- Email: [email protected]
Tissue fibrinolytic activity in the anterior segment of the eye, as related to aqueous outflow
Ex])tl Eye Rcs. (1970) 10, 297-30]
T ~ s u e Fibrinolytic Activity in the Anterior Segment of the Eye, As Related to Aqueous Outflow ~Li~,IIDA SA.IDUZZAFAR$
Department of Experimental Ophthalmology, Instit~tZe of Ophthalmology, University of Londo,~, Judd Street, London W.C.1, Er~gland (Received 6 May/1970, Lo~uton) Alto:rations in t h e n o r m a l f i b r i n o l y t i e p a t t e r n f o l l o w i n g i n t r a c a m e r a l i n j e c t i o n s o f p l a s m i n and an inhibitor of plasminogen activation, aminomethyl cyciohexane carboxylic acid (,~MCA), w e r e f o u n d t o c o r r e s p o n d w i t h t h e c h a n g e s in t h e f a c i l i t y o£ a q u e o u s out.flow ind u c e d b y t h e s e m~bstanm.~s in l i v i n g m o n k e y e y e s . :It is suggeste
1. Introduction
I'andbhq, Cocch~eri a n d A~strup (].962) f o u n d t h a t the s t r u c t u r e s of t h e eye varied in their fibrinolytic properties, a n d tlle fibrinolytic a c t i v i t y was shown to be due to plasminogen ,~ctiv~Ltor a c t i v i t y r a t h e r t h a n direct pro tease a c t i v i t y ( K w a a n a n d Astlaq), 1963; I~andolfi ~nd K w a a n , 1967); these a u t h o r s also f o u n d t h a t t h e endot h e l i u m of the canal of Sctdemm was fibrinolytically active in a d u l t h u m a n eyes, b u t inactive in the eyes of the h u m a n foetus a n d n e w b o r n infant. These observations, as well as the presence of plasminogen a n d p l a s m i n o g e n a c t i v a t o r a c t i v i t y in t h e aqueou~s h u m o u r of several a n i m a l species, ]ed t h e m to believe t h a t fibrinolysis m i g h t p l a y a physiological role in t h e meclm,~ism of aqueous outflow. Experirr/ents designed to d e t e r m i n e t h e effect of t h e p r o t e o l y t i c enzyme, plasmin, on t h e f a c i l i t y of outflow of living c y n o m o l g u s m o n k e y s h a v e shown t h a t i n t r a cameral h~jeetion of this substance causes a significant r e d u c t i o n in t h e outflow resistance (Perkins a n d Saiduzzafar, 1969), the m a g n i t u d e of t h e change being similar to t h a t observed in dead eyes (Pandolfi a n d Astrup, 1966; Pandolfi, 1967a). ttowevero it was felt t h a t these perfusion e x p e r i m e n t s did n o t p r o v i d e suflqcient evidence regarding the site or mode of action of t h e perfusing substances. T h e p r e s e n t s t u d y was therefore u n d e r t a k e n primariJy to t r y to r e l a t e t h e changes in outflow resistance t o t h e patterfi of tissue fibrinolytic a c t i v i t y i n t h e a n t e r i o r s e g m e n t of t h e eye. :Fresh frozen sections of n o r m a l c y n o m o l g u s m o n k e y eyes were e x a m i n e d b y fibrinolysis autog r a p h y , a n d were c o m p a r e d with similar sections of eyes pelffused a f t e r either p l a s m i n or i n h i b i t o r s of p l a s m i n o g e n a c t i v a t i o n h a d been placed in t h e a n t e r i o r chamber. 2. Experimental Procedure
Material Eyes removed from cynomolgus monkeys (21laca~ fa.scivularis), killed on completion of the perfusio n experiments, formed t h e bulk of the material. Normal unperfused cyaaomolgus m o n k e y eyes and eyes perfused with inert control solutions were examined for * Address for reprints: I n s t i t u t e of Ophthalmology, Gandhi E y e :l~ospital, Aligaxh, U.:P., India. 297
298
H. S A [ D U Z Z A F A R
c o m p a r i s o n . As a s u p p l e m e n t to t h e s t u d y t h e e y e s o f g u i n e a - p i g s a n d pigs, eiLhcr n o r m a l or t r e a t e d w i t h i n h i b i t o r s o l u t i o n s , w e r e e x a n f i n e d in t h e s a m e w a y . I n additio;~, a nun:tb e t o f n o r m a l a d u l t h u m a n e y e - b a n k e y e s o b t a i n e d w i t h i n 12--1,l. h r a f t e r d e a t h , as well as a f e w r a b b i t eyes, w e r e also s e c t i o n e d a n d e x a m i n e d b y f i b r i n o l y s i s a u t o g r a p h y .
3 l ethods T h e e n u c l e a t e d e y e w a s s n a p - f r o z e n in a t e s t - t u b e c o n t a i n i n g i s o p e n t a a e , p r e - c o o l e d t o - - 7 0 ° C in ~t flask c o n t a i n i n g a m i x t u r e of m e t h y l a t e d s p i r i t (or a c e t o n e ) a n d c a r d i c e . T h e i s o p e n t a n e w a s d e c a n t e d off a n d t h e f r o z e n eye was s e c t i o n e d i m m e d i a t e l y or s t o r e d in a s e a l e d c o n t a i n e r a t --20°C.
Preparation of fibrinolysis a utopral;h.¢ Solutions Fibri~mgen.. A COl./os o l u t i o n by. w e i g h t of A r m o u r B o v i n e Fil:rin(~gen, ]:'lasnm F r a c t i o n l, w a s p r e p a r e d in M i c h a e l i s buffer saline-azi(le, a(lju.~ted to p t l 7..t:-~_0.05 glas.s e l e c t r o d e (Biggs & M a e F a r l a n e , 1962). Thrombin. T h e c o n t e n t s of one vial of T o p i c a l T h r o m l ) i a ( [ ' a r k e D a v i s ) w e r e tli.~_,lved i~ 5 m2 of 500/0 g l y c e r o l to p r e p a r e a s t o c k s o l u t i o n (I(KK) u n i t s / r o l l , w h i c h wa.~ stor,:d in t h e d e e p freeze ( - - 2 0 ° C ) u n t i l r c q t t i r e d ; to p r e p a r e t h e w o r k i n g ~ohtt.io;~, i~ wm~ dilute(t w i t h b l i c h a e l i s b u f f e r to g i v e a c o u c e a t r a t i o n of 20 u n i t s / m l . ~;no.qm~ .-F"/brcsrr,bin r ~ T u : e
~.:Ce!iop~ne
!
G~e:,s ~ o ~
• ,. ~"
,,
'
~'
', ,' "
'
~----:
_~ L2"¢R!};P,g
$Ct"~
{o)
Uncoated border \
•
Fibrin coating
i'. ~-:~'.-:~'".:L::.:-:"--~'-"J (b)
Cellophane
~ . Uncoated horde turned down (c)
~-- Cover g.oss Tissue sec!<~n
FzQ. 1. Pl~paration of fibrirx-eoated cellophane. (~) Cross.~etion of plate c~rrying ~-~agulation mix ture. (b) Fibrin-coated cellophane strip s ~ n from above. (c) Cross-section of cover-gla~ carrying tissue ~c~ion and fibrin-coated cellophane.
Preparation of tissue sections T h e f r o z e n e y e w a s o u t i n h a f t t h r o u g h t h e e q u a t o r , a n d t h e a n t e r i o r s e g m e n t was suit,a b l y t r i m m e d a n d m o u n t e d on t h e t i s s u e h o l d e r ( c h u c k ) in o r d e r t o o b t a i n s a g i t t a l sect i o n s o f Vhe a n g l e o f t h e a n t e r i o r c h a m b e r . 5£ichaelis buffer s a l i n e (or t h e e m b e d d i n g m e d i n m , O.C.T.*) w a s u s e d for f r e e z i n g t h e s p e c i m e n b l o c k to t h e c h u c k . S e c t i o n s 7 - 8 tzm thick were cut on a cryostat, and each section was taken directly from the knife onto a coverslip. E a c h t i s s u e s e c t i o n w a s c o v e r e d w i t h a piece o f f i b r i n - c o a t e d C e l l o p h a n e , a n d p l a c e d o n a d a m p p l a s t i c s p o n g e i n a n i n c u b a t i o n c h a m b e r (Fig. 1). T h e f i b r i n - c o a t e d s e c t i o n s w e r e k e p t a t 4°C f o r 24 h r , f o l l o w e d b y s u i t a b l e i n c u b a t i o n ( u s u a l l y 15--30 rain) a t 37°C. A:fter t h e r e q u i r e d i n c u b a t i o n , t h e p r e p a r a t i o n s w e r e t r a n s f e r r e d to s t a i n l e s s s t e e l *
"Tissue-Tek" O.C.T. compound (Ames).
]"[..vrr.: ') 3l'+~nlo,v: eili~rv l~r>dv. (>'. 40.) Tnelll)~dion: 90 rain ~t ,~j C..
I'I,,~TI~ 3. 5 1 o l i k i ' ) ' :
iris. ( -, .t0.) lltclll,itli~ln
: 2ti iliili al 37 ('+,
I~LAmE4 . 5 I o n k e y eye: normal angle of anterior chambor. Incul.aticn: 30 rain at 3T (;.
I'r. x rt:
5..'~b,t,l, cv : all~l,', pq'rfi,.-~,,I af)er l~la.~j~;il~ injecl icon. (:< :10.) I Jlet~b~rtion : 2~t mitl at 37'-C.
Pl.t'rt+: t-;. ]"h+smJt+-pt,rf1~s¢.d mon|+t,.+" eye, Jnr.t+bated for 20 +~+ht at~ 37"C, with fibrin col>tainJn~ .ASICA+
])L,vr).: 7. M(mk(,v ~m.~h,: p(.r.fu.,,ed wiih l',ine('r',~ .~,)Imi,m. (. '..- .I,')..} Iu,,ub)~l io)~ • ?,i) mirl ;it ::)'; i'
I)I.AT~ 8, M o n k e y ungte: porfilsed after AMCA injeetion. (><40.) lneut, m t i o n : 20 mi~ a~,t a7"C'.
|'t,.~'l~l ,, 9, ~;ulnea-.Pi~ eye.: nocmal an~h., ~d'nnt~'riur ~-h:~nlber, (.. ,ffl.) [fl~mba~ h',l: 3{) rain at, 37 '(_:.
t's,,~T~: lfl. (]t~iae~-pig angh.: slm~ying absence, nf 13,,is e,rresponding t , absence of cnd~.~thcli~l cells.
(>:,10.) [J~cubal io~l" 30 rain at 37'~'C.
l'J...,t.r~,: i I, (; u i n,.:,~: pig a ~@ e . ( / ' 40. ) I n~ u !:~ l i~~~
:PLAT>: 12. G u i a e a , p i g iris: Ai~ICA placed in anterior chamber.: Incub~t-ion: 30 rain at 37"C.
I't...~'r~: 1:,~. "l.'i.~ :,x'c: n o r m a l ang:h: ~dame:"ic, r ehamb~_~r, (.,:4().) 11~eubat.ilm : 5 rain at. 37~"C.
I~LAT~:14: I)ig cox'ileal cadothelium. I~n6ubaLioI~: 5 mitl at 37'~0.
t'I,Aa'F, l(i. l~*~bl,ii, u)'o: noHllltl ~.4 lit" ~lt~ ,t ....t,,,
iiliglo (~f,ti~tei'ior
cll~tlll|)(:i-.
(,,~ ,Itri.) l tlotlhfllion:
:|(I lili~l ~lt :i7-(3 ~ftt-t"
PI.t"'~: t S..[T~nl~an e y e : norm~d a n ~ l e t~f ~mterior el:aml)er. ( >~ ,10°) I n e u l ) a l i~m : 0 - 5 rain a t ,, 1 C,.
~I~)[,A.'I'I ', 20. "l~[.Unl~'lll I?.';V~C eo~'nea : h u m a n
fibrin. I n e u h a t h m :
0 - 5 r a i n at, .~," ",~.t...-"
[~LATI'~ ~ | . l]tllll;ll~ ir~s: IJ,'~vint, fibrils', l~culmti~-,n: 0--5 miJi at. 3, C.
lClBItlNOLYTIC
ACT1VITY
AND AQUEOUS
OUTFLOW
299
cover-slip racks, fixed in neutral buffered formalin, stripped of Cellophane, and stained, dehydrated, cleared and mounted according to the method of Todd and Nunn (1967). 3. Results
3,[o~ke!/ eyes Section,s of tl~e ~t(,n-i~erfused normal ante r i ~ segment. The greatest fibrinolytic a c t i v i t y was generally seen a r o u n d the episclcral a n d c o n j u n c t i v a l vessels, as well as a r o u n d the canal of Schlemm and its collectors (Plate 1) ; there was m o d e r a t e a c t i v i t y in t h e ciliary b o d y (Plate 2) and little or none in the iris (Plate 3), while t h e t r a b e c u l a r meshwork, ciliary processes and lens were always inactive. I n c r e a s i n g the i n c u b a t i o n time to 30 rain at 37°C cal~.~cd cor, fluence of some areas of lysis (Plate 4).
Sections of perfused mo'n~'ey eyes following the :'ntraz.ameral injection of plasmin. A confluent " t r a c k " of fibrinolysis was seen e x t e n d i n g from t h e r e , o n of the canal of Schlomm towards the limbal vessels, and. bac!~vards to the ciliary b o d y (Plate 5). Where, however, AA[CA in a concentrat.iou of i0 ~a ,~[ was i n d o r p o r a t e d into the fibrin coating on the section, plasrnin lysis was inhibited (Plate 6).
A'ecd.ions ,~ nwT~:e!! eyes p~'rfu.~ed after intracamvral injections of control Ringer's sol uzfon. Some a t t e n u a t i o n of the nmrmai fibrinolytie p a t t e r n was observed, and no confluent arenas of lysis were seen after 30 rain i n c u b a t i o n a t 37°C (Plate 7).
Seaions of monkey eyes perfu.sed after intraeameral injection of A M C A . These fibrinolysis aut.ograpim showed an a l m o s t c o m p l e t e disappearance of the fibrinolytic a c t i v i t y n o r m a l l y seen in the anterior segment of the eye (Plate 8). Guinex~-pig eyes Non-.perfused ant~-ior seg memt sect.i(ms. Using tile same procedures, it was found t h a t a l n i n i m u m i n c u b a t i o n time of 30 min a t 37°0 was required to reveal clear areas of Iysis. A consistent a n d striking feature in t h e a n t e r i o r segment of the guinea-pig eye was a line of fibrinolytio a c t i v i t y along t h e i n t a c t corneal e n d o t h e l i u m (Plate 9), which disappeared a t places where the e n d o t h e l i a l cells h a d i n a d v e r t e n t l y been dislodged (Plate 10). The bloodvessels a t the angle of the a n t e r i o r c h a m b e r also showed m o d e r a t e a c t i v i t y (Plate 11).
An~ericrr segmeT~$seclio~$ of eyes where A M C A had been played in the anterior chamber. These sections showed a complete disappearance of t h e n o r m a l fibrinolytic p a t t e r n in the anterior s e g m e n t of t h e eye (Plate. 12).
Discrete a n d confluent areas of lysis were seen in sections of t h e anterior segment after i n c u b a t i o n a t 37°C for as s h o r t a t i m e as 5 rain (Plate 13). _i line of strong fibrino!ytic a c t i v i t y was seen i,i association w i t h the corneal e n d o t h e l h l m , similar to t h e a p p e a r a n c e f o u n d in t h e glllnea-pig eye (Plate 1 4 ) . / i l l a c t i v i t y disappeared following t h e i n t r a c a m e r a l injection of A2,ICA (Plate 15).
Rabbit eyes A n t e r i o r segment sections of non-pert'used r a b b i t eyes showed no fib rinolytic a c t i v i t y following t h e s t a n d a r d i n c u b a t i o n t i m e of 24 hr a t 4°C a n d 30 r a i n a t 37°C (Plate 16). A few sections which were k e p t for 7 2 h r a t 4°C a n d f o r 60 rain a t 37°C showed areas of lysis a t t h e angle of the a n t e r i o r c h a m b e r a n d a r o u n d a few of t h e iris vessels (Plate 171. However, prolongcd i n c u b a t i o n was f o u n d u n s a t i s f a c t o r y on
300
II. SAID UZZAFAB.
accotmt of the nmncrous spots of "peripheral lysis" which appeared in areas not connectcd with the tissue section. t l u m a n eyes
I~ibrinolysis autographs prepared from eye-bank material showed very strong fibrinolyti¢ a c t i v i t y in the anterior segment. I n c u b a t i o n for.0-5 rain at 37°63 was suiti.. cient to cause discrete ~nd confluent areas of lysis. The region of the canal of Sclflcmm and its collector vessels was ahvuys very active (Plate 18). Comparative studies, using b o v i n e a n d h u m a n fibrin, revealed an interesting feature. W h e n bovine fibrin was used, the l i m b a l region ~*as vcry active, but only doubtflfl a c t i v i t y w~s seen in association with the corneal epithelinm (Plate 19). Howdvcr, with truman fibrin, the whole of the intact corneal epithelium showed fibrinolytic a c t i v i t y (.Plate 20). The vessels of the iris were extremely active when either bovine or h u m a n fibrin was used (I/late 21). 4. D i s c u ~ i o n
Tiffs s t u d y has confirmed t h a t tissue fibrinolytic a c t i v i t y in the eye varies considera b l y from one species to another. J u d g e d by the m i n i m m n incubation time required to produce cle~Lrly defined areas of lysis, h u m a n eyes wer6fout~d to be the most active. Pig eyes were only slightly less active, followed by the m o n k e y and trainee-pig eyes. lgabbit eyes were coI~sistently poor in their fibrinolytic response. The fibrinoh'tic a c t i v i t y which was found a.~sociated with the corneal end.othcliuna of the gu-ne~:~-pig (Plate 9) a n d the pig (Plate 14) provides an exception to the usual assumption that fibrinolytic activi W in the various structures of the eye is specifically related to their v a s c u l a r i t y (Pandolfi, 1967b). Although tim reason for this unusual a c t i v i t y of the avascular endothelium r e m a i n s obscure, it resembles the activity found in the e.ndothelium of the systemic veins and capillaries (Todd, 1959). i t is perhaps significant t h a t guinea-pig and pig eyes do not h a v e a canal of Schlemm, while o~a the other h a n d m o n k e y and h u m a n eyes, which have a well-developed S c h l e m m ' s canal, showed no a c t i v i t y connected with the corneal endothelium. I n h m n a n eyes th~~ i n t a c t corneal e p i t h e l i u m showed fibrinoly~ic a c t i v i t y when i n c u b a t e d with h u m a n fibrin, b u t not with bovine fibrin (Plates 19 and 20). This selective a c t i v i t y of the avascular corneal epithelium, which does not appear to have been reported before, shows t h a t tissue activators of plasminogen m a y sometimes p o s s e ~ a species-specificity resembling t h a t of streptokinase. This is not the case w i t h all tissues; for example the h u m a n iris showed a high degree of fibrinoly~ic activity, and there was no detectable difference between the lysis of h u m a n or bovine fibrin ( P l a t e 2I) caused b y the tissue activators of the h u m a n iris. As reported in a n earlier s t u d y (Perkins and: Saiduzzafar, 1969), t h e injection of p l a s m i n into the anterior c h a m b e r of living m o n k e y eyes caused a significant increase in t h e facility o f outflow. Fibrinolysis autographs prepared from m o n k e y eyes treated w i t h p l a s m l n in this way shmved a distinct, t r a c k of massive fibrin lysis, while the trabectflar meshwork r e m a i n e d inactive. These findings suggest t h a t p l a s m i n reduces the resistance to aqueous outflow b y t h e enzymic lysis of fibrin in the outflow pathways and exerts its action on the endothelium of the c a n a l of Schlemm or the collector e" . This was also confirmed b y t h e disappearance of the fibrinolY%ie a c t i v i t y i l a r sections (from plasmin-perfused eyes) were i n c u b a t e d w i t h fibrin conta _dViCA.Pandolfi (1967a) also concluded from his experiments on enucleated
FII~I~INOLYT[(3 ACTIVITY AND AQUEOUS OUTFLOW
301
monl(ey eyes that the effect of plasmin on outflow resistance was a true enzyme action. When AMCA was injected into the anterior chamber of living monkeys, subsequent perfusions showed a reduction in the facility of outflow (Saiduzzafar, 1970). Fibrinolysis autog~'aphy of these perfused eyes showed a suppressiou of the normal pattern of fibrinolytic activity in the angle of the anterior chamber (:Plate 8). Tiffs confirmed that an inhibitiou of the normal plasminogen activation in the ocular tissues had occurred, :~nd that tile site (~f action of AMCA was the same as t h a t of plasmin, viz. the vascular part of the outflow p a t h w a y and the canal of Scldemm distal to the trabeeular meshwork. Since the amoun~ of endogenous plasmin available in the region of the angle of the anterior chamber is obviously small, any suppression of the tissue activator activity would cause only a small change in outflow facility when compared with the cffe('t caused by exogenot~ plasmin. Although, for practical reasons, the facility of outflow was not measured in the glfinca-pig and pig eyes, the supprcssiorl of the normal fibrinolytic pattern in the anterior segment was consistently observed (Plates ]2 and 15)after intracameral injection of A MCA. The control monkey eyes, where perfilsion experiments were peribrmed using Ringer's solution, showed no significant changes either in facility of outflow or in the patte~'n of fibrinolytic activity. The slight attenuation of the latter (Plate 7) could be attributed to a dilution or washing out of the tissue activators in the outflow pathways. In the 1LSng monkey eye there thus appears to be a component of the outflow resistance which lies distal to the trabecular meshwork and which is directly concerned with the removal by plasmin of fibrin in the 'outflow pathway. ACKNOWLEDGMENTS I am very gra~eful to Professor E. S. Perkins for his guidance and encouragement. I also wish to thank Dr A. S. Todd, of the Royal Infirmary, Duudee, for allowing me to leara tha technique in his laboratory. This work was supt~orted by a grant fi:om the Itayward Foundation. REFERENCES
:Biggs, R. and :hlacFarlane, II. G. (1962). Human Blood Coagulation and its .Dizorders. 3rd Ed. p. 370. :Blackwell, Oxford. Kwaan, H. C. and Astrup, T. (1963). Arch. PaIhol. 76, 595. Pandolfi, M. (1967a). Am. J. OphthalnwI. 64, 1141. Pazldolfi, ~L (1967b). Arch. Ophtha!rnol. (Chicago) 78, 512. Pandolfi, M. and Astrup, T. (1966)..Prec. Soc. Exp. Biol. ~l~ed. 121, 139. Pandolfi, ~I., Coccheri, S. and Astrup, T. (1962). Prec. Soc. Jrgxp. JBiol. _~led. 11}9, 159. Paadolfi, M. and Kwa~n, H. C. (1967). Arch. Ophthalmol. (Chicago)77, 99. Perkins, E. S. and Saiduzzafar, H. (1969). Exptl Eye Res. 8, 386. Saiduzzafar, H. (1970). Thesis ibr Ph.D., University of London. "2odd, A. S. (1959). J. PatlmL ~Bacteriol. 78, 281. Todd, A. S. and Numl, A. (1967). Prec. l~t Int: ~ymp. on Tissue l~acZ.ors in the Homeostasis of the Coaqulation.tVibrlnolysis System, pp. 57-78. Florence.
T ~ s u e Fibrinolytic Activity in the Anterior Segment of the Eye, As Related to Aqueous Outflow ~Li~,IIDA SA.IDUZZAFAR$
Department of Experimental Ophthalmology, Instit~tZe of Ophthalmology, University of Londo,~, Judd Street, London W.C.1, Er~gland (Received 6 May/1970, Lo~uton) Alto:rations in t h e n o r m a l f i b r i n o l y t i e p a t t e r n f o l l o w i n g i n t r a c a m e r a l i n j e c t i o n s o f p l a s m i n and an inhibitor of plasminogen activation, aminomethyl cyciohexane carboxylic acid (,~MCA), w e r e f o u n d t o c o r r e s p o n d w i t h t h e c h a n g e s in t h e f a c i l i t y o£ a q u e o u s out.flow ind u c e d b y t h e s e m~bstanm.~s in l i v i n g m o n k e y e y e s . :It is suggeste
1. Introduction
I'andbhq, Cocch~eri a n d A~strup (].962) f o u n d t h a t the s t r u c t u r e s of t h e eye varied in their fibrinolytic properties, a n d tlle fibrinolytic a c t i v i t y was shown to be due to plasminogen ,~ctiv~Ltor a c t i v i t y r a t h e r t h a n direct pro tease a c t i v i t y ( K w a a n a n d Astlaq), 1963; I~andolfi ~nd K w a a n , 1967); these a u t h o r s also f o u n d t h a t t h e endot h e l i u m of the canal of Sctdemm was fibrinolytically active in a d u l t h u m a n eyes, b u t inactive in the eyes of the h u m a n foetus a n d n e w b o r n infant. These observations, as well as the presence of plasminogen a n d p l a s m i n o g e n a c t i v a t o r a c t i v i t y in t h e aqueou~s h u m o u r of several a n i m a l species, ]ed t h e m to believe t h a t fibrinolysis m i g h t p l a y a physiological role in t h e meclm,~ism of aqueous outflow. Experirr/ents designed to d e t e r m i n e t h e effect of t h e p r o t e o l y t i c enzyme, plasmin, on t h e f a c i l i t y of outflow of living c y n o m o l g u s m o n k e y s h a v e shown t h a t i n t r a cameral h~jeetion of this substance causes a significant r e d u c t i o n in t h e outflow resistance (Perkins a n d Saiduzzafar, 1969), the m a g n i t u d e of t h e change being similar to t h a t observed in dead eyes (Pandolfi a n d Astrup, 1966; Pandolfi, 1967a). ttowevero it was felt t h a t these perfusion e x p e r i m e n t s did n o t p r o v i d e suflqcient evidence regarding the site or mode of action of t h e perfusing substances. T h e p r e s e n t s t u d y was therefore u n d e r t a k e n primariJy to t r y to r e l a t e t h e changes in outflow resistance t o t h e patterfi of tissue fibrinolytic a c t i v i t y i n t h e a n t e r i o r s e g m e n t of t h e eye. :Fresh frozen sections of n o r m a l c y n o m o l g u s m o n k e y eyes were e x a m i n e d b y fibrinolysis autog r a p h y , a n d were c o m p a r e d with similar sections of eyes pelffused a f t e r either p l a s m i n or i n h i b i t o r s of p l a s m i n o g e n a c t i v a t i o n h a d been placed in t h e a n t e r i o r chamber. 2. Experimental Procedure
Material Eyes removed from cynomolgus monkeys (21laca~ fa.scivularis), killed on completion of the perfusio n experiments, formed t h e bulk of the material. Normal unperfused cyaaomolgus m o n k e y eyes and eyes perfused with inert control solutions were examined for * Address for reprints: I n s t i t u t e of Ophthalmology, Gandhi E y e :l~ospital, Aligaxh, U.:P., India. 297
298
H. S A [ D U Z Z A F A R
c o m p a r i s o n . As a s u p p l e m e n t to t h e s t u d y t h e e y e s o f g u i n e a - p i g s a n d pigs, eiLhcr n o r m a l or t r e a t e d w i t h i n h i b i t o r s o l u t i o n s , w e r e e x a n f i n e d in t h e s a m e w a y . I n additio;~, a nun:tb e t o f n o r m a l a d u l t h u m a n e y e - b a n k e y e s o b t a i n e d w i t h i n 12--1,l. h r a f t e r d e a t h , as well as a f e w r a b b i t eyes, w e r e also s e c t i o n e d a n d e x a m i n e d b y f i b r i n o l y s i s a u t o g r a p h y .
3 l ethods T h e e n u c l e a t e d e y e w a s s n a p - f r o z e n in a t e s t - t u b e c o n t a i n i n g i s o p e n t a a e , p r e - c o o l e d t o - - 7 0 ° C in ~t flask c o n t a i n i n g a m i x t u r e of m e t h y l a t e d s p i r i t (or a c e t o n e ) a n d c a r d i c e . T h e i s o p e n t a n e w a s d e c a n t e d off a n d t h e f r o z e n eye was s e c t i o n e d i m m e d i a t e l y or s t o r e d in a s e a l e d c o n t a i n e r a t --20°C.
Preparation of fibrinolysis a utopral;h.¢ Solutions Fibri~mgen.. A COl./os o l u t i o n by. w e i g h t of A r m o u r B o v i n e Fil:rin(~gen, ]:'lasnm F r a c t i o n l, w a s p r e p a r e d in M i c h a e l i s buffer saline-azi(le, a(lju.~ted to p t l 7..t:-~_0.05 glas.s e l e c t r o d e (Biggs & M a e F a r l a n e , 1962). Thrombin. T h e c o n t e n t s of one vial of T o p i c a l T h r o m l ) i a ( [ ' a r k e D a v i s ) w e r e tli.~_,lved i~ 5 m2 of 500/0 g l y c e r o l to p r e p a r e a s t o c k s o l u t i o n (I(KK) u n i t s / r o l l , w h i c h wa.~ stor,:d in t h e d e e p freeze ( - - 2 0 ° C ) u n t i l r c q t t i r e d ; to p r e p a r e t h e w o r k i n g ~ohtt.io;~, i~ wm~ dilute(t w i t h b l i c h a e l i s b u f f e r to g i v e a c o u c e a t r a t i o n of 20 u n i t s / m l . ~;no.qm~ .-F"/brcsrr,bin r ~ T u : e
~.:Ce!iop~ne
!
G~e:,s ~ o ~
• ,. ~"
,,
'
~'
', ,' "
'
~----:
_~ L2"¢R!};P,g
$Ct"~
{o)
Uncoated border \
•
Fibrin coating
i'. ~-:~'.-:~'".:L::.:-:"--~'-"J (b)
Cellophane
~ . Uncoated horde turned down (c)
~-- Cover g.oss Tissue sec!<~n
FzQ. 1. Pl~paration of fibrirx-eoated cellophane. (~) Cross.~etion of plate c~rrying ~-~agulation mix ture. (b) Fibrin-coated cellophane strip s ~ n from above. (c) Cross-section of cover-gla~ carrying tissue ~c~ion and fibrin-coated cellophane.
Preparation of tissue sections T h e f r o z e n e y e w a s o u t i n h a f t t h r o u g h t h e e q u a t o r , a n d t h e a n t e r i o r s e g m e n t was suit,a b l y t r i m m e d a n d m o u n t e d on t h e t i s s u e h o l d e r ( c h u c k ) in o r d e r t o o b t a i n s a g i t t a l sect i o n s o f Vhe a n g l e o f t h e a n t e r i o r c h a m b e r . 5£ichaelis buffer s a l i n e (or t h e e m b e d d i n g m e d i n m , O.C.T.*) w a s u s e d for f r e e z i n g t h e s p e c i m e n b l o c k to t h e c h u c k . S e c t i o n s 7 - 8 tzm thick were cut on a cryostat, and each section was taken directly from the knife onto a coverslip. E a c h t i s s u e s e c t i o n w a s c o v e r e d w i t h a piece o f f i b r i n - c o a t e d C e l l o p h a n e , a n d p l a c e d o n a d a m p p l a s t i c s p o n g e i n a n i n c u b a t i o n c h a m b e r (Fig. 1). T h e f i b r i n - c o a t e d s e c t i o n s w e r e k e p t a t 4°C f o r 24 h r , f o l l o w e d b y s u i t a b l e i n c u b a t i o n ( u s u a l l y 15--30 rain) a t 37°C. A:fter t h e r e q u i r e d i n c u b a t i o n , t h e p r e p a r a t i o n s w e r e t r a n s f e r r e d to s t a i n l e s s s t e e l *
"Tissue-Tek" O.C.T. compound (Ames).
]"[..vrr.: ') 3l'+~nlo,v: eili~rv l~r>dv. (>'. 40.) Tnelll)~dion: 90 rain ~t ,~j C..
I'I,,~TI~ 3. 5 1 o l i k i ' ) ' :
iris. ( -, .t0.) lltclll,itli~ln
: 2ti iliili al 37 ('+,
I~LAmE4 . 5 I o n k e y eye: normal angle of anterior chambor. Incul.aticn: 30 rain at 3T (;.
I'r. x rt:
5..'~b,t,l, cv : all~l,', pq'rfi,.-~,,I af)er l~la.~j~;il~ injecl icon. (:< :10.) I Jlet~b~rtion : 2~t mitl at 37'-C.
Pl.t'rt+: t-;. ]"h+smJt+-pt,rf1~s¢.d mon|+t,.+" eye, Jnr.t+bated for 20 +~+ht at~ 37"C, with fibrin col>tainJn~ .ASICA+
])L,vr).: 7. M(mk(,v ~m.~h,: p(.r.fu.,,ed wiih l',ine('r',~ .~,)Imi,m. (. '..- .I,')..} Iu,,ub)~l io)~ • ?,i) mirl ;it ::)'; i'
I)I.AT~ 8, M o n k e y ungte: porfilsed after AMCA injeetion. (><40.) lneut, m t i o n : 20 mi~ a~,t a7"C'.
|'t,.~'l~l ,, 9, ~;ulnea-.Pi~ eye.: nocmal an~h., ~d'nnt~'riur ~-h:~nlber, (.. ,ffl.) [fl~mba~ h',l: 3{) rain at, 37 '(_:.
t's,,~T~: lfl. (]t~iae~-pig angh.: slm~ying absence, nf 13,,is e,rresponding t , absence of cnd~.~thcli~l cells.
(>:,10.) [J~cubal io~l" 30 rain at 37'~'C.
l'J...,t.r~,: i I, (; u i n,.:,~: pig a ~@ e . ( / ' 40. ) I n~ u !:~ l i~~~
:PLAT>: 12. G u i a e a , p i g iris: Ai~ICA placed in anterior chamber.: Incub~t-ion: 30 rain at 37"C.
I't...~'r~: 1:,~. "l.'i.~ :,x'c: n o r m a l ang:h: ~dame:"ic, r ehamb~_~r, (.,:4().) 11~eubat.ilm : 5 rain at. 37~"C.
I~LAT~:14: I)ig cox'ileal cadothelium. I~n6ubaLioI~: 5 mitl at 37'~0.
t'I,Aa'F, l(i. l~*~bl,ii, u)'o: noHllltl ~.4 lit" ~lt~ ,t ....t,,,
iiliglo (~f,ti~tei'ior
cll~tlll|)(:i-.
(,,~ ,Itri.) l tlotlhfllion:
:|(I lili~l ~lt :i7-(3 ~ftt-t"
PI.t"'~: t S..[T~nl~an e y e : norm~d a n ~ l e t~f ~mterior el:aml)er. ( >~ ,10°) I n e u l ) a l i~m : 0 - 5 rain a t ,, 1 C,.
~I~)[,A.'I'I ', 20. "l~[.Unl~'lll I?.';V~C eo~'nea : h u m a n
fibrin. I n e u h a t h m :
0 - 5 r a i n at, .~," ",~.t...-"
[~LATI'~ ~ | . l]tllll;ll~ ir~s: IJ,'~vint, fibrils', l~culmti~-,n: 0--5 miJi at. 3, C.
lClBItlNOLYTIC
ACT1VITY
AND AQUEOUS
OUTFLOW
299
cover-slip racks, fixed in neutral buffered formalin, stripped of Cellophane, and stained, dehydrated, cleared and mounted according to the method of Todd and Nunn (1967). 3. Results
3,[o~ke!/ eyes Section,s of tl~e ~t(,n-i~erfused normal ante r i ~ segment. The greatest fibrinolytic a c t i v i t y was generally seen a r o u n d the episclcral a n d c o n j u n c t i v a l vessels, as well as a r o u n d the canal of Schlemm and its collectors (Plate 1) ; there was m o d e r a t e a c t i v i t y in t h e ciliary b o d y (Plate 2) and little or none in the iris (Plate 3), while t h e t r a b e c u l a r meshwork, ciliary processes and lens were always inactive. I n c r e a s i n g the i n c u b a t i o n time to 30 rain at 37°C cal~.~cd cor, fluence of some areas of lysis (Plate 4).
Sections of perfused mo'n~'ey eyes following the :'ntraz.ameral injection of plasmin. A confluent " t r a c k " of fibrinolysis was seen e x t e n d i n g from t h e r e , o n of the canal of Schlomm towards the limbal vessels, and. bac!~vards to the ciliary b o d y (Plate 5). Where, however, AA[CA in a concentrat.iou of i0 ~a ,~[ was i n d o r p o r a t e d into the fibrin coating on the section, plasrnin lysis was inhibited (Plate 6).
A'ecd.ions ,~ nwT~:e!! eyes p~'rfu.~ed after intracamvral injections of control Ringer's sol uzfon. Some a t t e n u a t i o n of the nmrmai fibrinolytie p a t t e r n was observed, and no confluent arenas of lysis were seen after 30 rain i n c u b a t i o n a t 37°C (Plate 7).
Seaions of monkey eyes perfu.sed after intraeameral injection of A M C A . These fibrinolysis aut.ograpim showed an a l m o s t c o m p l e t e disappearance of the fibrinolytic a c t i v i t y n o r m a l l y seen in the anterior segment of the eye (Plate 8). Guinex~-pig eyes Non-.perfused ant~-ior seg memt sect.i(ms. Using tile same procedures, it was found t h a t a l n i n i m u m i n c u b a t i o n time of 30 min a t 37°0 was required to reveal clear areas of Iysis. A consistent a n d striking feature in t h e a n t e r i o r segment of the guinea-pig eye was a line of fibrinolytio a c t i v i t y along t h e i n t a c t corneal e n d o t h e l i u m (Plate 9), which disappeared a t places where the e n d o t h e l i a l cells h a d i n a d v e r t e n t l y been dislodged (Plate 10). The bloodvessels a t the angle of the a n t e r i o r c h a m b e r also showed m o d e r a t e a c t i v i t y (Plate 11).
An~ericrr segmeT~$seclio~$ of eyes where A M C A had been played in the anterior chamber. These sections showed a complete disappearance of t h e n o r m a l fibrinolytic p a t t e r n in the anterior s e g m e n t of t h e eye (Plate. 12).
Discrete a n d confluent areas of lysis were seen in sections of t h e anterior segment after i n c u b a t i o n a t 37°C for as s h o r t a t i m e as 5 rain (Plate 13). _i line of strong fibrino!ytic a c t i v i t y was seen i,i association w i t h the corneal e n d o t h e l h l m , similar to t h e a p p e a r a n c e f o u n d in t h e glllnea-pig eye (Plate 1 4 ) . / i l l a c t i v i t y disappeared following t h e i n t r a c a m e r a l injection of A2,ICA (Plate 15).
Rabbit eyes A n t e r i o r segment sections of non-pert'used r a b b i t eyes showed no fib rinolytic a c t i v i t y following t h e s t a n d a r d i n c u b a t i o n t i m e of 24 hr a t 4°C a n d 30 r a i n a t 37°C (Plate 16). A few sections which were k e p t for 7 2 h r a t 4°C a n d f o r 60 rain a t 37°C showed areas of lysis a t t h e angle of the a n t e r i o r c h a m b e r a n d a r o u n d a few of t h e iris vessels (Plate 171. However, prolongcd i n c u b a t i o n was f o u n d u n s a t i s f a c t o r y on
300
II. SAID UZZAFAB.
accotmt of the nmncrous spots of "peripheral lysis" which appeared in areas not connectcd with the tissue section. t l u m a n eyes
I~ibrinolysis autographs prepared from eye-bank material showed very strong fibrinolyti¢ a c t i v i t y in the anterior segment. I n c u b a t i o n for.0-5 rain at 37°63 was suiti.. cient to cause discrete ~nd confluent areas of lysis. The region of the canal of Sclflcmm and its collector vessels was ahvuys very active (Plate 18). Comparative studies, using b o v i n e a n d h u m a n fibrin, revealed an interesting feature. W h e n bovine fibrin was used, the l i m b a l region ~*as vcry active, but only doubtflfl a c t i v i t y w~s seen in association with the corneal epithelinm (Plate 19). Howdvcr, with truman fibrin, the whole of the intact corneal epithelium showed fibrinolytic a c t i v i t y (.Plate 20). The vessels of the iris were extremely active when either bovine or h u m a n fibrin was used (I/late 21). 4. D i s c u ~ i o n
Tiffs s t u d y has confirmed t h a t tissue fibrinolytic a c t i v i t y in the eye varies considera b l y from one species to another. J u d g e d by the m i n i m m n incubation time required to produce cle~Lrly defined areas of lysis, h u m a n eyes wer6fout~d to be the most active. Pig eyes were only slightly less active, followed by the m o n k e y and trainee-pig eyes. lgabbit eyes were coI~sistently poor in their fibrinolytic response. The fibrinoh'tic a c t i v i t y which was found a.~sociated with the corneal end.othcliuna of the gu-ne~:~-pig (Plate 9) a n d the pig (Plate 14) provides an exception to the usual assumption that fibrinolytic activi W in the various structures of the eye is specifically related to their v a s c u l a r i t y (Pandolfi, 1967b). Although tim reason for this unusual a c t i v i t y of the avascular endothelium r e m a i n s obscure, it resembles the activity found in the e.ndothelium of the systemic veins and capillaries (Todd, 1959). i t is perhaps significant t h a t guinea-pig and pig eyes do not h a v e a canal of Schlemm, while o~a the other h a n d m o n k e y and h u m a n eyes, which have a well-developed S c h l e m m ' s canal, showed no a c t i v i t y connected with the corneal endothelium. I n h m n a n eyes th~~ i n t a c t corneal e p i t h e l i u m showed fibrinoly~ic a c t i v i t y when i n c u b a t e d with h u m a n fibrin, b u t not with bovine fibrin (Plates 19 and 20). This selective a c t i v i t y of the avascular corneal epithelium, which does not appear to have been reported before, shows t h a t tissue activators of plasminogen m a y sometimes p o s s e ~ a species-specificity resembling t h a t of streptokinase. This is not the case w i t h all tissues; for example the h u m a n iris showed a high degree of fibrinoly~ic activity, and there was no detectable difference between the lysis of h u m a n or bovine fibrin ( P l a t e 2I) caused b y the tissue activators of the h u m a n iris. As reported in a n earlier s t u d y (Perkins and: Saiduzzafar, 1969), t h e injection of p l a s m i n into the anterior c h a m b e r of living m o n k e y eyes caused a significant increase in t h e facility o f outflow. Fibrinolysis autographs prepared from m o n k e y eyes treated w i t h p l a s m l n in this way shmved a distinct, t r a c k of massive fibrin lysis, while the trabectflar meshwork r e m a i n e d inactive. These findings suggest t h a t p l a s m i n reduces the resistance to aqueous outflow b y t h e enzymic lysis of fibrin in the outflow pathways and exerts its action on the endothelium of the c a n a l of Schlemm or the collector e" . This was also confirmed b y t h e disappearance of the fibrinolY%ie a c t i v i t y i l a r sections (from plasmin-perfused eyes) were i n c u b a t e d w i t h fibrin conta _dViCA.Pandolfi (1967a) also concluded from his experiments on enucleated
FII~I~INOLYT[(3 ACTIVITY AND AQUEOUS OUTFLOW
301
monl(ey eyes that the effect of plasmin on outflow resistance was a true enzyme action. When AMCA was injected into the anterior chamber of living monkeys, subsequent perfusions showed a reduction in the facility of outflow (Saiduzzafar, 1970). Fibrinolysis autog~'aphy of these perfused eyes showed a suppressiou of the normal pattern of fibrinolytic activity in the angle of the anterior chamber (:Plate 8). Tiffs confirmed that an inhibitiou of the normal plasminogen activation in the ocular tissues had occurred, :~nd that tile site (~f action of AMCA was the same as t h a t of plasmin, viz. the vascular part of the outflow p a t h w a y and the canal of Scldemm distal to the trabeeular meshwork. Since the amoun~ of endogenous plasmin available in the region of the angle of the anterior chamber is obviously small, any suppression of the tissue activator activity would cause only a small change in outflow facility when compared with the cffe('t caused by exogenot~ plasmin. Although, for practical reasons, the facility of outflow was not measured in the glfinca-pig and pig eyes, the supprcssiorl of the normal fibrinolytic pattern in the anterior segment was consistently observed (Plates ]2 and 15)after intracameral injection of A MCA. The control monkey eyes, where perfilsion experiments were peribrmed using Ringer's solution, showed no significant changes either in facility of outflow or in the patte~'n of fibrinolytic activity. The slight attenuation of the latter (Plate 7) could be attributed to a dilution or washing out of the tissue activators in the outflow pathways. In the 1LSng monkey eye there thus appears to be a component of the outflow resistance which lies distal to the trabecular meshwork and which is directly concerned with the removal by plasmin of fibrin in the 'outflow pathway. ACKNOWLEDGMENTS I am very gra~eful to Professor E. S. Perkins for his guidance and encouragement. I also wish to thank Dr A. S. Todd, of the Royal Infirmary, Duudee, for allowing me to leara tha technique in his laboratory. This work was supt~orted by a grant fi:om the Itayward Foundation. REFERENCES
:Biggs, R. and :hlacFarlane, II. G. (1962). Human Blood Coagulation and its .Dizorders. 3rd Ed. p. 370. :Blackwell, Oxford. Kwaan, H. C. and Astrup, T. (1963). Arch. PaIhol. 76, 595. Pandolfi, M. (1967a). Am. J. OphthalnwI. 64, 1141. Pazldolfi, ~L (1967b). Arch. Ophtha!rnol. (Chicago) 78, 512. Pandolfi, M. and Astrup, T. (1966)..Prec. Soc. Exp. Biol. ~l~ed. 121, 139. Pandolfi, ~I., Coccheri, S. and Astrup, T. (1962). Prec. Soc. Jrgxp. JBiol. _~led. 11}9, 159. Paadolfi, M. and Kwa~n, H. C. (1967). Arch. Ophthalmol. (Chicago)77, 99. Perkins, E. S. and Saiduzzafar, H. (1969). Exptl Eye Res. 8, 386. Saiduzzafar, H. (1970). Thesis ibr Ph.D., University of London. "2odd, A. S. (1959). J. PatlmL ~Bacteriol. 78, 281. Todd, A. S. and Numl, A. (1967). Prec. l~t Int: ~ymp. on Tissue l~acZ.ors in the Homeostasis of the Coaqulation.tVibrlnolysis System, pp. 57-78. Florence.