Ezptl Eye Res. (1969) 8, 386-396

The Effect of P lasmin on the Faci l i ty o f Outf low in Cynomolgus M o n k e y s

E . S. P E R K I N S * AND HASIIDA SAIDUZZAFARJ"

Institute of Ophthalmology, Univeraity of London, London, W.C.1, England

(Received ~O April 1969, London)

Plasmin , when placed in the an te r io r c h a m b e r of living cynomolgus monkeys , caused a sub- s t an t i a l and s ta t i s t ica l ly significant, increase in t he coefficient of faci l i ty o f outflow. H igh ly pur i f ied porc ine plasmin was as ef fec t ive as h u m a n plasmin in inc.re,~ing the faci l i ty o{" outf low, and did no t cause the rises in sys temic a r te r ia l pressure ob ta ined wi th the earlier ba tches of lysofibrin. T he re was no cons is ten t tKII in. the in t raoeu la r pressure c~'en whe,l large increases in fac i l i ty o f out f low occurred. This could be pa r t l y accomated for by " 'pseudo-fac i l i ty" . P la smin caused some var iab i l i ty o f the pupils with a t e n d e n c y towards pupi l lary , d i la ta t ion , b u t tim change was no t s ignif icant or consistent .

1. Introduction

Perfusion studies by Pandolf i and Ast rup (1966) on the ermcleated eyes of rhesus monkeys indicated t h a t t ryps in-ac t iva ted porcine plasmin ~was able to reduce tile resistance to aqueous outflow. Thus in 10 pairs of eyes in ~h ich saline was the per- fusion fluid in bo th eyes, the mean rat io of C values (coefficient of faci l i ty of outflow) of eye 1/eye 2 varied from 0-96 to 1-10. However, when plasmin was added to the perfusion fluid in eye 1, i t was found t h a t in 12 pairs of eyes the mean rat io eye l /eye 2 varied from 1-29 to 1-70, represent ing a significant increase in the faci l i ty of outflow in the eye perfused with plasmin.

:Further perfusion studies by Pandolfi (1967) on the excised eyes of rabbi ts and monkeys , using h u m a n as well as porcine plasmin, showed similar results in rhesus and vervet monkeys, bu t no t in rabbits . Pandolfi thus demonst ra ted t h a t the abi l i ty of p lasmin to lower the resistance to aqueous outflow varied fl'om species to species. This effect was considered to be related to the earlier findings by Kwaan and Astrup (1963), Pandolf i and K w a a n (1967) and Pandolf i and A$trup (1967) of the presence of f ibrinolytie ac t iv i ty in the region of the canal of Schlem and the collector vessels. The selective localization of fibrinolytic ac t iv i ty was shown using a modification of Todd 's his tochemical technique of fibrinolysis au tographs (Todd, 1959).

The present series of perfusion experiments was carried ou t in order to determine whether plasmin could al ter the faci l i ty of outflow in the eyes of l iving monkeys. The drug was injected in to the anter ior chamber and the faci l i ty of outflow determined by means of cons tan t pressure perfusion.

2. Experimental Procedure

Heal thy male cynomolgus monkeys (Macaca irus), weighing between 3.4 and 9 kg, were used in this study. The monkeys were carefully examined to exclude any general or

* Address for reprints: :E. S. :Perkins, Ins t i tu te of Ophthalmology, Judd Street, London, W.C.I., England.

T Supported by a Fellowship from the Hayward Foundation.

3 8 6

P L A S S I I N A N D I , ' A C I L I T k " O F O U T F L O W I N M O N K E Y S 387

ocular pathology. General anaesthesia was used in all experiments. Veterinary Nembutal , 60 mg/kg body weight, was injected intraperitoneally as the initial dose. This usually sufficed for the durat ion of the experiment, but iu some cases it had to be supplemented by further intravenous increments.

A femoral vein was cannulated and heparin (4000 units) was administered intraven- ously at the beginning of the experiment. Next, the femoral artery was cannula ted and connected to a strain-gauge pressure transducer to record the systemic arterial blood pressure. A thermistor in the left auditory canal gave a continuous recording of the head temperature, while the animal 's rectal temperature was noted periodically from a pre- cision thermometer , rmlding to 0-1°C.

TR

T| T 2

A~ A2

LE RE

s, I I I I s2

B5

B5 g 6

D B 4

B, I / I II I I s2

Z~ir bubble

FM

I,'xO. 1. Schemat ic d iagram of the perfusion appara tus .

The head of the animal was fixed in position, and the cornea kept moist by an inter- mi t ten t drip of 0"9% saline.

The flowmeter used in these experiments has previously been described by Edwards, Hal lman and Perkins (1967) and the perfusion apparatus is shown diagrammatica!ly in Fig. 1. The whole system was filled with physiological Ringer 's solution (B~rhny, 1964) except for t ha t par t of the fiowmeter occupied by Evans blue (T 1854).

Two hypodermic needles were inserted into the anterior chamber o f each eye. The first, a no. 17 (25 gauge), was connected to the flowmeter (~M) and its reservoirs (EBI~ and R.I'¢) by means of nylon and silicone rubber tubing through a 4-way glass junction. The second needle, a no. 30 (30 gauge), was connected by a T-junction (A ~, A 2) to:

388 E. S. P E R K I N S A N D H. S A I D U Z Z A F A R

(a) the Agla m i c r o m e t e r syr inges (S 1 a n d Se), a n d (b) a s t r a ln -gauge pressure t r a n s d u c e r (T 1 a n d T c) to p rov ide a con t i nuous record ing of

t h e i n t r a o c u l a r pressure. T h r e e - w a y t aps (D t nnd D ~) m o u n t e d on the t r ansduce r s a l lowed t h e m to be c o n n e c t e d to a reservoi r (TEl to p rov ide c , l ib ra t ion pres,uures.

Six bu l ldog a r t e r y forceps (B 1, B", B 3, B 4, B s a n d B ~) were cl ipped onto the soft s~licone r u b b e r tub ing . Select ive r e m o v a l of these clips e n a b l e d the flow of fluid in t he f lowmeter a n d its t ub ing to be con t ro l led as desired. Clips B ~ and B 8 were used to discom~ect the syr inges S 1 a n d S e while t h e y were n o t in use.

The b o d y t e m p e r a t u r e of t he m o n k e y was k e p t as c o n s t a n t as possible du r ing the e x p e r i m e n t b y the use of a r egu l a t ed hea t i ng p la te i nco rpo ra t ed in to the ope ra t i ng table.

H e a r t and r e sp i r a to ry ra tes were coun t ed d u r i n g e a c h set of perfusions as a check on the genera l condi t ion and v a s o m o t o r s tab i l i ty of the a n i m a l dur ing the e x p e r i m e n t .

Fo l lowing the inser t ion of t he two needles in to each anter io~ c h a m b e r the i n t r a o c u h r pressure was a l lowed to s tabi l ize for a few m i n u t e s unt i l , w h e n a : s t eady s t a t e w~Ls reached , pe r fus ion was begun . I n t h e first four exper imel l t s of this series per fus ion pressures of 4 a n d 6 m m H g above t h e p reva i l i ng i n t r a o c u l a r pressure were used. H o w e v e r , i t was sub- s e q u e n t l y f o u n d more sa t i s fac to ry to perfuse a t pressure differe,~tials of 4, 6 and 8 m m H g respec t ive ly , a n d th is was t h e p rocedure f , iJowed in t he s u b s e q u e n t n ine exper imen t s . Fua' ther, i t was cons idered useful to r a n d o m i z e t he side of the ini t ia l perfus ion.

:Following these in i t ia l per fus ions a t t h e p r e d e t e r m i n e d pressure different ials , t he eyes were a l lowed a few m i n u t e s to s tabi l ize before t h e in jec t ion of t,t)e d rug or cont ro l solut ion i n t o t he an t e r io r c h a m b e r . I n th is case, also, t,t~e select ion of eyes for i f i iect ion was ,:,n a r a n d o m basis, so t h a t in some e x p e r i m e n t s the cont ro l so lu t ion was in j ec ted first and i~ o thers the drug .

Tl~e m e t h o d of i n j ec t i on was as follows: The level of the R i n g e r reservoi r of the t l owmete r ( R R ) : w a s set to the i n t r aocu l a r

pressua-e of t he eye to be in jec ted , as shown on t h e recorder . I f for e x a m p l e t he r i gh t eye was to be in jec ted , bu l ldog chps B e, B 4, B 6 a n d B 8 were r e m o v e d in t h a t o rder a n d 50 #l of d r u g or cont ro l so lu t ion i n j e c t e d over 8-12 rain. This p rocedure enab led the i n t r a o c u l a r pressure to be m a i n t a i n e d close to its n o r m a l va lue while the eye rece ived the injectiola, a t t h e end of w h i c h the clips were rep laced in the reverse o rder (B 8, B 6, B a a n d BZ).

A t the end of t he in jec t ion t he in t ra .ocular pressures were observed for a few m i n u t e s and a l lowed to s tabi l ize before t he n e x t perfus ions were s t a r t ed . The p rocedu re was t hen re- p e a t e d for t h e o the r eye wh ich was s imi la r ly in j ec ted a n d t hen perfused. In order to al low sufficient t ime for t h e full ac t ion of t he d rug to become a p p a r e n t t he perfus ions were r e p e a t e d 2 or 3 t imes in each eye, so long as the cond i t ion of the a n i m a l r e m a i n e d satis- factoi~y.

The size of t he pupi ls was n o t e d a t the c o m m e n c e m e f f t of t he e x p e r i m e n t , a n d aga in a f t e r eve ry set of perfusions, as well as a f t e r the a d m i n i s t r a t i o n of the drug .

A t the end of t h e ex~periment t he m o n k e y was ki l led by an overdose of i n t r a v e n o u s N e m b u t a l . W h e n this was i nd i ca t ed t he eyes were r e m o v e d for s u b s e q u e n t his tology.

Drugs

Lysof ibr in (Novo) , w h i c h is a n ac t iva to r - f ree l y o p h i l h e d porc ine p lasmin , was suppl ied b y N o v o I ~ d u s t r i as a s ter i le f reeze-4r ied p o w d e r in bo t t l es c o n t a i n i n g 600 Novo uni t s (NU) pe r bot t le . B a t c h e s 1"06 a n d 4*35 were suppl ied in i t ia l ly , b u t la ter , when i t b e c a m e avai lab le , some " i m p r o v e d " lysof ibr in (no. 5*38) was used. The con t en t s of e a c h bot t le were dissolved in Owren ' s buffer (Biggs a n d Macfa r l ane , 1962) to o b t a i n a c o n c e n t r a t i o n of 20 NU/m] . This s tock so lu t ion was s to red in t h e deep freeze ( - -20°C) in smal l a m p o u l e s to prevent frequent thawing and refreezing of the whole of the stock solution. At the time of the experiment the contents of one ampoule were brought to room temperature and d i lu t ed w i t h ~ i n g e r ' s so lu t ion to a c o n c e n t r a t i o n of 10 N U / m l .

P" F,~' l jtJ uu!~,,j,n! ,...q! -~ f do~,~ ouo .qlu,~o.~(lo.t ,~u!o~:.rl .valoui.~xoll otl~ .1o .~uo!l.lorl I~lt,)z!.:o~{ llUUL~ o.~x I u~',.).~xaoq pt.x.v;)lu! ;uLi ̀ "sp.X:~l ~.ln~sa.ld oaJtll '1~: saA'a ,UoI l)~tu .lti~!J ,3111 jo i.l()!s~'lj.lad ~tl!.~lm|.,-; 'g;t!|)j();)a.i lu;)!,Lx'l u jo l.Jv([ "1 :lJ.~"ld

rj I

: P L A S h I I N A N D I ~ A C I L I ' I ? Y O F O U T . F L O W I N M O N K E Y S 389

Human plasmin was obtained from Kabi Plmrmaceuticals as a glycerol-activated freeze-dried powder in bottles contai~ing 25 casein units (CU) per bottle (Plasmin Kabi, Grade A, human lyophilized). This was dissolved in distilled water to obtain a solution of 10 CU/ml.

Control solutions

In exi)erimen'ts using lysofibrin the injection into the control eyc consisted of 50 t*l of a

solution containing equal parts of Owren's buffer and Ringer 's solution. Where Plasmin Kabi was used, the control injection was 50 tel of the Ringer 's solution.

Ccdculations

The pressure t ransducers for the blood pressure and the in t r aocu la r pressure were ca l ibra ted a t the begbln ing and end of each exper iment .

A typ ica l recording is seen in P la te 1. Each step of the f lowmeter recording repre- sented a volume of 1.82 tL1, a n d the t ime t a k e n for a n y ' g i v e n n u m b e r of s teps could be read off the t ime marke r a t the edge of the paper .

Thus the coefticient of fac i l i ty of outflow, C, could be ca lcu la ted for eve ry set of perfusions using the formula :

V C =l, xA15

where I.: --- vo lume of fluid (/A), T = t ime (rain),

A p = pressure different ial du r ing the perfusion (mmHg) . The volume (/A) was ca,lculated by mu l t i p ly ing the n u m b e r of s teps by 1,82.

Sources of error in the flowmetcr

(1) Var ia t ions in i l lumiuatior , of different par t s of the photocel l cause s l ight dif- ferences in sens i t iv i ty in par t s of the f lowmeter coil, so t h a t changes in the photocel l ou tpu t m a y not be qui te cons t an t over the whole coil. This however is no t s ignif icant because the d is tance from one m a r k to the n e x t on the record ing will st i l l represent a flow of ]-82 tel of fluid into the eye.

(2) Var ia t ions in calibre of the ny lon tube would cause errors, b u t p rovided t h a t the tune is coiled careful ly w i t h o u t undue tension, such errors are no t impor t an t .

(3) The res is tance to outflow in the tubing, t hough present , is small , so t h a t a rap id flow was ob ta ined from a pressure d i f ferent ia l of 0.25 manIIg be tween the two ends of the coil.

Results

The resul ts p resen ted here are based on the f indings f rom 13 cynomolgus monkeys . A few p re l im ina ry exper iments , which were carr ied ou t to es tabl i sh the o p t i m u m dose of N e m b u t a l anaes thes ia and the effective concen t ra t ion for the intJ:acameral p l a sndn injections, are n o t included.

The coefficient of fac i l i ty of outflow (C) i n / A ] m i n / m m H g was ca lcula ted f rom the recordings of each expe r imen t (P la te 1). The mean values for C before and af te r the injeet io~ of p lasmin , toge the r w i t h the i r s t a n d a r d dev ia t ions a n d s t a n d a r d errors of ~he m e a n for each of the pressure different ials are shown in Table I. The t t e s t of sigv-ificance between hl i t ia l C (X1) and final C (X2) in the plasnf in group was h igh ly

390 E . S . P E R K I N S A N D H . S A I D U Z Z A F A R

significant a t the 1% level for pressure differentials of z~P --- 4 and ziP -~ 6 mmHg, and a t the 5°/o level for LiP -- 8 mmHg. Table I I s imilar ly gives the mean values for in i t ia l and final C on the control side, where the t test showed no significant difference between them. Figures 2 and 3 are scat tergrams showing the relat ionship between all in i t i a l and final C values on the plasmin and control sides respectively. The distribu- t ion of the points in relat ion to the " l ine of no change" clearly shows the difference in response of the two eyes.

TABB~ I

Mean initial a , d final C ~alues (plasmin side)

P l a s m i n I n t i t i a l C (XI ) F i n a l C (X~)

~ Iean 3 I e a n ,_lp v a l u e s v a l u e s t t e s t

( m m H g ) n (C) S.D. S.Z.~I. (0 ) S.D. S.~..~L X l , X2 1"

4 13 0.43 0-18 0.05 0.76 0 .30 0-08 3.47 ~.0 .01 6 13 0.39 0.15 0 .04 0-72 0 . °6 0.07 3-92 <:0-01 8 9 0.44 0-15 0.05 0-73 0 .27 0.10 2.72 < 0 . 0 5

I n t h i s a n d t h e foUowing t a b l e s : n = n u m b e r o f r e a d i n g s f r o m w h i c h t h e m e a n s were ob ta ine~ l ; 8.D. ~ s t a n d a r d d e v i a t i o n ; s.w..M. ~--- s t a n d a r d e r r o r o f t h e m e a n ; A ~ ~ p r o c u r e d i f f e r en t i a | a t which per fus iorm were ca r r i ed o u t ; P = p r o b a b i l i t y .

TABBB I I

Mean initial and fi~al C values (control side)

Cont ro l I n i t i a l C (X1) F i n a l U (Xe)

~ I ean ~Iean A p v a l u e s v a l u e s t t e s t

( m m ~ I g ) n (0) S.D. S.~..~l. (C) s .o . S.~:..M. ~'1, X~ P

4 9 0-44 0"17 0-06 0-44 0 . I7 0-06 0-06 ~ 0 - 5 6 8 0-43 0-16 0-05 0-43 0"15 0.05 0"03 -~0-5 8 6 0-44 0-11 0"05 0-45 0.12 0.05 0-10 ~ 0 - 5

The changes wi thin each ind iv idua l eye are set ou t in Tables I I I , IV and V, and the s ta t i s t ica l analysis showed t h a t the difference between the response of the plasmb~ eye and the control eye was highly significant a t the 1~/o level for all the three pressure differentials. JFrom these tables and f rom ~igures 2 and 3 i t can be seen t h a t on the p lasmiu aide the values for final C were a lmost i nva r i ab ly higher t h a n for ini t ial C, whereas on the control side the differences were very small and no t consistent.

The rat ios between the mean values for C on the p lasmin side and the control side were also calculated. As seen in Table VI, the in i t ia l values (before injection} in the two eyes were very similar, and t h e ra t io of the outflow in the p lasmin eye to t h a t in the c o n ~ o i eye var ied between 0.92 and 9-99. However , after inject ion the plasmin

I ' L A S , M I N A N D F A C I L I T Y O F O U T F L O W I N M O N K E Y S

TABLE I l l

Effect of plasmin and control i,zjections on facility of outflow at z ip of 4 mmHg

391

J l " 4 m m I - I g

t ' l a s m i n s i d e F i n a l G m i n u s i n i t i a l G ( X 0

C o n t r o l s i d e F i n a l C m i n u s i n i t i a l G ( X 2 )

.n~--- 13

0 . 3 8 - - 0 - 0 5 0 -39 0 . 0 2 9 - 4 0 0 . 0 4 0 . 1 8 0 . 0 0-34 ~ 0 -03 0 -38 - - 0 . 1 6 0-04 - - 0 - 0 3 0"23 0"0 0 .33 0 . 0 5 0 .0 0 . 0 0 . 7 5 0 .0 0 . 6 0 0 -04 0"41 0 . 0

.Mean ~ 0-37 M e a n ~ - - 0 " 0 2 S.l) . ~ 0-18 s .D. == 0 . 0 7 s .~ .~I . ----- 0"05 s.>:..~. ~ 0 . 0 2 t te,~t v a l u e ---:- 6 . 9 9 t t e s t v a l u e ~ - - 0 - 6 1 t t e s t o f s i g n i f i c a n c e b e t w e e n X t, X 2 --~ 5-6:{

t " .< 0 .0 [

' ] ' a ?3 L~: I V

I'2ffect of plasmin a~l control injection,s o~ the facility of outflow at ZIP of 6 mmHg

A P (0 m m l t g )

L ' l a s m i n sidt~ F i n a l C minu.~ i n i t i a l U ( X 1 )

C o n t r o l s i d e F i n a ! C m i n u s i n i t i a l C ( X 2 )

n ---~ 13

0 . 4 0 0"13 0 . 3 2 0"04 0 -37 0 ' 0 5 0-21 0"0 0-41 - - 0 " 0 6 0 -29 - - 0 " 2 0 0 -13 0"03 0 -18 0 -0 0 -27 0 -0 0-27 0 . 0 0 . 8 4 0-0 0 . 4 9 - - 0 - 0 4 0 -16 0 -03

5 I e a n ---- 0 -33 M e a n -~-- - - 0 . 0 0 2 S.D. ~--- 0 - 1 9 S.D. ~ 0"10 S.~.~r. --~ 0 -05 S.E.~I. = 0 -03 t t e s ~ v a l u e ~ 6 . 4 9 t t e s t v a l u e ---- - - 0 - 0 7 t t e s t o f s i g n i k l c a n c e b e t w e e n X x , X 2 -~- 4 - 7 0

2 : ' < 0 . 0 1

392 E . S . P E R K I N S A N D t I . S A I D U Z Z A : F A R

TAl~ ".E V

Effect of pla.~m, in and control injections on the favility of outflow at .4p of 8 mmHff

~ P 8 m m H g

P l a s m i n s i d e F i n a l C m i n u s i n i t i a l C ( X 0

C o n t r o l s ide .Final C m i n u s i n i t i a l C ( X 2)

n~9

0.35 - - 0 . 0 O 0 .19 - - 0 - 1 2 0-0 0 .05 0-30 0-0 0 .19 0-06 0 .17 0 .0 0-7t) 0-0 0 .28 0-0 i 0 .16 0. I0

i~lean == 0-30 M e a n ------ 0.01 S.D. -~- 0.21 S J ) . --= 0-08 S.~.M. ~ 0-07 ,~.~.,~t. = 0.03 t test. v a l u e =--- 4-13 I t c , t v a l u e --.= 0--'20 t test; o f s i g n i f i c a n c e b e t w e e n X t , X2 --= 3 .28

P < 0 - 0 1

| ' 4 - • o

1 . 2 -

o

" T a t ,

• " : " / 1

/ ' : . / I

0 0 - 2 0 - 4 0 . 6 0 - 8

Initiol C - volue$ ( p i / n ~ n per mmHg)

F r o . 2, R e l a t i o n s h i p o f i n i t i a l C t o Final C o n t h e p l a s r n i n s ldo.

P L A S M 1 N A N D F A C I L I T Y O F O U T I " L O W i N M O N K E Y S 393

values were in each case higher than those of the controls so t h a t the ratios varied betwee, n 1-64 and 1.74.

. '~ 0.8: "I" E

0 . 6 e -

E

=" 0 . 4 t o

' 0"2

0

h

0

- O O_O O O ~ 0

o o

0 .2 0"4 0 - 6 0"7

Init ial C - v a l u e s ( p l / m i n per mmHg )

I, h c . 3. I l e l a t i o n s h i p of in i t i a l C (.o final C on the com.rol side.

Thr(mghout the exper iment the in t raoeular pressures were being recorded mane- metrically as seen in Plate 1. Table VII gives the mean values for the s teady-s ta te pressures (Po) on the plasmin side and t, hc control side, before injec.tion (initial) and after injection (fiual). together with the stat ist ical analysis. The table shows tha t al though plasmin caused a small rise in the overall in t raocular preasure, the rise was not s tat is t ical ly significant. On the control side the mean pressures were sl ightly lower after injection bur. the difference was not s tat is t ical ly significant.

TA BLII: Vii

Ratios of pla.~min C to control C before an.d after injectian

.Mean in i t i a l G Me.~n f inal C (Before i n j ec t i on ) (Af te r i n j e c t i o n )

R a t i o R a t i o J P Contzx)i eye P l a s m i n eye p l a s m i n / c o n t r o l Cont ro l eye P l a s m i n eye p l a s m i n / c o n t r o l

4 0-44 0"43 {)-96 0"44 0-76 1-74 6 0-43 0"39 0"92 0-43 0.7.'2 1 "69 8 0.44 0-44 0-99 0-45 0"73 1-64

I t was found t h a t the pupils usual ly contracted slightly after the ini t ia l perfusion; this was t hough t to be due to the cooling effect of the Ringer 's solution, which was a t room temperature,, several degrees below t h a t of the eye. Somewhat g-reater difl~erences in pupil size were noted after the plmsmin injections, and the pupi l lary diameters were therefore noted before and after the plasmin and control inject ions and were com- pared (Table VIII ) . The graphs (Figs 4 and 5) show t h a t there was a greater var ia t ion in pupil iary d iameter on the plasmin side, and the change was more often towards di la ta t ion t han constrict ion.

394 E . S . P E R K I I ~ S A N D H A M I D A S A I D U Z Z A F A R

TABLE V I I

M e a n initial and 'fi,,wd Po values on the 19lasmin and control sides, ,'espectively

, d P

P l a s m i n s ide I n i t i a l Po (Ix) Final Po (X,)

t t e s t n l~fe~n S.D. s . ~'-.M. ~ I e a n S.D. S.F,.M. .X~, ~X7 2 P

4 6 8

13 14-43 2-65 0-~4 15-89 4 .44 1-23 1-01 0-2 < P < 0 - 3 1:3 14.47 2-70 0.75 15-89 4.44 1-23 0-99 0 -3-< P <20"4

8 I3 -69 2-65 0-94 15-13 5-56 1.96 0"66 P < 0 . 5

C o n t r o l s ido 9 14"53 3-69 1.23 13-85 3 ' 8 5 1-28 0-38 1' > 0-5 9 14"53 3-69 1-23 13"85 3-85 1.28 0-38 P > 0-5 6 13.17 3-37 1-38 12-38 2"81 1-15 0-44 P > 0 - 5

5 . 0

4 . . 0

g

2 " 0

I - 0

0

• Q •

. . . . I i . . . . I , 1 . i ,

I - 0 2"0 ~'0 4"0 5"0

B e f o r e i n j e c t i o n ( m m )

P u p i l l a r y d i a m e t e r s be fo re a n d a f t e r p l a s m i n injee%ion.

4 -O

E 3-0 E t-

O

2-0 -~

0 I - 0 2 - 0

! 3-0 4;0 5-0

B e f o r e i n j e c t i o n ( r a m )

FIG. 5. P u p i l l a r y d i a m e t e r s b e f o r e a n d a f t e r c o n t r o l i n j e c t i o n .

P L A S M I N A N D F A C I L I T Y O F O U T F L O W I N M O N K E Y S

TA~r.~: V I I I

Pz~pillary diameter before and after injection of plasmin and control solution

395

S. NO.

P l a s m i n C o n t r o l :Before A f t e r B e f o r e A f t e r

i n j e c t i o n i n j e c t i o n i n j e c t i o n i n j e c t i o n ( r a m ) ( r a m ) ( m m ) ( r a m )

1 4 .0 3-5 4-0 3"5 2 2-5 3"5 - - - - 3 3 .0 1"6 3"5 2-2 4 3"0 2.8 2-8 3-0 5 3,2 3-8 - - - - 6 2-5 3"0 2 .5 2-6 7 2.'2 2-6 1 -S 1 -S 8 '2-2 3"4 2"0 2-2 9 3-6 2 .8 - - - -

10 3"2 2"8 - - - - 1 1 a 2"'2 2-6 - - - - 11 b 2"0 3"4 - - - - 12 3"4 3-4 2"8 2"6 13 3"0 4"4 3-2 3"8

3. Discuss ion

]n this series of experiments the coefficient of facil i ty of outflow was significantly increased in the eye which received the plasmin injection, while no such increase occurred in the control eye. In some of the prel iminary experiments perfusions were repeated every 15-20 rain in an a t t e m p t to follow the t ime course of the effect. However, dur ing- th is pro.c~ss tim enzyme was cons tan t ly being diluted as well as replaced by Ringer ' s solution coming into the anter ior chamber from ~he reservoir with each set of perfusions, and this may have affected the results. F rom the rap id i ty with w h i c h plasmin .in similar concent ra t ion began to exert its p re t ense ' ac t iv i ty in vitro o n fibrin plates i t was es t imated t h a t i t shotfld become effective approxi- mate ly 30 rain after t h e eye had stabilized fblIowing the injection. This, in fact, was found to be the case, and the g~eatest incr.eases were found to occ~ur 30 to '90 rain after the plasmin injection. Thereafter the effect was variable, largely depending on the condition of the monkey and the depth of the anaesthesia. The max imum durat ion of effectiveness in this series was 133 rain.

As was to be expected, 50 /~1 of Ringei-'s solution or buffer, or hea t - inac t iva ted plasmin, injected into the control eye had no significant dffeet on the facil i ty of out- flow. The ex~periment ~dth the heated plasmin, which had previously been tested on a fibrin Plate to ensure t h a t it had lost its proteolyt ic act ivi ty , was carried out in order to confirm t h a t the increase in faci l i ty of outflow was actual ly due to the fibrinolytic properties of the plasmin.

These experiments thus show t h a t the effect of plasmin on the faci l i ty of outflow. or outflow resistance, found by Pandolfi and Astrup (19.66) and by Pandolfi (1967) in eaucleated monkey eyes, occurs also in the eyes of l iving monkeys (Maraca irus), and the change appears to be of a similar maglfi tude (Table VI). The similar i ty in the act ion of plasmin on the living and dead eye is probably due to the fact t ha t the

396 E. S. PEI~]~INS AND H. S A I D U Z Z A F A P ~

fibrinolytic activity of tissues is known to survive following the death and degenera- tion of cells; this hsz been demonstrated in the ceils of the corneal epithelium and endothehum, which were inactive when intact but showed marked fibrinolytic activity foUowing injury (Pandolfi and Astrup, 1967).

I t was somewhat su~rprising, however, tha t in spite of corLsistent increases in the facility of outflow (C), plasmin did not cause any fall in intraocular pressure (Po), the mean values for which were in fact slightly higher in the plasmin eye (Table VII) ; this rise was not statistically significant, and was apparently due to the rise in systemic arterial pressure caused by the earlier batches of lysofibrin (Fig. 2) because it was less evident when improved lysofibrin was used (Fig. 3)~ and did not occur at all with the

k human plasmin (Fig. 4). However, even ~n the latter groups of monkeys plasmiu did not cause the lowering of intraocular pressure which might have been expected from the magnitude of the increase in facility of outflow. Although the exact mechanism of the plasmln effect is not known, a possible explanation may be theft the same process which caused a lowering of the outflow resistance caused a parallel increase in aqueous formation. Another possible explanation is that the increase in total fi~cility which is measured by this perft~sion technique may be due to an increase in "pseudo-filcility" (B~r~ny, 1966). Such an increase in pseudo-facility could only account for part of the increase in total facihty observed.

Changes in the diameter of the pupil following plssmin injecVions did not follow any regular pattern, though it was observed that pupillary dilatation occurred more often than constriction (Table VIII , Figs 4 and 5). Since the pupils were sometimes observed to dilate after the control injections also, no grea~ significance could be attached to this finding.

ACKNOWLEDGMENTS

We are very grateful to Messrs Novo Industri A/S, London, for their kind co-operation and generous supplies of lysofibrin and improved lysofibrin.

We wish to thank Mr g. :Edwards for his technieaI assistance, Mr B. F. Augier for the computer programming and statistical analysis, and Mrs K. Loosen for secretarial help.

R E F E R E N C E S

B~rAny, E. H. (1964). Invest. O ohthalmol. 3, 135. BA14ny, E. H. (1966). In G/aueoma. Tutzing Symposium. (Ed. by Leydhecker, W.) Karger, Basel,

1967. Biggs, R. and ]~Iacfarlane, R. G. (1962). Human Blood Coagulation. Blaekwell Scientific Publica-

tions, Oxford. Edwards, J., HaUman, V. L., and Perkins, E. S. (1967). Exptl Eye Ties., 6, 316. Kwa~n, H. (3. and Astrup, T. (1963). Arch. Pathol. 76, 595. Pandolfi, •. (1967). Am. J. Oph2halmoL 64, 1141. Pandolfi, M. and Astrup, T. (1966). Prov. 8oe. 2~xTtl ~iol. Med. 121, 139. Pandolfi, .hi. and Astrup, T. (1967). Arch. OavhthalmoL (Chicago) 77, 258. Pandolfi, M. and Kwaan, H. C. (1967). Arch. Ophthalmol. (Chicago) 77, 99. Todd, A. S. (1959). J. Pathol. Bacteriol. 78, 281.