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A new method for the determination of the swelling pressure of the corneal stroma in vitro
Exp. Eye Res. (1963) 2, 122-129
A N e w M e t h o d for t h e D e t e r m i n a t i o n o f the S w e l l i n g P r e s s u r e o f the C o r n e a l S t r o m a i n v i t r o * BENGT O. H]~D]3YS'~ .AND CLAES H. DOnLMA~N
CorJ~eal Research Unit, Depart~wnt of Clinical Eye Research, Institute of Biological and Medical Scie~wes, Reti~a Fou~Mation, Boston, Massachusetts, U.S.A. (Received 19 January .1963) A n e w m e t h o d f o r t.he d e t e r m i n a t i o n o f t h e s w e l l i n g p r e s s u r e o f t h e c o r n e a l s t r o m a a n d sclcr,% h a s b e e n d e v e l o p e d . A t i s s u e b u t t o n a t a n y d e g r e e o f h y d r a t i o n is p l a c e d b e t w e e n t w o g l a s s filters. T h e u p p e r f i l t e r is m o v a b l e a n d co,~.nected t o a capa, c i t a n c e t r a n s d u c e r ; t h e l o w e r filter is f i x e d . W h e n 0'9~/o iNaC1 is b r o u g h t in c o n t a c t w i t h t h e t i s s u e , s l i g h t s w e l l i n g o c c u r s a n d t h e f o r c e is r e c o r d e d . T h e s w e l l i n g is r e s t r i c t e d c o n s i d e r a b l y b y t h e t r a n s d u c e r . T h e r e f o r e , a s t e a d y f o r c e is r a p i d l y r e a c h e d , u s u a l l y w i t h i n 30 mh~. Experiments were carried out on steer, rabbit and human corneal stroma and on steer sclera o v e r a wide r a n g e o f laydrat.ions. T h e swelling p r e s s u r e o f t h e n o r m a l c o r n e a l s t r o m a , w a s f o u n d t o b e a b o u t 60 m m H g a n d t h a t o f t h e n o r m a l s c l e r a a b o u t 17 m m I:[g. T h e s w e l l i n g p r e s s u r e o f t h e s c l e r a s h o w e d a g r e a t e r d e p e n d e n c e o n h y d r a t i o n t h a n did that of the corneal stroma.
1. Introduction
3 I e a s u r e m e n t of t h e swelling presstn'e, i.e. tile mechallical pressure applied to tile e x t e r n a l surfaces of a tissue t h a t ]?revents it from swelling while in a solution, offers a useful a p p r o a c h to studies on corneal d e h y d r a t i o n . T h e swelling p r o p e r t i e s can t h u s be c h a r a c t e r i z e d b y a pressure r a t h e r t h a n b y t h e a m o u n t of w a t e r t h a t t h e tissue can t a k e up. These memx,,rements can be carried o u t a t all stages of swelling, a n d t h e y are f a c i l i t a t e d b y t h e f a c t t h a t t h e cornea swells o n l y in a direction p e r p e n d i c t d a r to its mLvfaees. F r o m t h e swelling pressure m e a s u r e m e n t s , t h e fluid u p t a k e a t various stages of h y d r a t i o n and, c o n s e q u e n t l y , t h e load on t h e d e h y d r a t i o n m e c h a n i s m can be calculated. :By c h a n g i n g t h e t e m p e r a t u r e , h y d r a t i o n , p H , ionic s t r e n g t h , etc., Jnformation can be o b t a i n e d a b o u t t h e n a t u r e of t h e forces i n v o l v e d in t h e swelling process. :In t h e first r e p o r t e d m e a s u r e m e n t s of swelling pressure ( K i n s e y a n d Coghn, 1942), corneal sweili,lg was r e s t r i c t e d b y plach~g v a r i o u s weights on t o p of t h e tissue durh~g i m m e r s i o n in aqueous solution. I n t h i s way, a pressl~re was f o u n d w h i c h k e p t t h e cornea a t i t s original h y d r a t i o n . I n t h e e x p e r i m e n t s b y P a u (] 954), t h e swelling of t h e cornea a n d sclera was res t r i c t e d b y t h e colloidal osmotic pressure of t h e solution in w h i c h t h e tissues were immersed. T h e osmotic pressm'e was r e g u l a t e d b y v a r y i n g t h e c o n c e n t r a t i o n of an i n e r t macromolecule: viz. p o l y v i n y l p y r o l l i d o n e (I~VP), in a saline solution. T h e swelling pressure of t h e tissue was a s s u m e d to equal t h a t colloidal osmotic pressure which did n o t change t h e h y d r a t i o n of t h e tissue, l~au's t e c h n i q u e was modified b y D o h l m a n a n d A n s e t h (1957), who s t u d i e d t h e w e i g h t c h a n g e s of isolated cortical s t r o m a a n d * This i n v e s t i g a t i o n was s u p p o r t e d b y a P H S r e s e a r c h g r a n t (B-2220) f r o m t h e N a t i o n a l I n s t i t u t e o f ~ e u r o l o g i c a l Diseases a n d :Blindness, U.S. Public H e a l t h Service. This i n v e s t i g a t i o n was c o n d u c t e d u n d e r a F i g h t F o r S i g h t F o s t - D o c t o r a esearch F e l l o w s h i p o f t h e N a t i o n a l Council to C o m b a t ]31indncss, I n c . , New Y o r k , X e w York. 122
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s c l e r a e n c l o s e d in d i a l y s i s b a g s s u b m e r g e d i~.~ d e x t r a n s o l u t i o n s o f v a r y i n g o s m o t i c pressure. The methods cited gave measurements of limited accuracy, and they were also t i m e - c o n s u m i n g , s i n c e i t o f t e n t o o k a s l o n g a s t w o d a y s b e f o r e a n e q u i l i b r i u m wa.s r e a c h e d b e t w e e n t h e t~ssue a n d t h e s o l u t i o n . A r a p i d a n d m o r e a c c u r a t e m e t h o d h a s therefore been designed for the determination of the swelling pressure.
2. M a t e r i a l s and M e t h o d s
PreparaHon of ,~pecimens C o r n e a l s t r o m a s f r o m s t e e r s a n d a l b i n o r a b b i t s w e i g h i n g °~-3 k g w e r e p r e p a r e d b y s c r a p i n g off t h e e p i t h e l i u m , e n d o t h e l i u m a n d 1 ) e s c e m e t ' s m e m b r a n e w i t h i n 4 h r a f t e r t h e d e a t h of t h e a n i m a l . T h e s t r o m a w a s t h e n u s e d i m m e d i a t e l y or w a s f r o z e n a t -- 15°C a n d s t o r e d ~b t h e s a m e t e m p e r a t u r e for u p t o 3 w e e k s . N o d i f f e r e n c e w a s f o u n d b e t w e e n t h e s w e l l i n g p r e s s u r e o f t h e t h a w e d t i s s u e a n d a n d t h a t o f t h e fresh. H u m a n c o r n e a s w e r e e x c i s e d f r o m e y e s t h a t h a d b e e n s t o r e d a t - - 7 9 ° C for a c o n s i d e r a b l e t i m e . The s t r o m a w a s p r e p a r e d in t h e s a m e m a n n e r as t h a t of t h e s t e e r s a n d r a b b i t s . V a r i o u s h y d r a t i o n v a l u e s w e r e o b t a i n e d b y e v a p o r a t i o n of t h e s t r o m a s a t r o o m t e m p e r a t u r e ( 2 0 - 2 5 ° C ) or b y d r o p p i n g 0 " 9 % NaC1 o n t h e s u r f a c e s . T h e t i s s u e s w e r e t h e n s t o r e d a t 4°C for a t l e a s t 5 h r t o p r o d u c e u n i f o r m h y d r a t i o n . P i e c e s of s c l e r a of e v e n t h i c k n e s s w e r e e x c i s e d f r o m t h e e y e s o f s t e e r s w i t h i n ,t hr a f t e r s l a u g h t e r , a n d were c a r e f u l l y s c r a p e d t o r e m o v e a n y a d h e r i n g r e m n a n t s o f t h e c h o r o i d . T h e y w e r e t h e n u s e d i m m e d i a t e l y or f r o z e n a n d s t o r e d in t h e s a m e m a n n e r as t h e c o r n e a l s t r o m a . Various h y d r a t i o n values were o b t a i n e d for t h e sclera using t h e m e t h o d e m p l o y e d for t h e c o r n e a l s t r o m a . B e f o r e t h e s w e l l i n g p r e s s u r e w a s m e a s u r e d , c i r c u l a r b u t t o n s , 13"5 m a n (steer s t r o m a a n d sclera), 9"0 m m ( h u m a n s t r o m a ) a n d 6"5 m m ( r a b b i t s t r o m a ) in d i a m e t e r , w e r e p u n c h e d o u t of t h e s p e c i m e n s . I n a c o n t r o l series o f e x p e r i m e n t s , d e s i g n e d t o c o m p a r e t h e effects of t h e b u t t o n sizes o u t h e p r e s s u r e m e a s u r e m e n t s , discs o f all t h r e e sizes w e r e p r e p a r e d from the steer stroma.
]'[ a~o meter A Sanborn Model 121B electromanometer,* a capacitance manometer, was used. The ~ransduc.er w a s m o u n t e d on a m i c r o s c o p e s t a n d (Fig. 1) a n d c o n n e c t e d t o t h e b r i d g e h o u s i n g w i t h a c a b l e a c c o r d i n g to t h e m e t h o d o f R a p p a p o r t , B l o c k a n d I r w i n (1959). The signals from the electromanometer were amplified by a Sanborn ])C Coupling Preanaplifier ( M o d e l 1 5 0 - 1 3 0 0 ) a n d w e r e r e c o r d e d w i t h a S a n b o r n R e c o r d e r ( M o d e l ] 5:~-100B). T h e t r a n s d u c e r h a d t w o p a r a l l e l m e t a l p l a t e s a b o u t 25g. a p a r t . T h e u p p e r p l a t e w a s i m m o b i l e , w h e r e a s t h e l o w e r o n e w a s s l i g h t l y flexible. W h e n p r e s s u r e w a s e x e r t e d o n t h e l o w e r p l a t e , i t b e n t t o w a r d t h e u p p e r , a n d t h e c a p a c i t a n c e of t h e . t r a n s d u c e r i n c r e a s e d . T h e c h a n g e s in c a p a c i t a n c e w e r e c o n v e r t e d b y t h e c i r c u i t r y i n t o v o l t a g e s p r o p o r t i o n a l to t h e p r e s s u r e s . T h e e l e c t r o n i c e q u i p m e n t r e m a i n e d s t a b l e for a l o n g t i m e . L i n e a r r e c o r d i n g s o f f o r c e s u p to t h e e q u i v a l e n t of 500 m m I-Ig p r e s s u r e w e r e o b t a i n e d w h e n c o r n e a l b u t t o n s 13-5 m m in d i a m e t e r w e r e u s e d . B e c a u s e o f p o s s i b l e d a m a g e t o t h e i n s t r u m e n t , h o w e v e r , t h i s t r a n s d u c e r is n o t r e c o m m e n d e d f o r m e a s u r e m e n t s o f f o r c e s e q u i v a l e n t to m o r e t h a n 350 m m :Fig p r e s s u r e u s i n g s p e c i m e n s of t h i s size.
Measurements of swelling pressure T h e t i s s u e b u t t o n w a s p l a c e d on a glass filter disc t o w h i c h a b r a s s r i n g w i t h a t h i c k n e s s o f 2-5 m m h a d b e e n g l u e d (Fig. 1). A s e c o n d glass filter disc, d e s i g n e d t o fit w i t h i n t h e b r a s s * Sanborn Company, Waltham, 5[ass.
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ring, was t h e n p l a c e d olt top of t h e tissue. Since tissue b u t t o u s o f t h r e e sizes w e r e used [n tl,ese exl)erimeJ~t~% t h e b,'~,ss ri,lgs a n d t h e u p p e r tilter discs h a d to be a v a i l a b l e in thi'ee sizes, so t h a t t h e b u t t o n s could be t i g h t l y enclosed. T h e liltevs w e r e filled w i t h 0 . 9 % NaCl i , t v u c ~ o a n d s t o r e d in t h e salil~e soluSion b e t w e e n c x p e r i m e n t s . T h e filters w i t h t h e e n c l o s e d tissue b u t t o n w e r e p l a c e d in t,. f l a t - b o t t o m e d d i s h o n t h e t a b l e of tlm m i c r o s c o p e s t a n d . A t h i n a l u m i , m m r o d w e i g h i n g 3 g w a s i n s e r t e d b e t w e e n t h e
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/ ~'io. l. Illustration of the system used to measurethe pressure. The corneal button (c) is placed between glass filters (f) and within a brass ring (m). The upper glass filter is connected to the lower of the 2 metal plates (a, b) of the transducer (T). An adaptor (A) holds the transducer and a metal spring (S) connects it to the coaxial cable (O).
u p p e r filter disp a n d t h e t r a n s d u c e r . T h e t r a n s d u c e r w a s l o w e r e d b y t u r n i n g t h e m i c r o m e t e r s c r e w of t h e m i c r o s c o p e u n t i l a v e r y s m a l l f o r c e was r e c o r d e d (Fig. 2). T h e d i s h w a s t h e n filled w i t h 0.9°/o INaC1 so t h a t t h e filters w e r e c o v e r e d . T h e swelling p r e s s u r e o f t h e t i s s u e was t h u s t r a n s m i t t e d b y t h e a l u r n i n i u m r o d t o t h e flexible p l a t e o f t h e t r a n s d u c e r a n d t h e force recorded. A f t e r t h e f o r c e h a d r e m a i n e d s t e a d y for 1 5 - 2 0 rain, t h e t r a n s d u c e r was q u i c k l y raised. T h e d i s t a n c e b e t w e e n t h e r e c o r d e d s t e a d y f o r c e a n d t h e b a s e line w a s m e a s u r e d , a n d t h e p r e s s u r e w a s c a l c t r l a t e d b y c o m p a r i s o n w i t h c a l i b r a t i o n steps. A f t e r c o m p l e t i o n o f t h e e x p e r i m e n t , t h e tissue b u t t o n w a s i m m e d i a t e l y r e m o v e d f r o m t h e glass filters, b l o t t e d b e t w e e n filter p a p e r s a n d w e i g h e d . T h e h y d r a t i o n of t h e s p e c i m e n was c a l c u l a t e d f r o m t h e dr)- w e i g h t of t h e tissue a f t e r d e s i c c a t i o n for 24 h r a t 5 5 ° 0 o v e r phosphorus pentoxide in vacuo. To c a l i b r a t e t h e i n s t r u m e n t , a b a l a n c e was m o d i f i e d so t h a t one o f its b e a m s c o u l d be
S\VELLING
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125
placed under the transducer and the metal rod could be inserted in between. By increasing the weights on the opposite beam, known forces were exerted on the transducer and recorded tFi- 2). T
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Fx(~. 2. G r a p h i c i l l u s t r a t i o n o f swelling p r e s s u r e m e a s u r e m e n t . A t a r r o w I, 0 . 9 % ~NaCI is b r o u g h t i n t o c o n t a c t w i t h t h e tissue. W h e n t h e t r a n s d u c e r is raised ( a r r o w I1L t h e r e c o r d e r pen m o v e s b a c k to t h e b a s e line. T h e d i s t a n c e b e t w e e n the s t e a d y force a n d the b a s e line is m e a s u r e d a n d c o m p a r e d w i t h t h e c a l i b r a t i o n s t e p s for t h e p r e s s u r e d e t e r m i n a t i o n s . :Each c a l i b r a t i o n s t e p c o r r e s p o n d s to l o g in this experiment. 3.
Results
T h e pressure m e a s u r e m e n t s could be carried o u t quickly, a n d the a p p a r a t u s was easy to handle. T h e r a p i d increase in pressure when t h e swelling solution was b r o u g h t in c o n t a c t w i t h t h e specimen soon levelled off a n d a s t e a d y force was usually o b t a i n e d w i t h i n 30 rain. W i t h d r y specimens, however, it t o o k up to 90 rain to reach a n equilibrium. 350 -F
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:FIe. 3. Swelling p r e s s u r e o f s t e e r s t r o m a (filled circles) a n d of s t e e r sclera (open circles) p l o t t e d a g a i n s t hydration.
Swelling ~'essure of corneal stro~w~ (a) Steer stroma The pressure d a t a o b t a i n e d were expressed in m m H g a f t e r correct i o n for t h e u n i t area (1 cm 2) of t h e tissue. The swelling pressure was p l o t t e d a g a i n s t t h e h y d r a t i o n (Fig. 3). A decrease in t h e h y d r a t i o n f r o m t h e n o r m a l resulted in a large rise in t h e swelling pressure. ~V]len t h e h y d r a t i o n increased, the pressure d r o p p e d to a lesser extent. A t a n o r m a l h y d r a t i o n of 3-45 mg water/rag d r y wt. (Duane, 1949), t h e swelling pressure was f m m d to be a b o u t 60 m m Hg.
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When the swelling pressure was p l o t t e d on a l o g a r i t h m i c scale a g a i n s t t h e h y d r a t i o n (Fig. ,1), the p o i n t s fell on a slightly curved line. The slope of t h e t a n g e n t a t n o r m a l h y d r a t i o n indicates t l m t t h e swelling pressure increases to a p p r o x i m a t e l y the 4 t h power of the h y d r a t i o n u n d e r these conditions.
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The swelling pressure was d e t e r m i n e d in tissue b u t t o n s of various diameters. Over the m e a s u r e d h y d r a t i o n r a n g e (2.0-4-5 mg water/rag d r y wt.), no difference could be f o u n d b e t w e e n t h e various sizes. I n 10 experiments, t h e swelling solution was cooled to 4-6°C b y add_ing cubes of frozen 0 . 9 % NaC1. The results showed swelling pressures similar to those o b t a i n e d at room temperature. To d e t e r m i n e t h e effect of a possible diffusion of macromolecu]cs f r o m the tissue, e x p e r i m e n t s were carried o u t in which dialysis m e m b r a n e s were placed b e t w e e n t h e tissue a n d t-he filters (Fig. 5). The results t h u s o b t a i n e d did n o t differ f r o m those f b u n d w i t h t h e regxdar procedure. Once the pressttre equilibrium h a d been reached, it r e m a i n e d s t e a d y for several hours. (b) R a b b i t stzo,nw~ W h e n t h e pressure values, expressed in m m I-Ig, were p l o t t e d a g a i n s t h y d r a t i o n (Fig. 6), a relationship v e r y similar to t h a t o b t a i n e d for t h e steer s t r o m a s was found, b u t t h e values were more scattered. A t a n o r m a l h y d r a t i o n of 3-40 mg w a t e r / m g d r y w%. (Duane, 1949), t h e swelling pressure was a p p r o x i m a t e l y 60 m m I-Ig, i.e. t h e same v a l u e as t h a t f o u n d for t h e steer stroma. (c) H u m a n s t r o m a The swelling pressures were p l o t t e d a g a i n s t t h e h y d r a t i o n (Fig. 6). Since o n l y a limited n u m b e r of s t r o m a s were a v a i l a b l e for m e a s u r e m e n t , fewer p o i n t s could be plotted, t h u s reduchlg t h e a c c u r a c y of t h e relationship. Neverthless, t h e points a p p e a r to fall close to t h e curve o b t a i n e d for t h e steer stroma. No value for
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the n o r m a l h y d r a t i o n of t h e h u m a n cornea is available. Assuming a h y d r a t i o n similar to t h a t of o t h e r m a m m a l s (steer, r a b b i t , cat), vie. a b o u t 3.40 mg water/rag d r y wt., t h e n o r m a l h u m a n s t r o m a should h a v e a swelling pressure of a p p r o x i m a t e l y 60 m m I~g. 350
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Hydration (mgwGterper mg dr:/wt.) l*~ta. 5. Swelling p r e s s u r e o f s t e e r s t r o m a in dialysis m e m b r a n e s p l o t t e d against; h y d r a t i o n . B r o k e T ~ swelling p r e s s u r e o f s t e e r s t r o m a r e c o r d e d w i t h o u t t h e use o f dialysis m e m b r a n e (see Fig. 3) included f o r c o m p a r i s o n .
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6 3 4 S Hydration (rag water per mg drywt.)
7
F r o . 6. Swelli_qg p r e s s u r e o f r a b b i t (filled circles) a n d h u m a n (open t r i a n g l e s ) s t r o m a p l o t t e d a g a i n s t h y d r a t i o n . . B r o k e n c u r v e : s t e e r s t r o m a (see :Fig. 3) i n c l u d e d f o r c o m p a r i s o n .
Swelli)~g l~ressure of steer sclera 2~ c o m p a r i s o n of t h e p r e s s u r e - h y d r a t i o n relationships of t h e steer sclera a n d corneal s~roma (]?ig. 3) reveals t h a t t h e swelling pressure of t h e sclera varies more w i t h h y d r a t i o n t h a n does t h a t of the stroma. :-~t a n o r m a l h y d r a t i o n of 2.14 rag w a t c r / m g d r y wt. (Duane, 19~9), the swelling pressure of t h e steer sclera is a b o u t 17 m m Hg.
128
BENG'I'
O. H E D D Y S
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CLAES
H.
DOHLSIAN
4. D i s c u s s i o n
A c a p a c i t a n c e t r a n s d u c e r was chosen for t h e swelling pressure determhlations. I t was f o u n d to combine a lfigh degree of a c c u r a c y w i t h a low volume d i s p l a c e m e n t a n d showed good electronic stability. The swelling of t h e specimen was considerably restricted b y the t r a n s d u c e r ; it allowed a 13-5 m m specfl~mn to swell only 8~ whcn a pressure of 100 m m H g was measm'ed. Such small v o h ~ n e changes m a d e r a p i d determ i n a t i o n s possible. The m e t a l ring restricted t h e s t r o m a f r o m swelling radially. :Furthermore, it has been shown ( H e d b y s a n d Mishima, 1962) t h a t t h e thickness of t h e excised cornea increases lh~early with t h e w a t e r u p t a k e within a h y d r a t i o n r a n g e of 1-0-4.5 m g w a t e r / r a g d r y wt. The pressure e x e r t e d b y t h e swelling cornea in a direction perpendicular to its surfaces can therefore be r e g a r d e d as its swe]|ing pressure when m e a s u r e d a t h y d r a t i o n s within this range. The values of t h e swelling pressure were not a~fected a p p r e c i a b l y b y t h e size of the tissue b u t t o n , indicath~g t h a t t h e efl:eet caused b y flattening of t h e corneal c u r v a t u r e was of nxinor importance. The swelling pressure of artificial polyelectro!yte gels (Katchalsk-y, 1954) a n d the osmotic pressure of colloidal solutions are p r o p o r t i o n a l to the absolute t e m p e r a t u r e . .,k dependence of t h e corneal swelling pressure on t h e t e m p e r a t u r e therefore seems possible. Lowering of t h e t e m p e r a t u r e from 20-25°C to 4-6°C should result in a press~re decrease of only 6 % which m i g h t not be d e t e c t e d w i t h t h e p r e s e n t method. The v a l u e for the swelling pressl~re a t n o r m a l h y d r a t i o n was found to be similar for steer, r a b b i t and, p r e s u m a b l y , for h u m a n corneas. ~klso, the p r e s s u r e - h y d r a t i o n relationship followed t h e same p a t t e r n over a wide r a n g e of h y d r a t i o n s . These findings suggest t h a t t h e physico-chelrdcal properties of t h e swelling m a t e r i a l are similar in these stromas. The swelling pressure found for t h e steer s t r o m a a t n o r m a l h y d r a tion, a p p r o x i m a t e l y 60 mzn H g , is in good a g r e e m e n t w i t h t h a t f o u n d b y Dohlma.n and A n s e t h (1957), viz. a b o u t 65 m m :~Ig. I t is significantly higher t h a n t h a t r e p o r t e d b y K i n s e y a n d Cogan (1942) for n o r m a l cat corneas, viz. 30 m m Hg, a n d slightly lower t h a n P a u ' s (1954) value for calf cornea, viz. 75 m m Hg. Maurice (1951) showed t h a t b y selecting o t h e r points in t h e curves of K i n s e y a n d Cogan a swelling pressure of 60 m m I:ig could be obtained. There is good evidence to suggest t h a t t h e swelling pressure is g e n e r a t e d in the interfibrillar substance, since t h e collagen fibrils do n o t h~crease in thiclcness u n d e r the conditions used a n d within the r a n g e of corneal h y d r a t i o n m e a s u r e d (Franpois , ]¢abaey a n d Vandermeersche, 195zi; Main'ice, 1957). I n addition to t h e colloidal osmotic effect of the freely diffusible macromolccules, one m u s t e m p h a s i z e t h e imp o r t a n c e of t h e electrostatic forces of t h e polyelectrolyies hx t h e interfibrillar space, shine conditions l~lown to alter t h e ionization in the cornea h a v e a m a r k e d effect ou its swelling (Y~insey a n d Cogan, 1942; Loeven, 1955). Therefore, a t t e n t i o n has been focused on t h e polysaceharides, which are polyelectro.]ytes w i t h a high charge density. Recently, one of us (Hedbys, 1961) offered strong evidence for their role in u n r e s t r i c t e d corneal swelling i n vitro. The swelling pressl~re of the sclera shows a g r e a t e r dependence on h y d r a t i o n t h a n t h a t of the cornea. A t n o r m a l h y d r a t i o n , the swelling pressure was f o u n d to be a b o u t 17 m m I-Ig. Tiffs value is in good ate'cement with t h a t of D o h ] m a n a n d A n s e t h (1957), b u t it is considerably lower t h a n t h a t r e p o r t e d b y :Pau (1954), viz. 75 m m Hg. The low swelling pressure of t h e sclera i~z v~ilro m a y result from a low concentration
S W E L L I N G PI~ESSU:RE OF CORNEAL STI?~O1MA
129
of polysaccharidcs, about 0'8% of the dry weight (Polat~fick, LaTcssa anel Katzin, 1957) or 1.2% of the dry weight (Smits, 1957), and fl'om the distinctly different fine structure of the collagen fibril network (Jakus, 1961). ACKNOWLEDG~IEI~TS The authors are greatly indebted to Drs. E. A. Balazs: D. ~[. Maurice and P. F. Curran for their helpful contributions through discussion. The skilful technical assistance of Miss ~E. 0berlander is gratefully acknowledged.
REFERENCES Dohlman, C. hi. and Aa~scth, A. (1957). Acla ophthal. Kbh. 35, 73. Duane, T. D. (1949). Amer. J. Ophthal. 32 (part II), 203. :Frangois, J., Rabaey, ~'I. and Vandermcersche, G. (1954). OphthalmoIogica, Basel 127, 74. Hedbys, .]3. 0. (1961). Exp. Eye _Res. 1, 81. Hedbys, ]3. 0. and ~Lishima, S. (1962). Exp. Eye Res. 1, 262. Jakus, M. A. (1961). In The Structure of the Eye, ed. by G. K. Smelscr, p. 365. Academic Press, New York. Katchalsky, A. (1954). In .Progress i~ Biophysics, ed. by J. A. V. Butler and J. T. Randall, Vol. IV, p. 1. Academic Press, New York. Kinsey, V. E. and Cogan, D. G. (I942). A.M.A. Arch. Ophthal. 28, 272. Locvcn, W. A. (1955). Acta anat. 24, 217. ~Iaurice, D. M. (1951). J. Physiol. 112, 367. Maurice, D. M. (1957). J.. Physiol. 136, 263. Pau, ]:i. (1954). v. Graefes Arch. Opl~thal. 154, 579. Pol~tnick, J., LaTessa, A. J. and Katzin, H. ]~'I. (1957). Biochim. biophys. Acta. 26, 361. l~appaport, I~'L]3., Block, E. H. and Irwin, J. W. (1959). J. al~pl. -physiol. 14, 651. Smits, G. (1957). Biochim. biophys. Acta 25, 542.
A N e w M e t h o d for t h e D e t e r m i n a t i o n o f the S w e l l i n g P r e s s u r e o f the C o r n e a l S t r o m a i n v i t r o * BENGT O. H]~D]3YS'~ .AND CLAES H. DOnLMA~N
CorJ~eal Research Unit, Depart~wnt of Clinical Eye Research, Institute of Biological and Medical Scie~wes, Reti~a Fou~Mation, Boston, Massachusetts, U.S.A. (Received 19 January .1963) A n e w m e t h o d f o r t.he d e t e r m i n a t i o n o f t h e s w e l l i n g p r e s s u r e o f t h e c o r n e a l s t r o m a a n d sclcr,% h a s b e e n d e v e l o p e d . A t i s s u e b u t t o n a t a n y d e g r e e o f h y d r a t i o n is p l a c e d b e t w e e n t w o g l a s s filters. T h e u p p e r f i l t e r is m o v a b l e a n d co,~.nected t o a capa, c i t a n c e t r a n s d u c e r ; t h e l o w e r filter is f i x e d . W h e n 0'9~/o iNaC1 is b r o u g h t in c o n t a c t w i t h t h e t i s s u e , s l i g h t s w e l l i n g o c c u r s a n d t h e f o r c e is r e c o r d e d . T h e s w e l l i n g is r e s t r i c t e d c o n s i d e r a b l y b y t h e t r a n s d u c e r . T h e r e f o r e , a s t e a d y f o r c e is r a p i d l y r e a c h e d , u s u a l l y w i t h i n 30 mh~. Experiments were carried out on steer, rabbit and human corneal stroma and on steer sclera o v e r a wide r a n g e o f laydrat.ions. T h e swelling p r e s s u r e o f t h e n o r m a l c o r n e a l s t r o m a , w a s f o u n d t o b e a b o u t 60 m m H g a n d t h a t o f t h e n o r m a l s c l e r a a b o u t 17 m m I:[g. T h e s w e l l i n g p r e s s u r e o f t h e s c l e r a s h o w e d a g r e a t e r d e p e n d e n c e o n h y d r a t i o n t h a n did that of the corneal stroma.
1. Introduction
3 I e a s u r e m e n t of t h e swelling presstn'e, i.e. tile mechallical pressure applied to tile e x t e r n a l surfaces of a tissue t h a t ]?revents it from swelling while in a solution, offers a useful a p p r o a c h to studies on corneal d e h y d r a t i o n . T h e swelling p r o p e r t i e s can t h u s be c h a r a c t e r i z e d b y a pressure r a t h e r t h a n b y t h e a m o u n t of w a t e r t h a t t h e tissue can t a k e up. These memx,,rements can be carried o u t a t all stages of swelling, a n d t h e y are f a c i l i t a t e d b y t h e f a c t t h a t t h e cornea swells o n l y in a direction p e r p e n d i c t d a r to its mLvfaees. F r o m t h e swelling pressure m e a s u r e m e n t s , t h e fluid u p t a k e a t various stages of h y d r a t i o n and, c o n s e q u e n t l y , t h e load on t h e d e h y d r a t i o n m e c h a n i s m can be calculated. :By c h a n g i n g t h e t e m p e r a t u r e , h y d r a t i o n , p H , ionic s t r e n g t h , etc., Jnformation can be o b t a i n e d a b o u t t h e n a t u r e of t h e forces i n v o l v e d in t h e swelling process. :In t h e first r e p o r t e d m e a s u r e m e n t s of swelling pressure ( K i n s e y a n d Coghn, 1942), corneal sweili,lg was r e s t r i c t e d b y plach~g v a r i o u s weights on t o p of t h e tissue durh~g i m m e r s i o n in aqueous solution. I n t h i s way, a pressl~re was f o u n d w h i c h k e p t t h e cornea a t i t s original h y d r a t i o n . I n t h e e x p e r i m e n t s b y P a u (] 954), t h e swelling of t h e cornea a n d sclera was res t r i c t e d b y t h e colloidal osmotic pressure of t h e solution in w h i c h t h e tissues were immersed. T h e osmotic pressm'e was r e g u l a t e d b y v a r y i n g t h e c o n c e n t r a t i o n of an i n e r t macromolecule: viz. p o l y v i n y l p y r o l l i d o n e (I~VP), in a saline solution. T h e swelling pressure of t h e tissue was a s s u m e d to equal t h a t colloidal osmotic pressure which did n o t change t h e h y d r a t i o n of t h e tissue, l~au's t e c h n i q u e was modified b y D o h l m a n a n d A n s e t h (1957), who s t u d i e d t h e w e i g h t c h a n g e s of isolated cortical s t r o m a a n d * This i n v e s t i g a t i o n was s u p p o r t e d b y a P H S r e s e a r c h g r a n t (B-2220) f r o m t h e N a t i o n a l I n s t i t u t e o f ~ e u r o l o g i c a l Diseases a n d :Blindness, U.S. Public H e a l t h Service. This i n v e s t i g a t i o n was c o n d u c t e d u n d e r a F i g h t F o r S i g h t F o s t - D o c t o r a esearch F e l l o w s h i p o f t h e N a t i o n a l Council to C o m b a t ]31indncss, I n c . , New Y o r k , X e w York. 122
S \ V I ~ L L I N G PR:I~SSUI%E OF C O R N E A L
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s c l e r a e n c l o s e d in d i a l y s i s b a g s s u b m e r g e d i~.~ d e x t r a n s o l u t i o n s o f v a r y i n g o s m o t i c pressure. The methods cited gave measurements of limited accuracy, and they were also t i m e - c o n s u m i n g , s i n c e i t o f t e n t o o k a s l o n g a s t w o d a y s b e f o r e a n e q u i l i b r i u m wa.s r e a c h e d b e t w e e n t h e t~ssue a n d t h e s o l u t i o n . A r a p i d a n d m o r e a c c u r a t e m e t h o d h a s therefore been designed for the determination of the swelling pressure.
2. M a t e r i a l s and M e t h o d s
PreparaHon of ,~pecimens C o r n e a l s t r o m a s f r o m s t e e r s a n d a l b i n o r a b b i t s w e i g h i n g °~-3 k g w e r e p r e p a r e d b y s c r a p i n g off t h e e p i t h e l i u m , e n d o t h e l i u m a n d 1 ) e s c e m e t ' s m e m b r a n e w i t h i n 4 h r a f t e r t h e d e a t h of t h e a n i m a l . T h e s t r o m a w a s t h e n u s e d i m m e d i a t e l y or w a s f r o z e n a t -- 15°C a n d s t o r e d ~b t h e s a m e t e m p e r a t u r e for u p t o 3 w e e k s . N o d i f f e r e n c e w a s f o u n d b e t w e e n t h e s w e l l i n g p r e s s u r e o f t h e t h a w e d t i s s u e a n d a n d t h a t o f t h e fresh. H u m a n c o r n e a s w e r e e x c i s e d f r o m e y e s t h a t h a d b e e n s t o r e d a t - - 7 9 ° C for a c o n s i d e r a b l e t i m e . The s t r o m a w a s p r e p a r e d in t h e s a m e m a n n e r as t h a t of t h e s t e e r s a n d r a b b i t s . V a r i o u s h y d r a t i o n v a l u e s w e r e o b t a i n e d b y e v a p o r a t i o n of t h e s t r o m a s a t r o o m t e m p e r a t u r e ( 2 0 - 2 5 ° C ) or b y d r o p p i n g 0 " 9 % NaC1 o n t h e s u r f a c e s . T h e t i s s u e s w e r e t h e n s t o r e d a t 4°C for a t l e a s t 5 h r t o p r o d u c e u n i f o r m h y d r a t i o n . P i e c e s of s c l e r a of e v e n t h i c k n e s s w e r e e x c i s e d f r o m t h e e y e s o f s t e e r s w i t h i n ,t hr a f t e r s l a u g h t e r , a n d were c a r e f u l l y s c r a p e d t o r e m o v e a n y a d h e r i n g r e m n a n t s o f t h e c h o r o i d . T h e y w e r e t h e n u s e d i m m e d i a t e l y or f r o z e n a n d s t o r e d in t h e s a m e m a n n e r as t h e c o r n e a l s t r o m a . Various h y d r a t i o n values were o b t a i n e d for t h e sclera using t h e m e t h o d e m p l o y e d for t h e c o r n e a l s t r o m a . B e f o r e t h e s w e l l i n g p r e s s u r e w a s m e a s u r e d , c i r c u l a r b u t t o n s , 13"5 m a n (steer s t r o m a a n d sclera), 9"0 m m ( h u m a n s t r o m a ) a n d 6"5 m m ( r a b b i t s t r o m a ) in d i a m e t e r , w e r e p u n c h e d o u t of t h e s p e c i m e n s . I n a c o n t r o l series o f e x p e r i m e n t s , d e s i g n e d t o c o m p a r e t h e effects of t h e b u t t o n sizes o u t h e p r e s s u r e m e a s u r e m e n t s , discs o f all t h r e e sizes w e r e p r e p a r e d from the steer stroma.
]'[ a~o meter A Sanborn Model 121B electromanometer,* a capacitance manometer, was used. The ~ransduc.er w a s m o u n t e d on a m i c r o s c o p e s t a n d (Fig. 1) a n d c o n n e c t e d t o t h e b r i d g e h o u s i n g w i t h a c a b l e a c c o r d i n g to t h e m e t h o d o f R a p p a p o r t , B l o c k a n d I r w i n (1959). The signals from the electromanometer were amplified by a Sanborn ])C Coupling Preanaplifier ( M o d e l 1 5 0 - 1 3 0 0 ) a n d w e r e r e c o r d e d w i t h a S a n b o r n R e c o r d e r ( M o d e l ] 5:~-100B). T h e t r a n s d u c e r h a d t w o p a r a l l e l m e t a l p l a t e s a b o u t 25g. a p a r t . T h e u p p e r p l a t e w a s i m m o b i l e , w h e r e a s t h e l o w e r o n e w a s s l i g h t l y flexible. W h e n p r e s s u r e w a s e x e r t e d o n t h e l o w e r p l a t e , i t b e n t t o w a r d t h e u p p e r , a n d t h e c a p a c i t a n c e of t h e . t r a n s d u c e r i n c r e a s e d . T h e c h a n g e s in c a p a c i t a n c e w e r e c o n v e r t e d b y t h e c i r c u i t r y i n t o v o l t a g e s p r o p o r t i o n a l to t h e p r e s s u r e s . T h e e l e c t r o n i c e q u i p m e n t r e m a i n e d s t a b l e for a l o n g t i m e . L i n e a r r e c o r d i n g s o f f o r c e s u p to t h e e q u i v a l e n t of 500 m m I-Ig p r e s s u r e w e r e o b t a i n e d w h e n c o r n e a l b u t t o n s 13-5 m m in d i a m e t e r w e r e u s e d . B e c a u s e o f p o s s i b l e d a m a g e t o t h e i n s t r u m e n t , h o w e v e r , t h i s t r a n s d u c e r is n o t r e c o m m e n d e d f o r m e a s u r e m e n t s o f f o r c e s e q u i v a l e n t to m o r e t h a n 350 m m :Fig p r e s s u r e u s i n g s p e c i m e n s of t h i s size.
Measurements of swelling pressure T h e t i s s u e b u t t o n w a s p l a c e d on a glass filter disc t o w h i c h a b r a s s r i n g w i t h a t h i c k n e s s o f 2-5 m m h a d b e e n g l u e d (Fig. 1). A s e c o n d glass filter disc, d e s i g n e d t o fit w i t h i n t h e b r a s s * Sanborn Company, Waltham, 5[ass.
12-I
B E N G T O. I i E D BYS A N D C L A E S H . : D O I i L M A N
ring, was t h e n p l a c e d olt top of t h e tissue. Since tissue b u t t o u s o f t h r e e sizes w e r e used [n tl,ese exl)erimeJ~t~% t h e b,'~,ss ri,lgs a n d t h e u p p e r tilter discs h a d to be a v a i l a b l e in thi'ee sizes, so t h a t t h e b u t t o n s could be t i g h t l y enclosed. T h e liltevs w e r e filled w i t h 0 . 9 % NaCl i , t v u c ~ o a n d s t o r e d in t h e salil~e soluSion b e t w e e n c x p e r i m e n t s . T h e filters w i t h t h e e n c l o s e d tissue b u t t o n w e r e p l a c e d in t,. f l a t - b o t t o m e d d i s h o n t h e t a b l e of tlm m i c r o s c o p e s t a n d . A t h i n a l u m i , m m r o d w e i g h i n g 3 g w a s i n s e r t e d b e t w e e n t h e
,L S-
C~
:-JA / C
'.
/ rn
/ ~'io. l. Illustration of the system used to measurethe pressure. The corneal button (c) is placed between glass filters (f) and within a brass ring (m). The upper glass filter is connected to the lower of the 2 metal plates (a, b) of the transducer (T). An adaptor (A) holds the transducer and a metal spring (S) connects it to the coaxial cable (O).
u p p e r filter disp a n d t h e t r a n s d u c e r . T h e t r a n s d u c e r w a s l o w e r e d b y t u r n i n g t h e m i c r o m e t e r s c r e w of t h e m i c r o s c o p e u n t i l a v e r y s m a l l f o r c e was r e c o r d e d (Fig. 2). T h e d i s h w a s t h e n filled w i t h 0.9°/o INaC1 so t h a t t h e filters w e r e c o v e r e d . T h e swelling p r e s s u r e o f t h e t i s s u e was t h u s t r a n s m i t t e d b y t h e a l u r n i n i u m r o d t o t h e flexible p l a t e o f t h e t r a n s d u c e r a n d t h e force recorded. A f t e r t h e f o r c e h a d r e m a i n e d s t e a d y for 1 5 - 2 0 rain, t h e t r a n s d u c e r was q u i c k l y raised. T h e d i s t a n c e b e t w e e n t h e r e c o r d e d s t e a d y f o r c e a n d t h e b a s e line w a s m e a s u r e d , a n d t h e p r e s s u r e w a s c a l c t r l a t e d b y c o m p a r i s o n w i t h c a l i b r a t i o n steps. A f t e r c o m p l e t i o n o f t h e e x p e r i m e n t , t h e tissue b u t t o n w a s i m m e d i a t e l y r e m o v e d f r o m t h e glass filters, b l o t t e d b e t w e e n filter p a p e r s a n d w e i g h e d . T h e h y d r a t i o n of t h e s p e c i m e n was c a l c u l a t e d f r o m t h e dr)- w e i g h t of t h e tissue a f t e r d e s i c c a t i o n for 24 h r a t 5 5 ° 0 o v e r phosphorus pentoxide in vacuo. To c a l i b r a t e t h e i n s t r u m e n t , a b a l a n c e was m o d i f i e d so t h a t one o f its b e a m s c o u l d be
S\VELLING
PEI~SSUt~E
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CORNEAL
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125
placed under the transducer and the metal rod could be inserted in between. By increasing the weights on the opposite beam, known forces were exerted on the transducer and recorded tFi- 2). T
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Fx(~. 2. G r a p h i c i l l u s t r a t i o n o f swelling p r e s s u r e m e a s u r e m e n t . A t a r r o w I, 0 . 9 % ~NaCI is b r o u g h t i n t o c o n t a c t w i t h t h e tissue. W h e n t h e t r a n s d u c e r is raised ( a r r o w I1L t h e r e c o r d e r pen m o v e s b a c k to t h e b a s e line. T h e d i s t a n c e b e t w e e n the s t e a d y force a n d the b a s e line is m e a s u r e d a n d c o m p a r e d w i t h t h e c a l i b r a t i o n s t e p s for t h e p r e s s u r e d e t e r m i n a t i o n s . :Each c a l i b r a t i o n s t e p c o r r e s p o n d s to l o g in this experiment. 3.
Results
T h e pressure m e a s u r e m e n t s could be carried o u t quickly, a n d the a p p a r a t u s was easy to handle. T h e r a p i d increase in pressure when t h e swelling solution was b r o u g h t in c o n t a c t w i t h t h e specimen soon levelled off a n d a s t e a d y force was usually o b t a i n e d w i t h i n 30 rain. W i t h d r y specimens, however, it t o o k up to 90 rain to reach a n equilibrium. 350 -F
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Hydration (ms water per mg dry wt.)
:FIe. 3. Swelling p r e s s u r e o f s t e e r s t r o m a (filled circles) a n d of s t e e r sclera (open circles) p l o t t e d a g a i n s t hydration.
Swelling ~'essure of corneal stro~w~ (a) Steer stroma The pressure d a t a o b t a i n e d were expressed in m m H g a f t e r correct i o n for t h e u n i t area (1 cm 2) of t h e tissue. The swelling pressure was p l o t t e d a g a i n s t t h e h y d r a t i o n (Fig. 3). A decrease in t h e h y d r a t i o n f r o m t h e n o r m a l resulted in a large rise in t h e swelling pressure. ~V]len t h e h y d r a t i o n increased, the pressure d r o p p e d to a lesser extent. A t a n o r m a l h y d r a t i o n of 3-45 mg water/rag d r y wt. (Duane, 1949), t h e swelling pressure was f m m d to be a b o u t 60 m m Hg.
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When the swelling pressure was p l o t t e d on a l o g a r i t h m i c scale a g a i n s t t h e h y d r a t i o n (Fig. ,1), the p o i n t s fell on a slightly curved line. The slope of t h e t a n g e n t a t n o r m a l h y d r a t i o n indicates t l m t t h e swelling pressure increases to a p p r o x i m a t e l y the 4 t h power of the h y d r a t i o n u n d e r these conditions.
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The swelling pressure was d e t e r m i n e d in tissue b u t t o n s of various diameters. Over the m e a s u r e d h y d r a t i o n r a n g e (2.0-4-5 mg water/rag d r y wt.), no difference could be f o u n d b e t w e e n t h e various sizes. I n 10 experiments, t h e swelling solution was cooled to 4-6°C b y add_ing cubes of frozen 0 . 9 % NaC1. The results showed swelling pressures similar to those o b t a i n e d at room temperature. To d e t e r m i n e t h e effect of a possible diffusion of macromolecu]cs f r o m the tissue, e x p e r i m e n t s were carried o u t in which dialysis m e m b r a n e s were placed b e t w e e n t h e tissue a n d t-he filters (Fig. 5). The results t h u s o b t a i n e d did n o t differ f r o m those f b u n d w i t h t h e regxdar procedure. Once the pressttre equilibrium h a d been reached, it r e m a i n e d s t e a d y for several hours. (b) R a b b i t stzo,nw~ W h e n t h e pressure values, expressed in m m I-Ig, were p l o t t e d a g a i n s t h y d r a t i o n (Fig. 6), a relationship v e r y similar to t h a t o b t a i n e d for t h e steer s t r o m a s was found, b u t t h e values were more scattered. A t a n o r m a l h y d r a t i o n of 3-40 mg w a t e r / m g d r y w%. (Duane, 1949), t h e swelling pressure was a p p r o x i m a t e l y 60 m m I-Ig, i.e. t h e same v a l u e as t h a t f o u n d for t h e steer stroma. (c) H u m a n s t r o m a The swelling pressures were p l o t t e d a g a i n s t t h e h y d r a t i o n (Fig. 6). Since o n l y a limited n u m b e r of s t r o m a s were a v a i l a b l e for m e a s u r e m e n t , fewer p o i n t s could be plotted, t h u s reduchlg t h e a c c u r a c y of t h e relationship. Neverthless, t h e points a p p e a r to fall close to t h e curve o b t a i n e d for t h e steer stroma. No value for
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127
STI~OMA
the n o r m a l h y d r a t i o n of t h e h u m a n cornea is available. Assuming a h y d r a t i o n similar to t h a t of o t h e r m a m m a l s (steer, r a b b i t , cat), vie. a b o u t 3.40 mg water/rag d r y wt., t h e n o r m a l h u m a n s t r o m a should h a v e a swelling pressure of a p p r o x i m a t e l y 60 m m I~g. 350
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F r o . 6. Swelli_qg p r e s s u r e o f r a b b i t (filled circles) a n d h u m a n (open t r i a n g l e s ) s t r o m a p l o t t e d a g a i n s t h y d r a t i o n . . B r o k e n c u r v e : s t e e r s t r o m a (see :Fig. 3) i n c l u d e d f o r c o m p a r i s o n .
Swelli)~g l~ressure of steer sclera 2~ c o m p a r i s o n of t h e p r e s s u r e - h y d r a t i o n relationships of t h e steer sclera a n d corneal s~roma (]?ig. 3) reveals t h a t t h e swelling pressure of t h e sclera varies more w i t h h y d r a t i o n t h a n does t h a t of the stroma. :-~t a n o r m a l h y d r a t i o n of 2.14 rag w a t c r / m g d r y wt. (Duane, 19~9), the swelling pressure of t h e steer sclera is a b o u t 17 m m Hg.
128
BENG'I'
O. H E D D Y S
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DOHLSIAN
4. D i s c u s s i o n
A c a p a c i t a n c e t r a n s d u c e r was chosen for t h e swelling pressure determhlations. I t was f o u n d to combine a lfigh degree of a c c u r a c y w i t h a low volume d i s p l a c e m e n t a n d showed good electronic stability. The swelling of t h e specimen was considerably restricted b y the t r a n s d u c e r ; it allowed a 13-5 m m specfl~mn to swell only 8~ whcn a pressure of 100 m m H g was measm'ed. Such small v o h ~ n e changes m a d e r a p i d determ i n a t i o n s possible. The m e t a l ring restricted t h e s t r o m a f r o m swelling radially. :Furthermore, it has been shown ( H e d b y s a n d Mishima, 1962) t h a t t h e thickness of t h e excised cornea increases lh~early with t h e w a t e r u p t a k e within a h y d r a t i o n r a n g e of 1-0-4.5 m g w a t e r / r a g d r y wt. The pressure e x e r t e d b y t h e swelling cornea in a direction perpendicular to its surfaces can therefore be r e g a r d e d as its swe]|ing pressure when m e a s u r e d a t h y d r a t i o n s within this range. The values of t h e swelling pressure were not a~fected a p p r e c i a b l y b y t h e size of the tissue b u t t o n , indicath~g t h a t t h e efl:eet caused b y flattening of t h e corneal c u r v a t u r e was of nxinor importance. The swelling pressure of artificial polyelectro!yte gels (Katchalsk-y, 1954) a n d the osmotic pressure of colloidal solutions are p r o p o r t i o n a l to the absolute t e m p e r a t u r e . .,k dependence of t h e corneal swelling pressure on t h e t e m p e r a t u r e therefore seems possible. Lowering of t h e t e m p e r a t u r e from 20-25°C to 4-6°C should result in a press~re decrease of only 6 % which m i g h t not be d e t e c t e d w i t h t h e p r e s e n t method. The v a l u e for the swelling pressl~re a t n o r m a l h y d r a t i o n was found to be similar for steer, r a b b i t and, p r e s u m a b l y , for h u m a n corneas. ~klso, the p r e s s u r e - h y d r a t i o n relationship followed t h e same p a t t e r n over a wide r a n g e of h y d r a t i o n s . These findings suggest t h a t t h e physico-chelrdcal properties of t h e swelling m a t e r i a l are similar in these stromas. The swelling pressure found for t h e steer s t r o m a a t n o r m a l h y d r a tion, a p p r o x i m a t e l y 60 mzn H g , is in good a g r e e m e n t w i t h t h a t f o u n d b y Dohlma.n and A n s e t h (1957), viz. a b o u t 65 m m :~Ig. I t is significantly higher t h a n t h a t r e p o r t e d b y K i n s e y a n d Cogan (1942) for n o r m a l cat corneas, viz. 30 m m Hg, a n d slightly lower t h a n P a u ' s (1954) value for calf cornea, viz. 75 m m Hg. Maurice (1951) showed t h a t b y selecting o t h e r points in t h e curves of K i n s e y a n d Cogan a swelling pressure of 60 m m I:ig could be obtained. There is good evidence to suggest t h a t t h e swelling pressure is g e n e r a t e d in the interfibrillar substance, since t h e collagen fibrils do n o t h~crease in thiclcness u n d e r the conditions used a n d within the r a n g e of corneal h y d r a t i o n m e a s u r e d (Franpois , ]¢abaey a n d Vandermeersche, 195zi; Main'ice, 1957). I n addition to t h e colloidal osmotic effect of the freely diffusible macromolccules, one m u s t e m p h a s i z e t h e imp o r t a n c e of t h e electrostatic forces of t h e polyelectrolyies hx t h e interfibrillar space, shine conditions l~lown to alter t h e ionization in the cornea h a v e a m a r k e d effect ou its swelling (Y~insey a n d Cogan, 1942; Loeven, 1955). Therefore, a t t e n t i o n has been focused on t h e polysaceharides, which are polyelectro.]ytes w i t h a high charge density. Recently, one of us (Hedbys, 1961) offered strong evidence for their role in u n r e s t r i c t e d corneal swelling i n vitro. The swelling pressl~re of the sclera shows a g r e a t e r dependence on h y d r a t i o n t h a n t h a t of the cornea. A t n o r m a l h y d r a t i o n , the swelling pressure was f o u n d to be a b o u t 17 m m I-Ig. Tiffs value is in good ate'cement with t h a t of D o h ] m a n a n d A n s e t h (1957), b u t it is considerably lower t h a n t h a t r e p o r t e d b y :Pau (1954), viz. 75 m m Hg. The low swelling pressure of t h e sclera i~z v~ilro m a y result from a low concentration
S W E L L I N G PI~ESSU:RE OF CORNEAL STI?~O1MA
129
of polysaccharidcs, about 0'8% of the dry weight (Polat~fick, LaTcssa anel Katzin, 1957) or 1.2% of the dry weight (Smits, 1957), and fl'om the distinctly different fine structure of the collagen fibril network (Jakus, 1961). ACKNOWLEDG~IEI~TS The authors are greatly indebted to Drs. E. A. Balazs: D. ~[. Maurice and P. F. Curran for their helpful contributions through discussion. The skilful technical assistance of Miss ~E. 0berlander is gratefully acknowledged.
REFERENCES Dohlman, C. hi. and Aa~scth, A. (1957). Acla ophthal. Kbh. 35, 73. Duane, T. D. (1949). Amer. J. Ophthal. 32 (part II), 203. :Frangois, J., Rabaey, ~'I. and Vandermcersche, G. (1954). OphthalmoIogica, Basel 127, 74. Hedbys, .]3. 0. (1961). Exp. Eye _Res. 1, 81. Hedbys, ]3. 0. and ~Lishima, S. (1962). Exp. Eye Res. 1, 262. Jakus, M. A. (1961). In The Structure of the Eye, ed. by G. K. Smelscr, p. 365. Academic Press, New York. Katchalsky, A. (1954). In .Progress i~ Biophysics, ed. by J. A. V. Butler and J. T. Randall, Vol. IV, p. 1. Academic Press, New York. Kinsey, V. E. and Cogan, D. G. (I942). A.M.A. Arch. Ophthal. 28, 272. Locvcn, W. A. (1955). Acta anat. 24, 217. ~Iaurice, D. M. (1951). J. Physiol. 112, 367. Maurice, D. M. (1957). J.. Physiol. 136, 263. Pau, ]:i. (1954). v. Graefes Arch. Opl~thal. 154, 579. Pol~tnick, J., LaTessa, A. J. and Katzin, H. ]~'I. (1957). Biochim. biophys. Acta. 26, 361. l~appaport, I~'L]3., Block, E. H. and Irwin, J. W. (1959). J. al~pl. -physiol. 14, 651. Smits, G. (1957). Biochim. biophys. Acta 25, 542.