0.1 tam ( L e a k a n d Burke., 1966, i96S) or ~)'2 t.tm (Kamagi.-,hi, 1961; F r a l e y a n d Wei~s, 1961) a n d me,'~sure several microns (Ix~.ak ~md ft~trke, 196.~-:) or u p to 6/xm a t t h e nucleus ( Y a m a g a s h i , 1961 ; Leak m~d Burke. 1966). In .~pitc.. ,.-.,f t h e a t t e n u a t e d n a t u r e of tb.e l y m p h a t i c endot.he!iun~: " p o r e s " or fe~,(:.~tn,.ti~.,~,s whi~d~ f r e q u e n t l y occur in blood vessels (Collin, 1969a) have. n~t. been o b s e r v e d in i y , n p h a t i e s ( Y a m a ~ s h i 1961 ; C a s l e y - S m i t h a n d F l o r e y , 1961 ; Casley-g~.,ith, 19,.3.t; l w a m o t o a n d Smelser, 1965). T h e nuclei m a y be f l a t t e n e d (Casley-Smith a n d ].,'lore)', 1.961) or large (Fraley a n d Weiss, 1961) a n d oval ( L e a k a n d :Burke, 1966) a n d f r e q u e n t l y p r o t r u d e into the l u m e n of t h e l y m p h a t i c vessel (Yamagishi, 196I ; F r a l e y a n d Weiss, 1.96 l ; C a s l e y - S m i t h a n d F l o r e y , 1961; Leak. and Bin:ks, 1966). T h e e l e c t r o n d e n s i t y of t h e l y m p h a t i c e n d o t h e l i u m is less titan t h a t of blood vascular e n d o t h e l i u m (Yamagishi, 1961; C a s l e y - S m i t h , 196,t). T h e lur~dnal stkrface is g e n e r a l l y smoot, h b u t c o n t a i n s some u n e v e n areas (Yamagishi, 1961) a n d l u m i n a l e n d o t h e l i a l p r o j e c t i o n s s i m i l a r to those seen in blood vessels ( F r a l e y
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a n d ~,Vek,'s, 1961.). The a l b m n i n a l e n d o t h e l i a l sm'face is also s m o o t h (Yamagishi, 1961) an~[ c o n t a i n s a l b u m i n a ! e n d o t h e l i a l p r o j e c t i o n s ( F r a l e y a n d Weiss, 1961; L e a k a n d Burke, 1963) which are n o t usu'tlly present in blood vessels. I n close associ,o)Aon w i t h the o u t e r e n d o t h e l i a l sur£ace h a v e been descril)ed collagen fibres (Yarna~stti, 1961; Casley-Smith am] FJorey, 19(;1) ibm b r a n c h i n g irregmlar fibres sin~ilar in a p p e a r a n c e to those seo.n insMe the cells (Cn.sley-Smith a n d Florey, 1.961) a n d " l y m p h a t i c a n c h o r ing tilamel~ts w|,ich c o n n e c t the small lymph.at.los to th.e s u r r o u n d i n g tissues a n d represen t- the bindil~g m e c h a n i s m t.h,~t is responsiMe for m a i n t a i n i n g t h e f i n n a t t a c h m e n t of I he i)',,qflt:~t.ic eal.,illary t.o the' a d j o i n i n g collagen fibres a n d cells of the connectiv,, lissu, .:,-,..,~ (l,~:ak and lh~rke, 1966 an~t I~(~8; Collin, 1969b). T.hese f i l a m e n t s arc nb,.m! 1(~!)a{ in wi,lth, .}rove :, holR)w profile, irregular beaded a p p e a r a n c e a n d insert on the out ~.r l,?aflct, of the t.rilaminar u n i t membra.ne (Leak a n d Burke, 1968). E a r l i e r l'~ralev a~=,! Wt-iss'(].q{;l ) were ltnable to show fibres of this t y p e a n d s t a t e d thai) there w a s " m ~ ex'i4I<,ncc o f d ire(:.b a r t a c h m e n t o f collagen to the lyn~.phatic wall". A I:,~sm:~,,nt. m,.~nbr:tn,:, surroun,lin~ tim l y m p h a t i c endoth.elium ha.s been r e p o r t e d to t~,a -,d~:ae:~t. (Yama~is}Ji..1961; Frale. 5, and "~Veiss, 1961) d i s c o n t i n u o u s (Leak a n d Burke, .19#;>1 m)t. d:.fini~iv(. (l,eak an(l |{m'k~', 1966) v e r y irregular or a b s e n t (CaMeySmith. 1'.*(;:1)or ~,ften vi~it,l,.~ b u t in m a n y I)laces appears broken up or absenl5 (GaMeySn,.ith ~md t."l,.~r,,)', I t~(;i : W a r , ko, ],loyd and M.a*Mmws, 196,t • I w a m o t o a n d Smelse:..-, I>::;.5). Wh,.n lJr~,:~:l~ the. m,.,r,~t,rane (:onsi:~ts of fine. tilarnents ( I w a m o t o a n d Smelsur, t {mS). I I P,'lix,-~.~ u,,,-r m',.,,s or ],~.,ric:vt~rs ,ire al.,scni. (Yamagishi, 1961: ]?raley a n d Weiss, i :}*;I • \Va~:ko, I.b~.',-,| and M~tt t i.:,:ws, 1}~6-t; I wamota) ,~nd Smelser, 1965) or r a r e l y seen ~,r.¢,t::~,.|. :-re:d} lvl*ipt~.'ttl,:.~. . ((.~>lev-Smith. . .. and Florc-v,. 1961). 'I'.im i:~t,-r<<::llu.tar j u n c t i o n s m a y hay,.' vau'ions a p p e a r a n c e s , viz. simple edge-to-edge {Frat,:'.y rim! W,:eiss. 19(;1) ov,:-rhtl)ping (Yamagishi, 1961.; F r a i e y a n d Weiss, 1961; l.-a.~: ~.,d ~.a:~k,,. !958) ma:: have i n d e n t a t i o n s (Yamagishi, 1961) or m a y be compl,.xl.v pli,.':~:,,d i;.~,.rdigiI..,:tin/ struct.ures ( F r a l e y a n d "Weiss% 1961. ; L e a k a n d 13urke, t °:~ .,t,,.}. ~ T h,- u:u,d (iista,tve }.,~:tx~e.en the I)l:)sn~a m e m b r a n e s oF'adjacent ceils is 100 to 2~."~ : k . . . . . ' . . ((..a:~:cy-N.u~hh and .Here.v, 19(}1). ,.%')metimes . the j m m t i o n s are partia.lly open (I,~-d< and llu:."k,'.. 19(;.S) amt h a v e localized s e p a r a t i o n s of 1500 to 2000 ]k (CasleyS m i i h aml I:lore)', t9~:;11. ()n o::e4,sions t h e lyn-:pha~io i n t e r c e l l u l a r j m m t i o n s m a y be compl,-.tel:," ,.q,.m (Yamagishi, 196t ; Casley,-Smith a n d Florey, 1961 ; L e a k a n d ~,"rke, 1966, 19~;s). I,y.-~,~pbati,: ju,~etions close ,o active muscles h a v e m a n y " o p e n " j u n c t i o n s , up to one in t.~vo to fi~'e j u n c t i o n s a n d th.e cells are often s e p a r a t e d b y considerable distance.s (0.5 to !0t~ ). In qtties~cent regions " o p e n " j u n c t i o n s o n l y occur once in 50 or 10t) jmmt ions (Caslcy-Sn,ith, 1964). A.dlmsion plates ,fr desmosomes at, the i n t e r c e l l u l a r j u n e t i m ~ of l y m p h a t i c endothelia,! cells are less conspict~olk~, t h a n in blood vessels (Casley-Smith a n d I~'lorey, ~. ~ ,9t5t) in w h i c h t.hcy are never a b s e n t (Fawcete,, 1963) a n d o f t e n are n o t visible a t all (Frah:.y and. ~V,:.iss, 1961 ; C a s l e y - S m i t h a n d glorey," 1961 ; Casley-Smith, 1964). :Leak a n d ]3u:rke (196.6) while obser~:ing t h a t a tight~ j u n c t i o n a l c o m p l e x was f r e q u e n t l y a b s e n t describes the " z o n u l a occludens", which occurs n e a r t h e l u m e n , t h e " m a c u l a a d h a e r e n s or desmo,.ome a n d " z o n u l a a(Lhaerens" all of w h i c h do occur in l y m p h a t i c e n d o t h c l i v m a n d are s i m i l a r tm those o b s e r v e d in blood v a s c u l a r e n d o t h e l i u r n in t h a t t h e y represen(~ n a n ' o w i n g of t;he i n t e r c e l l u l a r gap b e t w e e n cell surface m e m b r a n e s a n d .an increase in e l e c t r o n densil~y. AdJacsion devices h a v e also been o b s e r v e d b y I w a m o t o a n d Smelser (1965) in t h e eonju_nctiva.
2!2
If. B. COLLIN
L y m p h a t i c endothelial cells contain all of the usual organelles (C~sley-Smith, 1964 ; I w a m o t o and Smelser, 1965). Mitochondria q re usually elliptie~d in sect.ion with o mean d i a m e t e r of aboi~t 0-5/~m and usually contain from one to five cristae mit.ochondriales (Ca~]c~5~-Smith a n d Florey, 196l). The mitochondria arc n m s t l y situated near the nucleus (Yamagishi, 1961) as also is the Golgi a p p a r a t u s ((2asley-Smith and ]~'!orey, 1961; Fraley and Weiss 1961; Leak and Burke, 1966). Cytoplasmic vesicles have been reported by several" authors: pinocytotic vesicles 75-100 mt~ (Leak and Burke, 1966) vesicles and l u m i n a l and albmuinal caveolae intracellularcs 8 to ,10 mix (Fratey and Weiss, 1961) or 20 to 50 m/x (Ca'.sley-Smith and ]florcy, 1961) and coatc(| vesicles (Leak and Burke, 1966). :Few elements of smooth or rough tubnlar endophLsmic reticulum arc visiMe (Cash,'yS m i t h and. Florey, 1961; F r a l e y "rod Weiss, 1961; LeM¢ ~md Burke, 1966) while few (Casley-Smit.h and Florey, 1961) or numerous (Fraley and Weiss, 1961) free R N A granules are distributed t h r o u g h o u t the cytoplasm. A n o t h e r feature is the presence of fine branching irregular cytoplasmic fibre,,t or fil.'mmnts (Caslcy-Smith and Florey, 1961) which are 60 to 80-~, (Leak and Burke, 1968) a n d mostly run parMlel to the long axis of the cell (Leak and ].lurkc, 1965). These fibrillar structures were n o t observed by I~'r-~ley ~md Weiss ( l (.)61). The c o n t e n t s of the l y m p h a t i c vessel is of Some importance when dist:inction of blood a n d l y m p h a t i c vessels is considered, lgrytl~rocytes are not, usually present in l y m p h a t i c s (Iwamoto and Smeiser, 1965). In certa.in deep lylnphatics, e.g. of the colon, l y m p h o c y t e s are often present, b u t they have not been observed in Iymphatic.~ which did n o t drain lymphoid tissue (Casley--Smith and i?iorey, 1961). The m a t u r e corneal 15nnphatic vessels described show all of the i m p o r t a n t ch-m~cteristics wtfich have been a t t r i b u t e d to lymph vessels, including well-devetol)cd lwnp h a t i c anchoring filaments. The presence of these filament.s appears to be t.he only positive exiterion for ident.ifieation ill t h a t the other criteria are q u a n t i t a t i v c in nature or depend upon the absence of a feature such as a basement membrane, pericytes or e r y t h r o c y t e s and hence m u s t be consid.ered as negative criteria. Thus it is reasonable to conclude t h a t the vessels ddscribed arc true l y m p h a t i c vessels, lined b y a typical l y m p h a t i c endothelium. The claims of Aoki ( 1951 ), K a t a y a m a (1957) a n d Collin (1966a, b a n d c) t h a t a l y m p h vascularization of the cornea can occur in association with a blood vaseularization of the c o r n e a and t h a t the resulting 15nnphatics possess an endothelial lining (Collin, 1966c) are therefore confirmed. The presence of l y m p h a t i c vessels in the cornea m a y have very i m p o r t a n t effects on t h e m a i n t e n a n c e of the t r a n s p a r e n c y of the d a m a g e d corneL, the removal of foreign m a t e r i a l and fltdd from the corneal stroma, the spread of and defence against, infection a n d the initiation of the immunological reaction which results in the failure of some keratoplastie operations. These have been discussed elsewhere (Collin, I966a, b, 1970). ACKNOWLEDGMENTS As a R o y a l Society and Nuffield Commoawealth Foundation Bursar and holder of a British Council Travel Grant, I carried out the majority of this research at the University Laboratory of Physiology, Oxford and the Sir William D u n n School of l~athology, University of Oxford while on leave from m y present address. This research was also assisted in part by grant number 67/3467 from the National H e a l t h and Medical I~esearch Council of Australia. I ack-nowledge the assistance and encouragement given by the late Lord Florey, Provost
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IN :IJtlE VASCULAII, I Z E D B, A B B I T C O I I N E A
213
of Queen's College, Oxlbrd, by Professor R. D. Wright, Professor of Physiology, Universit,y of Melbourne, at whose suggestion this work was c o m m e n c e d and also the technical advice of Mr. 13. L. Shcppard. I wouhl also like to t h a n k i'rofessor D. Whitteridge, University L~tboratory of Physiology, Oxford, h~r the use of his d e p a r t m e n t ' s facilities including the A E I electron microscope; Professor I[. 1 [arris, The Sir William Dulm School of Pathology, Oxford, for use of facilities and Dr. G. Weddell, Dcpartmenb of l i u m a n Anatomy, University of Oxford for permis,~ion to use his ~l micas l',l,mskop 101 electroa microscope. REI"EI~ E N C E S Aoki, K. (1951). K~dbo-(/aku-Z~r.~st,i. 24, 142. BrmLs, P,. lt.. and I'aladc, (;. F,. (19{18). J. Cell Biol. 37, 244. (:a:~ley-Smi| h, ,J, 1',. ( ! !),14). A ~Lu. N. Y. ,|cad. ~ci. 116, 803. Cash.y.Smith, J. 1~.. and l,'lorcy, 1[. (19{;1). Quart. J. Exptl Physiol. 46, 101. Collin I1. IL (1,9(16a). l~re.~t. Optdhalmol. 5, 1. Coil in 11. ]~,. (196(;b). :lm. J. Optom. and Arch. Am. A oad. Optom. 43, 96. {.k)lJin I[. ].',. (196(;c). Inr,rsl. Opldhalrnol. S, 337. ())llin It. B. (tgt;!h}). Exptl Eye lte.~. 8, 16. Collin It. B. (19{19b). Exptl Eye Re~. 8, 102. CalVin H. B. (1970). hu'cst. Ophthalmol. 9, 1,16. Fawcett, D. W. (1959). In fl'he Microcirculation. (Ed. by Reynohl~, S. g. M. and Zweifach, B. M.) Un.iv(,rsity of I llirmis, llaltimore. Fawcett, D. W. (19f;3). /n The Peripheral Blood Vessels. (Ed. by Orbison, J. L. and Smith, I). E.) Williams and Wilkiruq. Baltimore. Flor~:y. ll. W. (1961). (,~uart..1. Exptl PhyMol. 44, 119. t"lorey, l,ord. (19~i6). Bril. ;lied. J. iJ, 487. Florcy, I,ord (196S). Quart. J. Exptl Physiol. 53, 1. Fralcy. E. E. and Wei.q.~. L. (1961). Am. J. Aura'. 109, 85. Itogan. M. J. and Fccn,;y, I.. (1961). :Ira. J. Ophthalmol. 5;1, 1084. lshikawa, T. (19~i:'). I~,,est. Ophthabuol. 2, 1. lwamoto, T. and Smel~r, G. K. (1965). Iu~:est. Ophlhalmol. 4, 815. Katayama. 11. (1957). Lymph
V e s s e l s in t h e V a s c u l a r i z e d Cornea
Rabbit
H . I~A]~P.Y COLLZN
The Victorian College of Optometry, University of Alelbourne, Melbourne, Victoria, Australia (Received 6 .~Iarch 1970, London) A carbon atmIm1~,3ion was injected into rabbit cornem~ vascularized as a result of silver rdtnzto application. Dot h light and electron microscopic techniques were used to study the c.,rrbon-filled corneal lylnph vessels. The corneal lymphatics have a structure similar to that of lymphatic vessels ira other sites. The endobhclium is thin, there are no pericytes and no basement membrane and anchoring filamenk~ are present. Hence, they am true corneal lymphatic ve.~uels.
1. Introduction I n p r e v i o u s p u b l i c a t i o n s (Collin, 1966a, b, c) it h a s b e e n show~l t h a t in a s s o c i a t i o n w i t h t h e g r o w t h of b l o o d vessels i n t o t h e m a m m a l i a n c o r n e a , t h e r e is f o r m e d i n t h e c o r n e a a s e t of c a n a l s w h i c h is q u i t e d i s t i n c t f r o m t h e n e w l y - g r o w n b l o o d vessels. T h e s e c a n a l s c a r r y p a r t i c u l a t e m a t e r i a l s u c h as c a r b o n i n t o t h e c o n j m m t i v a l l y m p h a t i c s y s t e m (Aoki, 1951; K a t a y a m a , 1957; Collin, 1 9 6 6 a ) . , n d p r o v i d e a n a v a i l a b l e r o u t e for i m m c d i a t e r e m o v a l of l a r g e m olectfles s u c h as J:~i-lab elled a l t m m i n f r o m a v a s c u l a r i z e d c o r n e a to t h e l y m p h n o d e s of t h e n e c k (Collin, 1970). As a r e s u l t of s i l v e r n i t r a t e s t a i n i n g of t-hese c a n a l s in w h o l e m o m l t p r e p a r a t i o n s , t h e y a p p e a r t o be l i n e d b y e n d o t h e l / u m in w h i c h t h e cell o u t l i n e s are s i n u o u s a n d s h o w a n i r r e g u l a r l y d e n t a t e p a t t e r n (Collin, 19'36c). A t . t e m p t s to d e m o n s t r a t e an e n d o t h e l i a l l i n i n g to t h e c o r n e a l l y m p h a t i c vessels u s i n g l i g h t m i c r o s c o p y a n d paraffin sections, h a v e b e e n unsuccesst'u] (Aoki, 1951; K a t a y a m a , 1957; Collin, 1966c). :It w a s t h e p u r p o s e of t h i s s t u d y to d e m o i m t r a t e , u s i n g b o t h l i g h t a n d e l e c t r o n m i c r o s c o p y , t h a t t h e c o r n e a l l y m p h vessels possess a c e l l u l a r e n d o t h e l i a l I i n i n g a n d to d e s c r i b e t h e ultr:,,stoact.ural c h a r a c t e r i s t i c s of t h e s e c o r n e a l l y m p h a t i c vessels. I n o r d e r to i d e n t i f y t h e c o r n e a l l y m p h a t i c vessels t h e y w e r e l a b e l l e d w i t h a n e l e c t r o n o p a q u e rr, a r k e r , viz. c a r b o n . O n l y vessels c o n t a i n i n g c a r b o n w e r e c o n s i d e r e d i u t h i s i m : e s t i g a t i o n a n d no vessels c o n t a i n i n g c a r b o n b u t r e c o g n i z a b l e as b l o o d vessels w e r e c o n s i d e r e d . I n fact, c a r b o n w a s n e v e r f o u n d w i t h i n t h e c o r n e a l b l o o d vessels.
2. Method The left ~ye of each of four a d u l t albino rab b its was a n a e s t h e t i z e d w i t h 0.4~/o Novesine (Wander) aLd silver n i ~ a t e stick was applied to t h e c e n t r e of t h e cornea for approxim a t e l y 2 see. Am area 4 to 5 m m in d i a m e t e r was d a m a g e d . I n t h e following d a y s ~he corneas b e c a m e h a z y a n d o e d e m a t o u s a n d t he r e was a p m ' u l e n t conjunctivitis. Blood vessels grew i n t o t h e corneas. After periods of 7 weeks (two animals) a n d 12 weeks (two animals) t h e r a b b i t s were a n a e s t h e t i z e d ~with i n t r a v e n o u s N e m b u t a l (Abbott) a n d a n in~ection of a c a r b o n suspension (1% in sterile isotonic saline of 100 rag/eros--from G u e n t h e r Wag n er, l-Iannover,
207
208
tt[. B. COLLIN
G e r m a n y . N u m b e r CI1/1431a) was m a d e into t.he c e n t r e of e a c h c o r n e a using a 30 g u a g e needle. I n one a n i m a l of each p a i r t,he c a r b o n was s.een to e n t e r ~t n e t w o r k of corneal c a m d s an,! t h e n c e flow i n t o the c o n j u n c t i w d l y m p h . l t i e vessels. T h e con~ea was flooded w i t h ice-cold buffered g l u t a r a l d e h y d e fixative consisting of 2 . 5 ~ g l u t a r a l d e h y d e in 0.067 ~t s o d i u m e a e o d y l a t e buffer at p H 7-4. Tim a n i m a l was t h e n killed with a n overdose of N e m b u t a l a n d t h e corneal tissue w.ls r e m o v e d a n d pla(:e:l in t h e fixative for 1 hr. The tissues were w a s h e d and f u r t h e r disscctc,1 i n t o om'~ll piee,:s in buffer of the s a m e m o l a r i t y and t h e n post-fixed in o s m i u m tet.roxide in 0.067 .~l s o d i m n eaeodylal.e ata p H 7-4. ( S h e p p a r d , p e r s o n a l communic.,~iion.) T h e tissues were d e h y d r a t e d in a c e t o n e and embe,.hled in Aral,lite. Urar~)'i steel:de (0"50/0 in 750'0 a c e t o n e ) bli,(rk s t a i n was followed 153, lead c i t r a t e (l~ev:mMs. 19~;3) atilt u r a n y l a c e t a t e ( 1°'/o aqueous). T h i n sections were cut (m ~tn l', l( 13 l.:ltr()t~:me [ using, glass. k n i v e s a n d e x a m i n e d on ::n A E I E,M t3B (Figs I nnd 3) or a S i e m e n s E l m i s k o p 101 ole,'tr,,n mieroscol)e. T h i c k sections were s~ained w i t h llichards,.),x's .~,tain (lli,:har~[:.;on..lar,.'£-tt an,I F i n k e , 1960) a m i x t u r e of A z u r e I I ~ 3 I e t h y h . m e blue :l u,l light mi~:ro;gr:lphu were t a k e n w i t h a Leica ,35 m m c a m e r a a t t a c h e d t.o a Zei.ss T r i n o c u l a r mierosvo]:~.
3. R e s u l t s
Seven weeks All o f t h e s e vessels a r e in tile l)eril)heral are.'~ o!:-the c o r n e a in -.he re,,a,ion 0.5 t(: 1"0 m m D o r a t h e l i , n b u s . As a r e s u l t all o b s e r v e d vessels w e r e q u i t e tart,., (Pl,~t,.s t and 2). T h e e n d o t h e l i a l cells are e l o n g a t e d a n d t h i n ( I " [ , t e 3) w ~ r y i n g f r o m a r o u n d 04!71"8/x ~md u p to 4.5 l* a.t tim n u c l e u s w h i c h is m o s t l y ilatt:e.ne,:t. The. (:ytopla.-.r,,~ .'nay b~: le~s d e n s e l y s t a i n i n g that), n e a r b y b l o o d v a s c u l a r e n d a t heiial c)'*<~plasm. "l'h(- ~mcl,,.n.s Ires v e r y e x t e n s i v e l i g h t s t a i n i ~ g n u c l e o p l ' t s m , nulp.t.r(sus nuc!erar l~(sres a m ! fr,:~tu.:mt! / o n e o r t, wo p r o m i n e n t - n u c l e o l i ( P l a t e 1). B o t h lumi,~al a n d a l b m n i n a l e n d o d l e l i a i projec~cions a r e present, b u t n e i t h e r a r e l i u m e r o u s , t:im: t)-m!,hat, ic. all(:ht)ri;2g fi.lalllt:tlt.-~ a r e p r e s e n t in s o m e a r e a s o f t h e a d v e n t i t i . d eel I surfatc(~s a n d are. quit,.., well ,:i,~v,dopt.d. T h e l m n e n o f t h e vessel m a y c o n t a i n c a r b o n am{ l i t t l e (..I~: ( P l a t e 1) ,)r oc,:~,.~ionMiy it m a y be l o o s e l y p a c k e d w i t h cells t h e m a j o r i t . y o f w h i c h r e s e m b l ~ lynq)l:ov3"tr.'; (Plate 2). T h e c y t o p l a s m o f t h e l y m p h a t i c e n d o t h e l i a l celbs cont~,ins b o t h r o u n d (0.2 1'-) a~ut e l o n g a t e d (0~8/a,) m i t o c h o n ( l r i a w h i c h a r e d a r k l y s t . a i n i n g w i t h w e ! l - d e f i a e d c r i s t a e and t h e y d o n o t a p p e a r to be r e s t r i c t e d ix) t h e r e g i o n n e a r t h e n u c l e u s . T t m r e is s o m e l o n g t u b u l a r r o u g h s u r f a c e d e n d o l ) l a s m i c r e t . i c u l m n a n d s o m e r i b o s o m e s o c c t t r r i n g s i n g l y or i n c l u s t e r s , b u t n e i t l l e r o f t h e s e is v e r y plcnt.ifu!. C)'totsla.smic fibrils a r e p r e s e n t a n d in s o m e a r e a s pinocygot-ic vesicles, a p p r o x i m a t e l y 67 n m in d i a m e t e r , a r e e x t r e m e l y n u m e r o u s ( P l a t e 3). C o a t e d v e s i c l e s a r e a l s o f:',ir]y e o m n t o n . The i n t e r c e l l u l a r j u n c t i o n s a r e m o s t l y c l o s e d a l t h o u g I t a f e w o p e n j u n c t i o n s a r e presenl;. I ) e s m o s o m e s a r e r a r e l y found and w h e n p r e s e n t a r e not, w e l l - d e v e l o p e d . A b a s e m e n t m e m b r a n e and pericytes are not present.
Twelve wee]as F o l l o w i n g carbon injection l y m p h a t i c vessels were f o u n d to be present in the cornea to a distance of 6 m m from the limbus. The ly~nphatic v e s s e l s are slightly larger than ~he blood vessels of the same section, the l y m p h vessels often measuring as m u c h as 55/~ b y 10 ix. T h e wall is c o m p o s e d solely of a single layer of endothelial cells w h i c h , n a y be e x t r e m e l y a t t e n u a t e d and
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10 l'L.~'J'r: (.). E n d o t h e l i a l w a l l of a c o r n e a l 13"J:lpnatie. N ' u m e r o u s ,'esicles tLIlcl c y t o t ~ l a s m i c .fit~ril.-." at,:: l)t.csent, l , y t ~ q ) h a t i e a n c h o r i n z /ilazt:cnts (2%.F) e x t e n d f r o n l t h e albttn~i~lal endotAlelial sux'/'ace to ~:he e 0 1 1 a g ~ t i b r e s (Co). 1,, _[~ttm<:t~. (x.10,tXJ0) ]?r,.,,Tr: I0. l : ] n d o t h c l i a | ~vall o f corJ~eal l y m p h ; x t i c , b e t w e e n 2 a n d 3 m m fvon~ t h e l i m b u s a h o w i n g a n c h o / ' i n g f i l a m e n t s (.-kJF) a~¢l t h r e e eot~+ed v e s i c l e s (G'~,'). Co, C o l l a g e n ;I5, l u m e n . ( x 40,000)
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209
regions as thin as d00 to 500/~ arc f r e q u e n t l y seen (:Plates 5 a n d 6). T h e cell tlfickness is u s u a l l y :rather c o n s t m l t e x c e p t a t t h e area of t h e n u c l e u s w h e n t h e thic.kness is m o s t l y in t h e r a n g e 1.5-2-3 tL w i t h a s i m i l a r t h i c k n e s s tbr t h e c y t o p l a s m n e a r t h e n u c l e u s or in regions w h i c h a p p e a r to be c u t m o r e obliqucly. T h e o t h e r e l e m e n t s w h i c h n o r m a l l y c o m p r i s e a b[ood vessel wall are n o t p r e s e n t ; advcnti~ial ceils w h e t h e r of a n m s c l e cell type, or p e r i c y t e s are n e v e r observed. On occasions w h e n c y t o p l a s m i c p o r t i o n s of cells were i b u n d o u t s i d e a n d a d j a c e n t to t h e e n d o t h e l i a l ('.ells, careful e x a m i n a t i o n r e v e a l e d t h a t t h e s e were p a r t s of fibroblasts, k e r a t o c y t c s or k e r a t o b l a s t s . A c o n t i n u o t m b a s e m e n t m e m b r a n e is n e v e r seen a r o m l d the Iyml)lJatic e n d o t h e l i u m . H o w e v e r , on rare occasions a s m a l l q u a n t i t y of e l e c t r o n (lense m a t e r i a l r e s e m b l i n g a s e g m e n t of b a s e m e n t m e m b r a n e ~.nd m e a s u r i n g a b o u t 2 i~ in l e n g t h was observed. T h e nuclei are. m o s t l y oval in sl,al)e a n d in g e n e r a l are flatter t h a n in blood v a s c u l a r endot.heli~un. T h e r e is e x t e n s i v e l i g h t staining, c e n t r a l m m l e o p l a s m a n d d e n s e peripheral c h r o m a t i n w h i c h is s o m e t i m e s m o r e d a r l d y s t a i n i n g t h a n in b l o o d v a s c u l a r en(lobhelial nuclei. A s l i g h t space is s o m e t i m e s p r e s e n t a r o u n d t h e n u c l e u s b e t w e e n t h e miclcar nmmlJra, nc an(t t h e c y t o p l a s m a n d this m a y be d u e to s h r i n k a g e or artifact. N u m e r o u s / m c l e a r pores are p r e s e n t a n d in these regions close c o n t a c t is always m a i n t a i n e d b e t w e e n t.he n u c l e u s a n d t h e c y t o p l a s m . O c c a s i o n a l nucleoli are seen, C'e[1 jul~(:tions v a r y front close a p p o s i t i o n of cells witl La set)aration of a r o u n d 100 ]k ( P l a t e 7) t h r o u g h t h e s l i g h t l y open jmmbions to w i d e l y s e p a r a t e d cells or gaps of up to 2.5/* b e t w e e n endotllelial cells. W e l l - d e v e l o p e d d e s m o s o m e s are n o t o b s e r v e d , b u t a n a r r o w i n g of the int.er,,'ellular space a n d a s l i g h t increase in t h e e l e c t r o n d e n s i t y of t h e tissue in t h e region were seen on s e v e r a l occasions. T h e s e r e p r e s e n t s o m e f o r m of ad]icsion device or t i g h t juuctio_u. C y t o p l a s m i c projeceions e x t e n d i n g i n t o t h e l u m e n of t h e l y m p h a t i c vessel in t h e region of the cell j u n c t i o n s are s o m e t i m c s p r e s e n t . The), c o n s i s t of t h i n " f m g e r l i k e " exgclJsions usually a b o u t 600 ~_ in w i d t h a n d v a r y i n l e n g t h up to 0-5/~ or more. T h e c y t o p l a s m is d e v o i d of organelles. A b l u m i n a l endotheli~d p r o j e c t i o n s are present. T h e s e are larger b r o a d - b a s e d e x t e n sions of t h e c:.'toplasm wil;h d i m e n s i o n s of 0.4-0-7/x in w i d t h a n d a b o u t 2.5 t x in l e n g t h . T h e s e e x t e n s i o n s c o n t a i n q u i t e n u m e r o u s r i b o s o m e s a n d m i t o c h o n d r i a are s o m e t i m e s p r e s e n t ( P l a t e 8). V e r y n u m e r o u s l y m p h a t i c a n c h o r i n g filaments, a p p r o x i m a t e l y 60 ~ hi d i a m e t e r are present~ on t h e a d v e n t i t i a l side of t h e e n d 0 t h e l i a l cells. T h e y e x t e n d f r o m t h e cell m e m b r a n e to t h e n e a r b y collagen fibres. T h e y a p p e a r to be c o n t i n u o u s w i t h b o t h of these s t r u c t u r e s b u t a c t u a l a t t a c h m e n t s to t h e cells or t h e c o l l a g e n c o u l d n o t be i d e n t i fied ( P l a t e s 9 a n d 10.) Similar fibrils are also seen w i t h i n t h e c y t o p l a s m of t h e e n d o t h e l i a l cells. T h e s e are fh~er a n d are s o m e t i m e s o r i e n t a t e d into b u n d l e s . W i t h i n t h e c y t o p l a s m t h e m i t o c h o n d r i a are m a i n l y c o n f i n e d to tile areas n e a r t h e nucleus. T h e s h a p e of t h e mitoch.ondria v a r i e s f r o m rom~d or o v a l w i t h a d i a m e t e r of a b o u t 0.25/x to q u i t e e l o n g a t e d s h a p e s m e a s u r i n g u p to 1-1 ~L in l e n g t h . S o m e m i t o chondria are v e r y e l e c t r o n d e n s e w i t h w e l l - d e v e l o p e d cristae. O t h e r s h a v e c e n t r a l u n s t a i n e d or p a l e areas a n d less p r o m i n e n t cristae. S o m e of t h e s e give t h e i m p r e s s i o n t h a t t h e y are h o l l o w or d i s r u p t e d . E l e m e n t s of e n d o p l a s m i c r e t i c u l u m are o n l y o c c a s i o n a l l y o b s e r v e d a n d c o n s i s t o f a few s h o r t strips or t u b u l a r e l e m e n t s w h i c h are p a r t l y r o u g h a n d p a r t l y s m o o t h , arid m o s t l y s i t u a t e d n e a r t h e nuclmts. S a m e seganents of r o u g h - s u r f a c e d e n d o p l a s m i e D
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retic~dum h a v e e n l a r g e d c i s t e r n a e a n d n m n e r o u s a t t a c h e d ribosomes. T h e n u m b e r of ribosomes varies g r e a t l y . I n some areas t h e y are quite sparse, while in o t h e r s t h e y are v e r y n u m e r o u s . T h e y m a y occur in s m a l l clusters or e v e n l y d i s t r i b u t e d over an area of cell cytopla.sm. Various t y p e s of vesicles are f o u n d w i t h i n t h e cell c y t o p l a s m . (1) Pinocy-totie vesicles are p r e s e n t in quite large n u m b e r s a n d are s o m e t i m e s very n u m e r o u s ( P l a t e s S, 9 a n d 10). T h e y are l~ypically r o u n d or oval and a b o u t ,100 :~. in d i a m e t e r w i t h occasional la.rger vesicles of a b o u t I000 .~. Of these vesicle.~ (ealle,.t " p l a s m a l e m m a l vesicles" b y B r a n s a n d P a l a d e , 19138) most. lie free wii-|,in the cytoplasm, while some open on t.o either t h e l u m i n ' d or the mlve,,titial surface of the en,.tot h e l i m n (called caveolae intra¢:ellutares b y various author.% e.g.t."l¢~r,.'y, 1966). (2) A few " c o a t e d " vesicles are present. :lJhese are more t)r~m~inent in t.hat th,,v a p p e a r i~ h a v e a dense lining a n d a c o a t of fine radiMly placed bri,,t.les. T h e y arc, ,,i s i m i l a r d i m e n s i o n s t.o the p i n o c y t 0 t i c vesicle.~ (Plate. l [1). (3) Occasionally, a mult.ivesic~dar b(xty is .-.'cc.~ and l n a v lm apl)roximat~';iy 0--t. }~ in d i a m e t e r . 4. Discussion
I d e n t i f i c a t i o n of the terminal ves~cl.~ de~,,(:ril)ed in t.hi-~ l)ap..-r .~ true 15",nl;h~tic. vessels is dependent, u p o n t h e uhr~t.ructur:~] characic.,i.~tAc.~ of th," ~.',:~-~,.}s e.~;,i th,~ir d i f f e r e n t i a t i o n from the new grown blcK~d vcs~-x.~Isof the coru,:.a. TI~e ii,,~ ~tructure ,..,f blood vessels has been reviewed on n u m e r o u s ¢.x'..c~L~io~s {l.'~w~'t:':.t.," 1959. 1')'-:;3; F t o r e y , i961, 1966, 1968; P a l a d e , 196I; It[o~m a.t~d .i,'eetz~;r, 19~11: I.-~hika;va. 1~33; P_,ru.us a n d I)alade, 1968) but. !he lit.<-n~ture o:) lyml)h-'-)ti,.: ~ t r u c t u r c i~. far Jlmr~-~limit(~t and will l)e s u m m a r i z e d here. T h e l y m p h a t i c c a p i l l a r y has a wide Imn,m (Yama.~..,.i.-:.|li, 196!; t;'ra., alld Wei:~.~.: 1 9 6 l ; L e a k a n d Burke, 1966, t968) which is t-.v:~ t.<~ Iive t.im~-:s ~.l,at: of bt(~:,,t capillaries (Casley-Smit}.b 196-t) a l t h o u g h the vc~s~:ls l e a d to exi.~t in a s~.,.mi-e,:dlal,:,~,,i s t a t e ( F r a l e y a n d Vi'eiss, J961.) Tb.e endol]ael.ial cells are t h i n and attenuate,:t (Suzuki, 1956; 'faniag{zhi. 1961; I w a m o t o a n d Smelser, 1965) and flat.t.ened (lx;ak and Burke, l~,'ff;(;) thimt~r ttmn m u s c l e blood capillaries (C,~.sley-Smit:h a n d .Florey, 1961) ......all,hough (.h~l,::y-S~nit.t.~ (1964) claims t h a t l y m p h a t i c e n d o t h e l i u m is ummlly rath,:.r ~hick~.'r---:md h a v e ~ m i n i m u m t h i c k n e s s of 0.1 tam ( L e a k a n d Burke., 1966, i96S) or ~)'2 t.tm (Kamagi.-,hi, 1961; F r a l e y a n d Wei~s, 1961) a n d me,'~sure several microns (Ix~.ak ~md ft~trke, 196.~-:) or u p to 6/xm a t t h e nucleus ( Y a m a g a s h i , 1961 ; Leak m~d Burke. 1966). In .~pitc.. ,.-.,f t h e a t t e n u a t e d n a t u r e of tb.e l y m p h a t i c endot.he!iun~: " p o r e s " or fe~,(:.~tn,.ti~.,~,s whi~d~ f r e q u e n t l y occur in blood vessels (Collin, 1969a) have. n~t. been o b s e r v e d in i y , n p h a t i e s ( Y a m a ~ s h i 1961 ; C a s l e y - S m i t h a n d F l o r e y , 1961 ; Casley-g~.,ith, 19,.3.t; l w a m o t o a n d Smelser, 1965). T h e nuclei m a y be f l a t t e n e d (Casley-Smith a n d ].,'lore)', 1.961) or large (Fraley a n d Weiss, 1961) a n d oval ( L e a k a n d :Burke, 1966) a n d f r e q u e n t l y p r o t r u d e into the l u m e n of t h e l y m p h a t i c vessel (Yamagishi, 196I ; F r a l e y a n d Weiss, 1.96 l ; C a s l e y - S m i t h a n d F l o r e y , 1961; Leak. and Bin:ks, 1966). T h e e l e c t r o n d e n s i t y of t h e l y m p h a t i c e n d o t h e l i u m is less titan t h a t of blood vascular e n d o t h e l i u m (Yamagishi, 1961; C a s l e y - S m i t h , 196,t). T h e lur~dnal stkrface is g e n e r a l l y smoot, h b u t c o n t a i n s some u n e v e n areas (Yamagishi, 1961) a n d l u m i n a l e n d o t h e l i a l p r o j e c t i o n s s i m i l a r to those seen in blood vessels ( F r a l e y
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a n d ~,Vek,'s, 1961.). The a l b m n i n a l e n d o t h e l i a l sm'face is also s m o o t h (Yamagishi, 1961) an~[ c o n t a i n s a l b u m i n a ! e n d o t h e l i a l p r o j e c t i o n s ( F r a l e y a n d Weiss, 1961; L e a k a n d Burke, 1963) which are n o t usu'tlly present in blood vessels. I n close associ,o)Aon w i t h the o u t e r e n d o t h e l i a l sur£ace h a v e been descril)ed collagen fibres (Yarna~stti, 1961; Casley-Smith am] FJorey, 19(;1) ibm b r a n c h i n g irregmlar fibres sin~ilar in a p p e a r a n c e to those seo.n insMe the cells (Cn.sley-Smith a n d Florey, 1.961) a n d " l y m p h a t i c a n c h o r ing tilamel~ts w|,ich c o n n e c t the small lymph.at.los to th.e s u r r o u n d i n g tissues a n d represen t- the bindil~g m e c h a n i s m t.h,~t is responsiMe for m a i n t a i n i n g t h e f i n n a t t a c h m e n t of I he i)',,qflt:~t.ic eal.,illary t.o the' a d j o i n i n g collagen fibres a n d cells of the connectiv,, lissu, .:,-,..,~ (l,~:ak and lh~rke, 1966 an~t I~(~8; Collin, 1969b). T.hese f i l a m e n t s arc nb,.m! 1(~!)a{ in wi,lth, .}rove :, holR)w profile, irregular beaded a p p e a r a n c e a n d insert on the out ~.r l,?aflct, of the t.rilaminar u n i t membra.ne (Leak a n d Burke, 1968). E a r l i e r l'~ralev a~=,! Wt-iss'(].q{;l ) were ltnable to show fibres of this t y p e a n d s t a t e d thai) there w a s " m ~ ex'i4I<,ncc o f d ire(:.b a r t a c h m e n t o f collagen to the lyn~.phatic wall". A I:,~sm:~,,nt. m,.~nbr:tn,:, surroun,lin~ tim l y m p h a t i c endoth.elium ha.s been r e p o r t e d to t~,a -,d~:ae:~t. (Yama~is}Ji..1961; Frale. 5, and "~Veiss, 1961) d i s c o n t i n u o u s (Leak a n d Burke, .19#;>1 m)t. d:.fini~iv(. (l,eak an(l |{m'k~', 1966) v e r y irregular or a b s e n t (CaMeySmith. 1'.*(;:1)or ~,ften vi~it,l,.~ b u t in m a n y I)laces appears broken up or absenl5 (GaMeySn,.ith ~md t."l,.~r,,)', I t~(;i : W a r , ko, ],loyd and M.a*Mmws, 196,t • I w a m o t o a n d Smelse:..-, I>::;.5). Wh,.n lJr~,:~:l~ the. m,.,r,~t,rane (:onsi:~ts of fine. tilarnents ( I w a m o t o a n d Smelsur, t {mS). I I P,'lix,-~.~ u,,,-r m',.,,s or ],~.,ric:vt~rs ,ire al.,scni. (Yamagishi, 1961: ]?raley a n d Weiss, i :}*;I • \Va~:ko, I.b~.',-,| and M~tt t i.:,:ws, 1}~6-t; I wamota) ,~nd Smelser, 1965) or r a r e l y seen ~,r.¢,t::~,.|. :-re:d} lvl*ipt~.'ttl,:.~. . ((.~>lev-Smith. . .. and Florc-v,. 1961). 'I'.im i:~t,-r<<::llu.tar j u n c t i o n s m a y hay,.' vau'ions a p p e a r a n c e s , viz. simple edge-to-edge {Frat,:'.y rim! W,:eiss. 19(;1) ov,:-rhtl)ping (Yamagishi, 1961.; F r a i e y a n d Weiss, 1961; l.-a.~: ~.,d ~.a:~k,,. !958) ma:: have i n d e n t a t i o n s (Yamagishi, 1961) or m a y be compl,.xl.v pli,.':~:,,d i;.~,.rdigiI..,:tin/ struct.ures ( F r a l e y a n d "Weiss% 1961. ; L e a k a n d 13urke, t °:~ .,t,,.}. ~ T h,- u:u,d (iista,tve }.,~:tx~e.en the I)l:)sn~a m e m b r a n e s oF'adjacent ceils is 100 to 2~."~ : k . . . . . ' . . ((..a:~:cy-N.u~hh and .Here.v, 19(}1). ,.%')metimes . the j m m t i o n s are partia.lly open (I,~-d< and llu:."k,'.. 19(;.S) amt h a v e localized s e p a r a t i o n s of 1500 to 2000 ]k (CasleyS m i i h aml I:lore)', t9~:;11. ()n o::e4,sions t h e lyn-:pha~io i n t e r c e l l u l a r j m m t i o n s m a y be compl,-.tel:," ,.q,.m (Yamagishi, 196t ; Casley,-Smith a n d Florey, 1961 ; L e a k a n d ~,"rke, 1966, 19~;s). I,y.-~,~pbati,: ju,~etions close ,o active muscles h a v e m a n y " o p e n " j u n c t i o n s , up to one in t.~vo to fi~'e j u n c t i o n s a n d th.e cells are often s e p a r a t e d b y considerable distance.s (0.5 to !0t~ ). In qtties~cent regions " o p e n " j u n c t i o n s o n l y occur once in 50 or 10t) jmmt ions (Caslcy-Sn,ith, 1964). A.dlmsion plates ,fr desmosomes at, the i n t e r c e l l u l a r j u n e t i m ~ of l y m p h a t i c endothelia,! cells are less conspict~olk~, t h a n in blood vessels (Casley-Smith a n d I~'lorey, ~. ~ ,9t5t) in w h i c h t.hcy are never a b s e n t (Fawcete,, 1963) a n d o f t e n are n o t visible a t all (Frah:.y and. ~V,:.iss, 1961 ; C a s l e y - S m i t h a n d glorey," 1961 ; Casley-Smith, 1964). :Leak a n d ]3u:rke (196.6) while obser~:ing t h a t a tight~ j u n c t i o n a l c o m p l e x was f r e q u e n t l y a b s e n t describes the " z o n u l a occludens", which occurs n e a r t h e l u m e n , t h e " m a c u l a a d h a e r e n s or desmo,.ome a n d " z o n u l a a(Lhaerens" all of w h i c h do occur in l y m p h a t i c e n d o t h c l i v m a n d are s i m i l a r tm those o b s e r v e d in blood v a s c u l a r e n d o t h e l i u r n in t h a t t h e y represen(~ n a n ' o w i n g of t;he i n t e r c e l l u l a r gap b e t w e e n cell surface m e m b r a n e s a n d .an increase in e l e c t r o n densil~y. AdJacsion devices h a v e also been o b s e r v e d b y I w a m o t o a n d Smelser (1965) in t h e eonju_nctiva.
2!2
If. B. COLLIN
L y m p h a t i c endothelial cells contain all of the usual organelles (C~sley-Smith, 1964 ; I w a m o t o and Smelser, 1965). Mitochondria q re usually elliptie~d in sect.ion with o mean d i a m e t e r of aboi~t 0-5/~m and usually contain from one to five cristae mit.ochondriales (Ca~]c~5~-Smith a n d Florey, 196l). The mitochondria arc n m s t l y situated near the nucleus (Yamagishi, 1961) as also is the Golgi a p p a r a t u s ((2asley-Smith and ]~'!orey, 1961; Fraley and Weiss 1961; Leak and Burke, 1966). Cytoplasmic vesicles have been reported by several" authors: pinocytotic vesicles 75-100 mt~ (Leak and Burke, 1966) vesicles and l u m i n a l and albmuinal caveolae intracellularcs 8 to ,10 mix (Fratey and Weiss, 1961) or 20 to 50 m/x (Ca'.sley-Smith and ]florcy, 1961) and coatc(| vesicles (Leak and Burke, 1966). :Few elements of smooth or rough tubnlar endophLsmic reticulum arc visiMe (Cash,'yS m i t h and. Florey, 1961; F r a l e y "rod Weiss, 1961; LeM¢ ~md Burke, 1966) while few (Casley-Smit.h and Florey, 1961) or numerous (Fraley and Weiss, 1961) free R N A granules are distributed t h r o u g h o u t the cytoplasm. A n o t h e r feature is the presence of fine branching irregular cytoplasmic fibre,,t or fil.'mmnts (Caslcy-Smith and Florey, 1961) which are 60 to 80-~, (Leak and Burke, 1968) a n d mostly run parMlel to the long axis of the cell (Leak and ].lurkc, 1965). These fibrillar structures were n o t observed by I~'r-~ley ~md Weiss ( l (.)61). The c o n t e n t s of the l y m p h a t i c vessel is of Some importance when dist:inction of blood a n d l y m p h a t i c vessels is considered, lgrytl~rocytes are not, usually present in l y m p h a t i c s (Iwamoto and Smeiser, 1965). In certa.in deep lylnphatics, e.g. of the colon, l y m p h o c y t e s are often present, b u t they have not been observed in Iymphatic.~ which did n o t drain lymphoid tissue (Casley--Smith and i?iorey, 1961). The m a t u r e corneal 15nnphatic vessels described show all of the i m p o r t a n t ch-m~cteristics wtfich have been a t t r i b u t e d to lymph vessels, including well-devetol)cd lwnp h a t i c anchoring filaments. The presence of these filament.s appears to be t.he only positive exiterion for ident.ifieation ill t h a t the other criteria are q u a n t i t a t i v c in nature or depend upon the absence of a feature such as a basement membrane, pericytes or e r y t h r o c y t e s and hence m u s t be consid.ered as negative criteria. Thus it is reasonable to conclude t h a t the vessels ddscribed arc true l y m p h a t i c vessels, lined b y a typical l y m p h a t i c endothelium. The claims of Aoki ( 1951 ), K a t a y a m a (1957) a n d Collin (1966a, b a n d c) t h a t a l y m p h vascularization of the cornea can occur in association with a blood vaseularization of the c o r n e a and t h a t the resulting 15nnphatics possess an endothelial lining (Collin, 1966c) are therefore confirmed. The presence of l y m p h a t i c vessels in the cornea m a y have very i m p o r t a n t effects on t h e m a i n t e n a n c e of the t r a n s p a r e n c y of the d a m a g e d corneL, the removal of foreign m a t e r i a l and fltdd from the corneal stroma, the spread of and defence against, infection a n d the initiation of the immunological reaction which results in the failure of some keratoplastie operations. These have been discussed elsewhere (Collin, I966a, b, 1970). ACKNOWLEDGMENTS As a R o y a l Society and Nuffield Commoawealth Foundation Bursar and holder of a British Council Travel Grant, I carried out the majority of this research at the University Laboratory of Physiology, Oxford and the Sir William D u n n School of l~athology, University of Oxford while on leave from m y present address. This research was also assisted in part by grant number 67/3467 from the National H e a l t h and Medical I~esearch Council of Australia. I ack-nowledge the assistance and encouragement given by the late Lord Florey, Provost
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of Queen's College, Oxlbrd, by Professor R. D. Wright, Professor of Physiology, Universit,y of Melbourne, at whose suggestion this work was c o m m e n c e d and also the technical advice of Mr. 13. L. Shcppard. I wouhl also like to t h a n k i'rofessor D. Whitteridge, University L~tboratory of Physiology, Oxford, h~r the use of his d e p a r t m e n t ' s facilities including the A E I electron microscope; Professor I[. 1 [arris, The Sir William Dulm School of Pathology, Oxford, for use of facilities and Dr. G. Weddell, Dcpartmenb of l i u m a n Anatomy, University of Oxford for permis,~ion to use his ~l micas l',l,mskop 101 electroa microscope. REI"EI~ E N C E S Aoki, K. (1951). K~dbo-(/aku-Z~r.~st,i. 24, 142. BrmLs, P,. lt.. and I'aladc, (;. F,. (19{18). J. Cell Biol. 37, 244. (:a:~ley-Smi| h, ,J, 1',. ( ! !),14). A ~Lu. N. Y. ,|cad. ~ci. 116, 803. Cash.y.Smith, J. 1~.. and l,'lorcy, 1[. (19{;1). Quart. J. Exptl Physiol. 46, 101. Collin I1. IL (1,9(16a). l~re.~t. Optdhalmol. 5, 1. Coil in 11. ]~,. (196(;b). :lm. J. Optom. and Arch. Am. A oad. Optom. 43, 96. {.k)lJin I[. ].',. (196(;c). Inr,rsl. Opldhalrnol. S, 337. ())llin It. B. (tgt;!h}). Exptl Eye lte.~. 8, 16. Collin It. B. (19{19b). Exptl Eye Re~. 8, 102. CalVin H. B. (1970). hu'cst. Ophthalmol. 9, 1,16. Fawcett, D. W. (1959). In fl'he Microcirculation. (Ed. by Reynohl~, S. g. M. and Zweifach, B. M.) Un.iv(,rsity of I llirmis, llaltimore. Fawcett, D. W. (19f;3). /n The Peripheral Blood Vessels. (Ed. by Orbison, J. L. and Smith, I). E.) Williams and Wilkiruq. Baltimore. Flor~:y. ll. W. (1961). (,~uart..1. Exptl PhyMol. 44, 119. t"lorey, l,ord. (19~i6). Bril. ;lied. J. iJ, 487. Florcy, I,ord (196S). Quart. J. Exptl Physiol. 53, 1. Fralcy. E. E. and Wei.q.~. L. (1961). Am. J. Aura'. 109, 85. Itogan. M. J. and Fccn,;y, I.. (1961). :Ira. J. Ophthalmol. 5;1, 1084. lshikawa, T. (19~i:'). I~,,est. Ophthabuol. 2, 1. lwamoto, T. and Smel~r, G. K. (1965). Iu~:est. Ophlhalmol. 4, 815. Katayama. 11. (1957). Lymph