- Email: [email protected]
Corneal epithelial Cl-dependent pump quantified
.EyeRes.{l
6) 43,7
Corneal
~11
ithelial Cl-dependent . H. BEEKHUIS
tte
Eye
P u n a p Q u a n t i"f i e d
A ~ D B. E . M ~ C A R E Y
spiral, Rotte m, e therla , a ory iversity*, A t l a n t a , U . S . A .
E y e Center,
Pairs of rabbit corneas were perfused and corneal thickness monitored. The epithelial side of the preparation, and after ~abi|ization also the endothelial Bide, was covered with silicone oil. A constant thinning of the cornea was observed at a r a ~ of I 1.1 pm hr -~ ( ~ 1-0 pm L h-il cm -s) s.v. 2-1, n ~ 9 . is t|linning was also present after endothelial removal- 12-3 pm hr -l, s.D. 1"6 (n = 5). Epithelial abr ion virtually abolish~ the thinning (2-5 pm hr -l, s.v. 1-89, n = 4), did cooling from B4 to 0°C. hen the preparation was ~ r f u s e d with a CI- e solution (SO4~- in ad of CI-, corrected for osmolarity with sucrose) no significant thinning of the preparation was observed (2"4 pm hr -1, S.D. 2"49, n ~ 4) a r covering both su ces with silicone oil. This simple set o f e x ~ r i m e n t s quantified the epithelial pump mechanism. The epithelial pump rate of about 1,2 pm L hr -~ cm -~ h ~ to be taken into account when endothelial pump r a ~ s are measured in the in vitro preparation with intact epithelium. y words" cornea- epithelium; active fluid pump.
1. Introduction S w e l l i n g f o r c e s in t h e c o r n e a l s t r o m a a r e o p p o s e d b y a c t i v e ion p u m p s in t h e e n d o t h e i i u r n ( D i k s t e i n a n d M a u r i c e , 1 9 7 2 ; M a u r i c e , 1972) a n d e p i t h e l i u m ( K l y c e , 1975, 1977) as well as b y t h e i n t r a o c u l a r p r e s s u r e ( M a u r i c c , 1972) a n d o s m o t i c m o v e m e n t o f fluids f r o m t h e c o r n e a t o t h e o v e r l y i n g h y p e r t o n i c t e a r f i ! m (in t h e o p e n eye condition) (Mishima and Mauriee, 196!). Endothelial pu~p activity of the rabbit cornea has been studied in vitro by monitoring stromal thickness with the laboratory specular microscope. In these e x p e r i m e n t s t h e excised c o r n e a w a s c l a m p e d in a eha ber a n d cove d w i t h silicone oil t o p r e v e n t fluid e v a p o r a t i o n f r o ~ t h e e p i t h e l i a l s u r f a c e . All c h a n g e s in c o r n e a l t h i c k n e s s m e a s u r e d d u r i n g t h e s e e x p e r i m e n t s h a v e b e e n a s c r i b e d t o fluid t r a n s p o r t a t t h e p e r f u s e d , e n d o t h e l i a l side o f t h e p r e p a r a t i o n { D i k s t e i n a n d M a u r i c e , 1 9 7 2 ; E d e l h a u s e r , G o n n e r i n g a n d V a n H o r n , 1978; E d e l h a u s e r , H a n n i k e n , P e d e r s e n a n d V a n H o r n , 1 9 8 1 ; M c C a ~ y , E d e l h a u s e r a n d V a n H o r n , 19731. In our experiments have quantified the active o v e m e n t o f fluid b y t h e e p i t h e l i u m o f t h e r a b b i t co e a in v i t r o .
2. Materials
Using the pe
and
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sion s y s t e m described b y D i k s t e i n a n d Maurice (1972) 31 pairs~of eiy:es of
an artificial a n ~ r i o r c h a m b e r . , T h i s \ c o r n e a w a s k t at:34°C, e x c e p t where n d. I n . ~ a 0 s t e x p e r i m e n t s t h e e p i t h e ! i M su c e o f the c 0 r n e a w a s covered w i t h Silicone oil 20 cS (Dow P l e ~ e send reprint reques~ to W. H: Beekhuis, M.D., Eye Hospital, Schiedamsevest 180, 3011 BH R o t ~ r d a m , The therlands. I
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FIo. I. (1), Silicone oil bubble 3 mm diame~r in perl~Jsion chamber'; (2)silicone oil bubble 7'5 mm diame~r in pex1"usion chamber, endothelium in~ct, epithelium intact; ( 3 ) a s in exF~riment (2), endothelium intact, epithelium removed; (4) as in e x ~ r i m e n t (2), endothelium removed, ~ithelium intact; (5) ~ in e x ~ ~ m e n t (2), a r ~ sion 1 hr with Cl--fr~e solution; (6)as in experiment (2), with ~ m p e r a t u ~ sMtch m 34 ~ 0°C a r 3 hr following "introduction of silicone oil in the pe~mfon chamber. ing) p r e y t su e v a p o ti a n d pe it viewing of t h e co ca. t h ugh the ~ecular icrosc (McCarey, 1979). T h e tificial a n ~ r i o r c h a m b e r w pe d witl~ a R~nger u t i o n w i t h g l u ~ t h i o n e a n d adenosine simil the o described b y D i k s ~ i n a ~ d ri ,. a r a ~ of 0-061 ml in -1. T h e h y d r o s t ~ i c p r e s s u r e in t h e ~ r i o r cha bet W m a i n t a i n e d in all e x p e ~ m e n t s a~ 20 m m H g by e l e v a t i n g t h e pe sion o u t l e t tube. m e a l thickness was d e ~ ined b y focusing the s p e c u l a r microscope a t t h e o u t e r stroma] su ~ and s u b ~ q u e n t l y at the dothelium d re ing t h e di rence Jn focal d ~ t b iYdm the microscope m i c r o m e t e r . D u r i n g t h e e x p e r i m e n t s thickness readings w e ~ pe rmed eve~ 15 rain. W h e n five Con~3cuti me u ~ m e n t s (1 hr) w e ~ w i t h i n 10 p m range t h e thickness w a s considered stable a n d t h e ex m e n t was c o n t i n u e d r changing of t h e p a r a m e t e r s . Six sets o f e x F ~ N m e n ~ s ~ p e r f o r m e d ( F i g . 1). I n i t i a l l y we t e s ~ d the i n f l u e n ~ on c o ~ e a l thickness of a 3 m m d i a m e t e r silicone oil bubble a g a i n s t the+endothelium (1). I n s u b ~ q u e n t e x p e r i m e n t s t h e total endothelial su ee was b l o c k e d b y silicone oil. m e a ~ d changes in ~t o t a l ' s t r o m a l thickness With b o t h t.he endothelial a n d epithelial I er i n t a c t (2), w i t h o u t e n d o t h e l i u m (3), w i t h o u t e p i t h e l i u m (4), d~ r pe sion o f b o t h c o , c a ! surfaces w/~h a CI-e ~~sion fluid for ! h r (Sl:+'A CI-fluid w a s p ~ p a r e d b y s u b s t i ~ t i o n of SO~ ~- for CI-- i ~ ~ of CI, KCI a n d
E P I T H E L I A L PUMP Q U A N T I F I E D
709
CaCI~.2HzO we added Na2SO4, KeSO4 and SO4.2HaO in such weights, that the number of Na, K d Ca ions mained unch ted. T h e molarity of such a solution of 226 mosmol l-l w cot' c ~ d ~ 305 osmol 1-x by ding suc~) . In the I t ex~riment" (6) changes in thickness during a ~ m p e r a t u m shift from 34 to 0°C were observed in a silicone-enclosed cornea with intact endothelium and epithelium. Corneal thickness m e ~ u r e m e n t s in all experiments were s u b j e c ~ d to linear regression analysis to d e ~ r m i n e the rates of thickness change.
3. R e s u l t s T h e results a g phically su ~ a r i z e d in Fig. I. A small (3 m m d i a m e t e r ) bubble of silicone oil does n o t affect corneal t h i c k n e s s o1" an i n t a c t perfused p r e p a r a t i o n . I n Fig. 1, g r a p h I it is shown t h a t corneal t h i c k n e s s does n o t change significantly a r i n t r o d u c t i o n o f a small q u a n t i t y of silicone oil forming a 3 m ~ d i a m e t e r oil bubble. T h e r a t e of thickness c h a n g e was 1-52 ~:2"02 p ~ hr -~ ( c a n ±S.D., n ~ 5). T h e v a l u e for r e ( s q u a r e d gression coefficient for t h e i n d i v i d u a l ex r i m e n t s w i t h i n this series) ranged m 0. I0 to 0"73. I n the second e x p e r i m e n t (Fig. 1, g h 2) the t h i n n i n g r a t e o f an i n t a c t cornea b e t w e e n layers of silicone oil, o b s e r v e d for 6 llr, was 11.1 :t: 2.16 prn hr -1 (n -- 9). T h e value for r ~ r a n g e d m 0.88 to 0'99. T h e t h i r d gr h in Fig. I illust ~ s t h e corneal t h i n n i n g r a t e in a silicone-enclosed cornea a r oval of t h e e othelial 1 er. T h e t h i n n i n g r a ~ was 12-3 ± I:'67 p m hr - t (n ~ 5). T h e v a l u e for r e ra ed from 0"59 to 0"97. T h e e ct of epithelial r e m o v a l on the t h i n n i n g of the cornea t h a t was covered w i t h silicone oil on b o t h sides is shown in the f o u r t h g r a p h of Fig. 1. T h e t h i n n i n g seen in t h e p r e v i o u s e x p e r i m e n t s is abolished. T h i n n i n g was 2.54_+ 1"89 p.m hr -t (n = 4), w i t h a r 2 value b e t w e e n 0-07 a n d 0"89. T h e p r e p a r a t i o n was p e r f u s e d w i t h a chloride-free solution a t t h e e n d o t h e l i a l side a n d ' t h e i n t a c t e p i t h e l i u m covered w i t h t h e sa e solution, which was c h a n g e d e v e r y 10 min. This was foUowed a f t e r 1 hr b y covering b o t h surfaces h silicone oil. A fairly c o n s t a n t s t r o m a l thickness of t h e C | - - d e p l e t e d cornea resulted. T h e t h i n n i n g r a t e o b s e r v e d was 2.44 ± 2.49/zm hr - l (n ~ 4) with r 2 r a n g i n g from 0"(~3 to 0.90 (see Fig. 1, g r a p h 5). T h e last experi e n t e x a m i n e d t h e ct of a ~ pe tu c h a n g e f r o ~ 34 to 0°C" on t h e t h i n n i n g r a ~ of an i n t a c t silicone oil-cove d p p a tion. I n th e co eas thUS t a ~ d the t h i n n i n g te a t 3 C m e a s u r e d 8-46:t=0"41/z h r -1 (n ~ 3) w i t h r21 r~nge of 0-8 "94 (Fig. 1, g r a p h 6). C h a n g i n g t h e t e m p e t u r e to 0°C abolished t h e t h i n n i n g duri t h e following 2 hr of o b s e r v a t i o n . T h e t h i n n i n g r a ~ s observed in e x p e r i m e n t s 2 a n d 3 we significantly different from the thinni r a ~ s of e x p e r i m e n t s 1,'4 a n d 5 ( P < 0"05, p a i r e d S t u d e n t ' s t test). o
4. D i s c u s s i o n T h e first e x p e r i m e n t (see Fig. 1) showed t h a t t h e i n t r o d u c t i o n of silicone oil in t h e perfusion c h a m b e r does n o t in itself h a v e a n y effect on corneal ~thickness a n d t h a t silicone oil does n o t h a v e , a n e ct on t h e e n d 0 helial-cell laI~er its m e t a b o l i s m during this short-~r .obs r a t i o n . B y specul ic Scope t h e endotl~blial cell pat~rn re~ains unchanged. I n t h e following e x p e r i m e n t s leaks on t h e endothelial side as well as on t h e epithelial side o f t h e p r e p a r a t i o n were sealed by silicone o i l . T h e t h i n n i n g obser~/ed in experinie'nt "
"
t
i
710
W. H. B E E K H U I S
A N D B. E. M c C A R E Y
(2) w i t h i n t a c t e p i t h e l i a l and e n d o t h e l i a l layers s first t h o u g h t to be due to an ongoing e n d o t h e l i a l p u m p m e c h ism. S e p a r a ~ d m t h e e n e r g y resources o f t h e a q u e o u s h u m o u r t h e active p u p mech ism corn to a full s t within inu~s (Fischbarg, 1973). The only o t h e r glucose source for the e n d o t h e l i u m would theoretically be t h e epithelial glycogen stores. E x p e r i m e n t s ( 3 ) a n d (4) p r o v e t h a t the t h i n n i n g o b s e r v e d is a n epithelial ~ e c h is w i t h t, dothelial involve ant. T h e e p i t h e l i u m can m a i n t a i n active p u m p a c t i v i t y for 6 {Riley a n d Maurice, 1970) to 9 (Klyce, 1977) hr. T h e s i m u l a t e d i n t r a o c u l a r p r e s s u r e of 20 m m H g was k e p t c o n s t a n t d u r i n g t h e s t a b i l i z a t i o n phase as well as a f t e r i n t r o d u c t i o n of t h e silicone-oil b u b b l e in t h e p e r f u s i o n cha bar. T h e t h i n n i n g ob r v e d following t h e i n ~ o d u ~ i o n of t h e silicone oil t h u s ca ot be ascribed to a n influence of t h e s i m u l a t e d in ocular pressure. sion o f t h e ou d cot a w i t h a chloride- e s o l u t i o n to eli i n a t e CI-n s p o r t a t t h e apical epithelial ~ e r n b r a n e (Klyce, 1 9 7 7 ) a b o l i s h e d t h e t h i n n i n g of t.he silicone oil-sealed corneal p r e p a r a t i o n . W h e n t h e ~ r n p e r a t u r e of t h e c l a m p e d cornea was c h a n g e d m 34 to O°C the corneal t h i n n i n g s t o p p e d . These o b s e r v a t i o n s w r a n t t h e conclusion t h a t t h e b b i t cor al e p i t h e l i u m c o n t a i n s a C l - - d e p e n d e n t , a c t i v e p u m p mecb ism t h a t m o v e s fluid fro t h e s t r o m a t o w ds t h e te s r e s u l t i n g in a t h i n n i n g rathe of a b o u t 1 2 / t i n h r -1 (Klyce, 1975). This corresponds to a w a t e r t r a n s p o r t of 1"2/tin l cm -2 hr -~. Considering t h a t t h e p o r t e d e n d o t h e l i a l p u m p r a t e is6-7 ,urn I c -2 hr-~ (Bau , M a u r i c e a n d McCarey, 1984), t h e e p i t h e l i u o f t h e r a b b i t cornea p a y s a n o t a b l e c o n t r i b u t i o n to t h e t o t a l corneal d e h y d ring ~ e c h a n i s m . T h e Cl--flux t h a t c o r r e s p o n d s w i t h a w a t e r flux of 1-2/tin ] cm -~ h r -i is 0"lS/zM (NaCl) cm -2 hr -1 (Klyce, 1975). W e h a v e s h o w n t h a t 15 ~/o of the w a ~ r t h a t leaves t h e s ~ o m a is re e y e d by epithelial pu ping, a n d t h a t epithelial p u m p a c t i v i t y ust be t a k e n into a c c o u n t when in v i t r o studies of t h e i n t a c t r a b b i t cornea are being performed. ACKNOWLEDGMENTS This work w supported by the Flieringa Foundation, The Netherlands and by IOLAB Corporation, U.S.A. REFERENCES Baum, J. P., Maurice, D. M. and McCarey, B. E. (1984). The active and passive transport of w a ~ r ac as the corneal endothelium. . Eye Res. 39, 33 2. Dik in, S. and Maurice, D. M. (1972). The me~abo/ic basis to the fluid pump in the cornea. d. Pltysiol. 22t, 2 1" Edelhauser, H . F . , (~onnering, R . and van Horn, D . L . (1978). intraocular irrigating lutions. Arch. Ophthal 1. 96, 51£-20. E d e l h a u ~ r , H. F., Hanniken, A. M., Pedersen, H . J . d van Horn, D. L. (!981). Osmotic tolerance of rabbit and human corneal endothelium. Arch. Ophthal~,~)l. 99, 1281-7. Fischbarg, J. (1973). Active and p~ssive properties of the rabbit corneal endothelium. . Eye Res. 15, 615--38, Kly , S. D. (1975) ~T nsport of Na, (21 and w a ~ r by the rabbit co eal epithelium at the resting potential. A m . J . ~iol. 228, 1 2. Klyce, S. D. (1977). Enhancing fluid eretion by the corneal epithelium. Invest. Ophthalmol. 16, 96 3. Mauri~,:D: M: (1972). The location of the fluid pump in the cornea: J. 221, 4 ~ 5 4 . McC ~," B. E. (1979). Specular microscopy: practical aspects for the researcher and the clinician" .Co ts:Op~d~Sz l~ 6, 174-91. McC , B. E.; Edelhau r,:E. F. d v a n H o r n , D. L. (1983). Functional and structural
EPITHELIAL I)U~IP QUANTIF]EI)
M
changes in the eal endothelium during in vitro pe sion. Invest. Ophthalmol. 12, 410-17. Mishima, S. anti Maurice, D. M. (1961). The effect of normal evaporation on the eye. .
Eye R~. I, 4
2.
Riley, M. V. and Maurice, D. M. (1970). In Biochemistry of b~e Eye. (t~d. Oraymore, C. N.). Pp. 37. Academic Press" London.
6) 43,7
Corneal
~11
ithelial Cl-dependent . H. BEEKHUIS
tte
Eye
P u n a p Q u a n t i"f i e d
A ~ D B. E . M ~ C A R E Y
spiral, Rotte m, e therla , a ory iversity*, A t l a n t a , U . S . A .
E y e Center,
Pairs of rabbit corneas were perfused and corneal thickness monitored. The epithelial side of the preparation, and after ~abi|ization also the endothelial Bide, was covered with silicone oil. A constant thinning of the cornea was observed at a r a ~ of I 1.1 pm hr -~ ( ~ 1-0 pm L h-il cm -s) s.v. 2-1, n ~ 9 . is t|linning was also present after endothelial removal- 12-3 pm hr -l, s.D. 1"6 (n = 5). Epithelial abr ion virtually abolish~ the thinning (2-5 pm hr -l, s.v. 1-89, n = 4), did cooling from B4 to 0°C. hen the preparation was ~ r f u s e d with a CI- e solution (SO4~- in ad of CI-, corrected for osmolarity with sucrose) no significant thinning of the preparation was observed (2"4 pm hr -1, S.D. 2"49, n ~ 4) a r covering both su ces with silicone oil. This simple set o f e x ~ r i m e n t s quantified the epithelial pump mechanism. The epithelial pump rate of about 1,2 pm L hr -~ cm -~ h ~ to be taken into account when endothelial pump r a ~ s are measured in the in vitro preparation with intact epithelium. y words" cornea- epithelium; active fluid pump.
1. Introduction S w e l l i n g f o r c e s in t h e c o r n e a l s t r o m a a r e o p p o s e d b y a c t i v e ion p u m p s in t h e e n d o t h e i i u r n ( D i k s t e i n a n d M a u r i c e , 1 9 7 2 ; M a u r i c e , 1972) a n d e p i t h e l i u m ( K l y c e , 1975, 1977) as well as b y t h e i n t r a o c u l a r p r e s s u r e ( M a u r i c c , 1972) a n d o s m o t i c m o v e m e n t o f fluids f r o m t h e c o r n e a t o t h e o v e r l y i n g h y p e r t o n i c t e a r f i ! m (in t h e o p e n eye condition) (Mishima and Mauriee, 196!). Endothelial pu~p activity of the rabbit cornea has been studied in vitro by monitoring stromal thickness with the laboratory specular microscope. In these e x p e r i m e n t s t h e excised c o r n e a w a s c l a m p e d in a eha ber a n d cove d w i t h silicone oil t o p r e v e n t fluid e v a p o r a t i o n f r o ~ t h e e p i t h e l i a l s u r f a c e . All c h a n g e s in c o r n e a l t h i c k n e s s m e a s u r e d d u r i n g t h e s e e x p e r i m e n t s h a v e b e e n a s c r i b e d t o fluid t r a n s p o r t a t t h e p e r f u s e d , e n d o t h e l i a l side o f t h e p r e p a r a t i o n { D i k s t e i n a n d M a u r i c e , 1 9 7 2 ; E d e l h a u s e r , G o n n e r i n g a n d V a n H o r n , 1978; E d e l h a u s e r , H a n n i k e n , P e d e r s e n a n d V a n H o r n , 1 9 8 1 ; M c C a ~ y , E d e l h a u s e r a n d V a n H o r n , 19731. In our experiments have quantified the active o v e m e n t o f fluid b y t h e e p i t h e l i u m o f t h e r a b b i t co e a in v i t r o .
2. Materials
Using the pe
and
IVlethods
sion s y s t e m described b y D i k s t e i n a n d Maurice (1972) 31 pairs~of eiy:es of
an artificial a n ~ r i o r c h a m b e r . , T h i s \ c o r n e a w a s k t at:34°C, e x c e p t where n d. I n . ~ a 0 s t e x p e r i m e n t s t h e e p i t h e ! i M su c e o f the c 0 r n e a w a s covered w i t h Silicone oil 20 cS (Dow P l e ~ e send reprint reques~ to W. H: Beekhuis, M.D., Eye Hospital, Schiedamsevest 180, 3011 BH R o t ~ r d a m , The therlands. I
8 3 5 / 8 6 / ! 10707 + 05 $03.
/0
~) ! 9 8 6 Academic
ss Inc. (
ndon) m i t e d
W. H , B E E K H U I S
708
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MEAN THIN~O RATE ,11.l~/ht SO Z ~ N g
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FIo. I. (1), Silicone oil bubble 3 mm diame~r in perl~Jsion chamber'; (2)silicone oil bubble 7'5 mm diame~r in pex1"usion chamber, endothelium in~ct, epithelium intact; ( 3 ) a s in exF~riment (2), endothelium intact, epithelium removed; (4) as in e x ~ r i m e n t (2), endothelium removed, ~ithelium intact; (5) ~ in e x ~ ~ m e n t (2), a r ~ sion 1 hr with Cl--fr~e solution; (6)as in experiment (2), with ~ m p e r a t u ~ sMtch m 34 ~ 0°C a r 3 hr following "introduction of silicone oil in the pe~mfon chamber. ing) p r e y t su e v a p o ti a n d pe it viewing of t h e co ca. t h ugh the ~ecular icrosc (McCarey, 1979). T h e tificial a n ~ r i o r c h a m b e r w pe d witl~ a R~nger u t i o n w i t h g l u ~ t h i o n e a n d adenosine simil the o described b y D i k s ~ i n a ~ d ri ,. a r a ~ of 0-061 ml in -1. T h e h y d r o s t ~ i c p r e s s u r e in t h e ~ r i o r cha bet W m a i n t a i n e d in all e x p e ~ m e n t s a~ 20 m m H g by e l e v a t i n g t h e pe sion o u t l e t tube. m e a l thickness was d e ~ ined b y focusing the s p e c u l a r microscope a t t h e o u t e r stroma] su ~ and s u b ~ q u e n t l y at the dothelium d re ing t h e di rence Jn focal d ~ t b iYdm the microscope m i c r o m e t e r . D u r i n g t h e e x p e r i m e n t s thickness readings w e ~ pe rmed eve~ 15 rain. W h e n five Con~3cuti me u ~ m e n t s (1 hr) w e ~ w i t h i n 10 p m range t h e thickness w a s considered stable a n d t h e ex m e n t was c o n t i n u e d r changing of t h e p a r a m e t e r s . Six sets o f e x F ~ N m e n ~ s ~ p e r f o r m e d ( F i g . 1). I n i t i a l l y we t e s ~ d the i n f l u e n ~ on c o ~ e a l thickness of a 3 m m d i a m e t e r silicone oil bubble a g a i n s t the+endothelium (1). I n s u b ~ q u e n t e x p e r i m e n t s t h e total endothelial su ee was b l o c k e d b y silicone oil. m e a ~ d changes in ~t o t a l ' s t r o m a l thickness With b o t h t.he endothelial a n d epithelial I er i n t a c t (2), w i t h o u t e n d o t h e l i u m (3), w i t h o u t e p i t h e l i u m (4), d~ r pe sion o f b o t h c o , c a ! surfaces w/~h a CI-e ~~sion fluid for ! h r (Sl:+'A CI-fluid w a s p ~ p a r e d b y s u b s t i ~ t i o n of SO~ ~- for CI-- i ~ ~ of CI, KCI a n d
E P I T H E L I A L PUMP Q U A N T I F I E D
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CaCI~.2HzO we added Na2SO4, KeSO4 and SO4.2HaO in such weights, that the number of Na, K d Ca ions mained unch ted. T h e molarity of such a solution of 226 mosmol l-l w cot' c ~ d ~ 305 osmol 1-x by ding suc~) . In the I t ex~riment" (6) changes in thickness during a ~ m p e r a t u m shift from 34 to 0°C were observed in a silicone-enclosed cornea with intact endothelium and epithelium. Corneal thickness m e ~ u r e m e n t s in all experiments were s u b j e c ~ d to linear regression analysis to d e ~ r m i n e the rates of thickness change.
3. R e s u l t s T h e results a g phically su ~ a r i z e d in Fig. I. A small (3 m m d i a m e t e r ) bubble of silicone oil does n o t affect corneal t h i c k n e s s o1" an i n t a c t perfused p r e p a r a t i o n . I n Fig. 1, g r a p h I it is shown t h a t corneal t h i c k n e s s does n o t change significantly a r i n t r o d u c t i o n o f a small q u a n t i t y of silicone oil forming a 3 m ~ d i a m e t e r oil bubble. T h e r a t e of thickness c h a n g e was 1-52 ~:2"02 p ~ hr -~ ( c a n ±S.D., n ~ 5). T h e v a l u e for r e ( s q u a r e d gression coefficient for t h e i n d i v i d u a l ex r i m e n t s w i t h i n this series) ranged m 0. I0 to 0"73. I n the second e x p e r i m e n t (Fig. 1, g h 2) the t h i n n i n g r a t e o f an i n t a c t cornea b e t w e e n layers of silicone oil, o b s e r v e d for 6 llr, was 11.1 :t: 2.16 prn hr -1 (n -- 9). T h e value for r ~ r a n g e d m 0.88 to 0'99. T h e t h i r d gr h in Fig. I illust ~ s t h e corneal t h i n n i n g r a t e in a silicone-enclosed cornea a r oval of t h e e othelial 1 er. T h e t h i n n i n g r a ~ was 12-3 ± I:'67 p m hr - t (n ~ 5). T h e v a l u e for r e ra ed from 0"59 to 0"97. T h e e ct of epithelial r e m o v a l on the t h i n n i n g of the cornea t h a t was covered w i t h silicone oil on b o t h sides is shown in the f o u r t h g r a p h of Fig. 1. T h e t h i n n i n g seen in t h e p r e v i o u s e x p e r i m e n t s is abolished. T h i n n i n g was 2.54_+ 1"89 p.m hr -t (n = 4), w i t h a r 2 value b e t w e e n 0-07 a n d 0"89. T h e p r e p a r a t i o n was p e r f u s e d w i t h a chloride-free solution a t t h e e n d o t h e l i a l side a n d ' t h e i n t a c t e p i t h e l i u m covered w i t h t h e sa e solution, which was c h a n g e d e v e r y 10 min. This was foUowed a f t e r 1 hr b y covering b o t h surfaces h silicone oil. A fairly c o n s t a n t s t r o m a l thickness of t h e C | - - d e p l e t e d cornea resulted. T h e t h i n n i n g r a t e o b s e r v e d was 2.44 ± 2.49/zm hr - l (n ~ 4) with r 2 r a n g i n g from 0"(~3 to 0.90 (see Fig. 1, g r a p h 5). T h e last experi e n t e x a m i n e d t h e ct of a ~ pe tu c h a n g e f r o ~ 34 to 0°C" on t h e t h i n n i n g r a ~ of an i n t a c t silicone oil-cove d p p a tion. I n th e co eas thUS t a ~ d the t h i n n i n g te a t 3 C m e a s u r e d 8-46:t=0"41/z h r -1 (n ~ 3) w i t h r21 r~nge of 0-8 "94 (Fig. 1, g r a p h 6). C h a n g i n g t h e t e m p e t u r e to 0°C abolished t h e t h i n n i n g duri t h e following 2 hr of o b s e r v a t i o n . T h e t h i n n i n g r a ~ s observed in e x p e r i m e n t s 2 a n d 3 we significantly different from the thinni r a ~ s of e x p e r i m e n t s 1,'4 a n d 5 ( P < 0"05, p a i r e d S t u d e n t ' s t test). o
4. D i s c u s s i o n T h e first e x p e r i m e n t (see Fig. 1) showed t h a t t h e i n t r o d u c t i o n of silicone oil in t h e perfusion c h a m b e r does n o t in itself h a v e a n y effect on corneal ~thickness a n d t h a t silicone oil does n o t h a v e , a n e ct on t h e e n d 0 helial-cell laI~er its m e t a b o l i s m during this short-~r .obs r a t i o n . B y specul ic Scope t h e endotl~blial cell pat~rn re~ains unchanged. I n t h e following e x p e r i m e n t s leaks on t h e endothelial side as well as on t h e epithelial side o f t h e p r e p a r a t i o n were sealed by silicone o i l . T h e t h i n n i n g obser~/ed in experinie'nt "
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A N D B. E. M c C A R E Y
(2) w i t h i n t a c t e p i t h e l i a l and e n d o t h e l i a l layers s first t h o u g h t to be due to an ongoing e n d o t h e l i a l p u m p m e c h ism. S e p a r a ~ d m t h e e n e r g y resources o f t h e a q u e o u s h u m o u r t h e active p u p mech ism corn to a full s t within inu~s (Fischbarg, 1973). The only o t h e r glucose source for the e n d o t h e l i u m would theoretically be t h e epithelial glycogen stores. E x p e r i m e n t s ( 3 ) a n d (4) p r o v e t h a t the t h i n n i n g o b s e r v e d is a n epithelial ~ e c h is w i t h t, dothelial involve ant. T h e e p i t h e l i u m can m a i n t a i n active p u m p a c t i v i t y for 6 {Riley a n d Maurice, 1970) to 9 (Klyce, 1977) hr. T h e s i m u l a t e d i n t r a o c u l a r p r e s s u r e of 20 m m H g was k e p t c o n s t a n t d u r i n g t h e s t a b i l i z a t i o n phase as well as a f t e r i n t r o d u c t i o n of t h e silicone-oil b u b b l e in t h e p e r f u s i o n cha bar. T h e t h i n n i n g ob r v e d following t h e i n ~ o d u ~ i o n of t h e silicone oil t h u s ca ot be ascribed to a n influence of t h e s i m u l a t e d in ocular pressure. sion o f t h e ou d cot a w i t h a chloride- e s o l u t i o n to eli i n a t e CI-n s p o r t a t t h e apical epithelial ~ e r n b r a n e (Klyce, 1 9 7 7 ) a b o l i s h e d t h e t h i n n i n g of t.he silicone oil-sealed corneal p r e p a r a t i o n . W h e n t h e ~ r n p e r a t u r e of t h e c l a m p e d cornea was c h a n g e d m 34 to O°C the corneal t h i n n i n g s t o p p e d . These o b s e r v a t i o n s w r a n t t h e conclusion t h a t t h e b b i t cor al e p i t h e l i u m c o n t a i n s a C l - - d e p e n d e n t , a c t i v e p u m p mecb ism t h a t m o v e s fluid fro t h e s t r o m a t o w ds t h e te s r e s u l t i n g in a t h i n n i n g rathe of a b o u t 1 2 / t i n h r -1 (Klyce, 1975). This corresponds to a w a t e r t r a n s p o r t of 1"2/tin l cm -2 hr -~. Considering t h a t t h e p o r t e d e n d o t h e l i a l p u m p r a t e is6-7 ,urn I c -2 hr-~ (Bau , M a u r i c e a n d McCarey, 1984), t h e e p i t h e l i u o f t h e r a b b i t cornea p a y s a n o t a b l e c o n t r i b u t i o n to t h e t o t a l corneal d e h y d ring ~ e c h a n i s m . T h e Cl--flux t h a t c o r r e s p o n d s w i t h a w a t e r flux of 1-2/tin ] cm -~ h r -i is 0"lS/zM (NaCl) cm -2 hr -1 (Klyce, 1975). W e h a v e s h o w n t h a t 15 ~/o of the w a ~ r t h a t leaves t h e s ~ o m a is re e y e d by epithelial pu ping, a n d t h a t epithelial p u m p a c t i v i t y ust be t a k e n into a c c o u n t when in v i t r o studies of t h e i n t a c t r a b b i t cornea are being performed. ACKNOWLEDGMENTS This work w supported by the Flieringa Foundation, The Netherlands and by IOLAB Corporation, U.S.A. REFERENCES Baum, J. P., Maurice, D. M. and McCarey, B. E. (1984). The active and passive transport of w a ~ r ac as the corneal endothelium. . Eye Res. 39, 33 2. Dik in, S. and Maurice, D. M. (1972). The me~abo/ic basis to the fluid pump in the cornea. d. Pltysiol. 22t, 2 1" Edelhauser, H . F . , (~onnering, R . and van Horn, D . L . (1978). intraocular irrigating lutions. Arch. Ophthal 1. 96, 51£-20. E d e l h a u ~ r , H. F., Hanniken, A. M., Pedersen, H . J . d van Horn, D. L. (!981). Osmotic tolerance of rabbit and human corneal endothelium. Arch. Ophthal~,~)l. 99, 1281-7. Fischbarg, J. (1973). Active and p~ssive properties of the rabbit corneal endothelium. . Eye Res. 15, 615--38, Kly , S. D. (1975) ~T nsport of Na, (21 and w a ~ r by the rabbit co eal epithelium at the resting potential. A m . J . ~iol. 228, 1 2. Klyce, S. D. (1977). Enhancing fluid eretion by the corneal epithelium. Invest. Ophthalmol. 16, 96 3. Mauri~,:D: M: (1972). The location of the fluid pump in the cornea: J. 221, 4 ~ 5 4 . McC ~," B. E. (1979). Specular microscopy: practical aspects for the researcher and the clinician" .Co ts:Op~d~Sz l~ 6, 174-91. McC , B. E.; Edelhau r,:E. F. d v a n H o r n , D. L. (1983). Functional and structural
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changes in the eal endothelium during in vitro pe sion. Invest. Ophthalmol. 12, 410-17. Mishima, S. anti Maurice, D. M. (1961). The effect of normal evaporation on the eye. .
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Riley, M. V. and Maurice, D. M. (1970). In Biochemistry of b~e Eye. (t~d. Oraymore, C. N.). Pp. 37. Academic Press" London.