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Hard contact lens-induced metabolic changes in rabbit corneas
Exp. Eye Res (1989) 49~ 769-775
Hard Contact L e n s - I n d u c e d M e t a b o l i c C h a n g e s In Rabbit Corneas KAZUO TSUBOTA* t , K E N N E T H R. KENYON t AND H o N G - M I N G
CHENG*~
* Howe Laboratory of Ophthalmology, Harvard Medical School and the Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, MA 02114, U.S.A. and ?Eye Research Institute, 20 Staniford Street, Boston, MA 02114, U.S.A. (Received 14 September 1988 and accepted in revised form 20 June 1989) The biochemistry of contact lens-cornea interaction is not well understood, although previous studies have suggested that corneal metabolic changes may be the underlying factor in morphological alterations. Using a rabbit model, this interaction has been examined with 3~p nuclear magnetic resonance (NMR) spectroscopy, which detects signals principally from the epithelium. The examination was supplemented with electron microscopy and histochemistry. Polymethylmethacrylate lenses caused reversible changes, including activation of anaerobic glycolysis and disturbance of membrane metabolite levels. These changes were far more severe than those occurring during prolonged eye closure. There appears to be an association between cellular deterioration and loss of membrane metabolites. On the other hand, oxygen-permeable silicone lenses allowed maintenance of nearly normal metabolic patterns. These results show multifaceted corneal response to hard contact lens wear. Key words: contact lens ; cornea ; Dk ; NMR ; glycolysis; membrane nmtabolism.
1. I n t r o d u c t i o n Most studies on c o r n e a - c o n t a c t lens (CL) i n t e r a c t i o n h a v e e m p h a s i z e d m o r p h o l o g i c a l aspects. I t is well k n o w n t h a t h a r d c o n t a c t lenses can alter corneal thickness a n d e n d o t h e l i a l cell s h a p e / d e n s i t y , a n d cause epithelial i n j u r y (see, e.g. Z a n t o s a n d H o l d e n , 1977 ; S h e r r a r d , 1978 ; B a r r a n d Schoessler, 1980; B e g m a n s o n a n d Chu, 1982 ; M a c R a e , M a t s u d a a n d Yee, 1985; M a t s u d a , M a c R a e , I n a b a a n d Manabe, 1989). These changes are m o s t l y t r a n s i e n t ; nevertheless, l o n g - t e r m h a r d CL wear can lead to corneal complications, p a r t i c u l a r l y u l c e r a t i v e k e r a t i t i s (Honan, 1979; Alfonso, M a n d e l b a u m , F o x a n d F o r s t e r , 1986). The b i o c h e m i c a l basis of C L - i n d u c e d changes, on t h e o t h e r hand, is n o t as well d o c u m e n t e d . Burns, R o b e r t s a n d Rich (1971) found an increase of A T P a n d l a c t a t e a n d a decrease of glycogen a n d glucose following p o l y m e t h y l m e t h a c r y l a t e (PMMA) lens wear. T h o f t a n d F r i e n d (1975) r e p o r t e d similar results, e x c e p t t h a t A T P decreased with a c o n c o m i t a n t increase of A D P . The reason for this d i s c r e p a n c y is unclear, a l t h o u g h a loss in A T P can b e t t e r e x p l a i n declined corneal functions such as t h e ionic p u m p s . K l y c e (1981) p r o p o s e d t h a t a c c u m u l a t i o n of l a c t a t e m a y be the osmotic basis of corneal edema, whereas B a r r a n d Schoessler (1980) suggested t h a t h a r d CLs m a y a d v e r s e l y affect the e n d o t h e l i a l b i c a r b o n a t e p u m p a n d therefore corneal deturgescence. Conflicting d a t a a n d differing v i e w p o i n t s n o t w i t h s t a n d i n g , it is conceivable t h a t C L - i n d u c e d change in corneal m e t a b o l i s m m a y be t h e basis of m o r p h o l o g i c a l complications. B o t h aerobic a n d a n a e r o b i c glycolysis are a c t i v e in the cornea (Riley, 1969a,b; Olson, Hoffert a n d F r o m m , 1970; Thoft a n d F r i e n d , 1972). The cornea r e s p o n d s to t r a u m a a n d h y p o x i a b y v a r y i n g the p r o p o r t i o n of these two modes (Thoft a n d F r i e n d , $* To whom correspondence should be addressed, at the Howe Laboratory of Ophthalmology. 0014-4835/89/110769+07 $03.00/0 26
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1975). T r a u m a causes an initial decrease b u t e v e n t u a l r e c o v e r y of A T P a n d glycogen levels. H y p o x i a , on t h e o t h e r hand, results in decreased A T P a n d glycogen, increased l a c t a t e , a n d d i m i n i s h e d o x y g e n u p t a k e . T h e e x t e n t of t r a u m a t i c i n j u r y a n d h y p o x i a therefore d e t e r m i n e t h e final corneal t o l e r a n c e to CLs. I n this p a p e r , t h e i n t e r a c t i o n b e t w e e n corneal e p i t h e l i u m a n d the h a r d CL is e x a m i n e d . As m o s t ( > 9 5 % ) a l p signals, e x c e p t t h a t of inorganic p h o s p h a t e Pi, originate from t h e e p i t h e l i u m (Greiner, B r a u d e a n d Glonek, 1985), the all) n u c l e a r m a g n e t i c resonance (NMR) a p p r o a c h is ideal for this purpose. A d d i t i o n a l l y , electron m i c r o s c o p y was used to e x a m i n e the a s s o c i a t e d m o r p h o l o g y , a n d periodic a c i d - S c h i f f (PAS) staining was used to e s t i m a t e t h e glycogen content. The N M R results a n d t h e i r m o r p h o l o g i c a l correlations are r e p o r t e d here.
2. M a t e r i a l s a n d M e t h o d s New Zealand albino rabbits, weighing 2-5 kg, were fitted with hard contact lenses in one eye, and the contralateral eye served as the control. The lenses were made of either polymethylmethacrylate (PMMA) or oxygen-permeable silicone lenses (Dk -- 12-5 or 55-5, manufacturer's specification). They were the same in design, each having a diameter of 13 mm and a base curve of 7"8 ram. This design permitted total coverage of the cornea (diameter l0 ram). The lens curvatures were steeper than those of the corneas as determined with fluorescein stain. After 12-, 24-, and 49 hr of continuous wear, the lenses were removed, the animals (eight in each group) were killed, and the eyes were enucleated and immediately frozen in liquid nitrogen. The corneas were excised and extracted with perchloric acid. In the eye closure experiment, designed to provide information on corneal metabolism under baseline hypoxia, the closure period was either 12- or 24 hr. The eyes/corneas were processed as described above. Extreme care had been taken to ensure that the final samples contained the same corneal wet weight per unit volume, at 2'5 g m1-1 NMR solution (20 % DsO + 20 mM EDTA), so the signal intensities from different samples (e.g. Fig. 1) could be compared quantitatively. F o r all samples, the alP-NMR spectroscopy was conducted at 109"3 MHz, using a Bruker HX270 spectrometer in the Fourier-transform mode. The NMR parameters were pulse angle 60 ~ (14 #see pulse duration), sweep width +_2500 Hz, recycle delay 0"5 see, line broadening 2 Hz, and 160000 acquisitions per spectrum. The resonances were identified according to previously described procedures (Cheng and Gonzglez, 1986). Electron microscopy was performed on corneas fixed in 4 % glutaraldehyde in 0"1 M eaeodylate (pH 7"2) and post-fixed in 1% OsO 4. Sectioning was achieved using a Sorvall MT2B mierotome on samples embedded in Epon 812. PAS staining for polysaccharides (Davis, Dulbeceo, Eisen and Grisherg, 1984) was performed to estimate glycogen content in the corneas.
3. R e s u l t s a l P - N M R s p e c t r a of the perchloric acid (PCA) e x t r a c t s of r a b b i t corneas are shown in Fig. 1, which includes the control corneas (A), corneas fitted w i t h P M M A lenses for 48 hr (B), corneas allowed to recover for 24 hr following 24 hr of P M M A lens wear (C), a n d corneas fitted w i t h o x y g e n - p e r m e a b l e silicone lenses (D/c---55-5) (D). These e x p e r i m e n t s were r e p e a t e d twice with t h e s a m e results. A c o m p a r i s o n between Fig. I(A) a n d (B) clearly shows t h a t w i t h P M M A lenses (1) A T P a n d glycerop h o s p h o r y l c h o l i n e (GPC) levels b o t h decreased b y 50 %, (2) p h o s p h o r y l c h o l i n e (PC) a n d g l y c e r o p h o s p h o r y l e t h a n o l a m i n e (GPE) d i s a p p e a r e d , a n d (3) a - g l y c e r o p h o s p h a t e (GP) doubled. A l t h o u g h cell loss occurred d u r i n g CL wear, this w o u l d n o t in itself affect t h e r e l a t i v e p r o p o r t i o n of the cellular m e t a b o l i t e s . These changes progressed after 12 hr of lens wear (Table I).
HARD CONTACT LENS-INDUCED
CORNEAL
METABOLIC
I
CHANGES
771
~ \l\
I GPE GPC
C
A
~ A T P ~
o
-Io
-;s
-5o
6(ppm) FIG. 1. atp NMR spectra of the PCA extracts of rabbit corneas. (A) Normal rabbit corneas, resonances include ccPOEO-glycerophosphate (GP), sugar phosphates (SP), phosphorylcholine (PC), inorganic phosphate (the most intense resonance), glycerophosphorylethanolamine (GPE), glycerophosphorylcholine (GPC), dinucleotides (N), ADP and ATP. (B) After 48 hr of PMMA CL wear. (C) After 24 hr of PMMA CL wear, followed by 24 hr of recovery; the appearance of the PCr resonance is noteworthy. (D) After 48 hr of silicone CL (Dk = 55"5) wear ; the appearance of an unknown resonance (X) is noteworthy. E v e n t h o u g h corneas fitted with PMMA lenses showed abnormal 31p-profiles after 12 hr, t h e y were able to recover. A 24-hr period appeared adequate [see Fig. 1 (C) and Table I]; however, the level of metabolites, except PC, was twice as high as in the control. This ' o v e r s h o o t ' phenomenon was also observed in corneas recovering from epithelial wounds. I t should be noted t h a t phosphocreatine (PCr) also became a p p a r e n t [Fig. 1 (C)]. The hypoxic effect can be diminished somewhat by increasing oxygen permeability, as with high oxygen-permeable silicone lenses ( D k = 55"5), although an u n k n o w n resonance [{Fig. 1 (D)] in the phosphomonoester region appeared. F u r t h e r study is necessary to investigate the nature of this metabolic change. E y e closure caused changes in the 31P-profile, particularly an increase in G P and decrease in A T P which a p p r o x i m a t e d t h a t of corneas fitted with PMMA lenses for 24 hr (Table I). I t should be noted, however, t h a t PC, G P E and GPC levels stayed relatively constant. The electron micrographs are shown in Fig. 2. PMMA-CL fitted corneas suffered severe d a m a g e after 24 hr, whereas eyes with oxygen-permeable CL or lid closure had minimal change. The morphological abnormalities are accompanied by a decrease in PC, G P E and GPC, suggesting a possible correlation between these compounds and cell structure. PAS staining of PMMA-CL fitted corneas showed m u c h less intense staining t h a n the control, indicating extensive loss of glycogen in the former. 26-2
772
K. T S U B O T A E T AL. TABLE 1
Change of P-31 profiles of rabbit corneas under various conditions. % Phosphorus signal
GP SP PC P1 GPE GPC PCr ATP ADP+N+DS
Normal
PMMAI2
PPMA24
PMMA48
Recov
0p48
2"1 8"4 3"2 15.8 3-2 3"2 0"0 50'5 13-7
5"4 9"7 2"7 12-4 2"7 3"2 0"0 45-4 18"4
5"5 14.8 0"9 22"2 1.9 1-9 0-0 33"3 19.4
3"8 6-4 0"0 38"5 0"0 1"3 0"0 11"5 38"5
2"5 .15"4 2"6 10"3 1"3 3"8 2"6 46"2 15"4
1"7 8-5 1"6 16"1 3.4 3-4 1"7 45"8 17"8
LidC12 LidC24 3"8 9"2 2.3 15"4 2-3 3"8 0'0 39"2 23"8
4"6 11-5 2-2 15"3 2"3 3"8 0"0 36"6 23'7
Phosphorus signal intensity was estimated by integrating the areas under each resonance. Values shown are the mean of duplicate experiments (eight corneas per experiment). PMMA12, 24, 48: PMMA lenses worn for 12-; 24-; and 48 hr, respectively. Recov : Recovery after 24 hr of PMMA lens wear. Op48 : Oxygen-permable lenses worn for 48 hr. LidC12, 24: Lid closure ibr 12- and 24 hr, respectively. Abbreviations : GP : a-glycerophosphate ; SP : sugar phosphates ; PC : phosphoryieholine ; P~ : inorganic phosphate ; GPE : glycerophosphorylethanolamine ; GPC : glycerophosphoryleholine ; PCr : phosphocreatine; ATP: a-, /?-, and y-ATP; ADP: a- and /?-ADP; N: dinucleotids; DS: nueleoside diphosphosugar.
4. D i s c u s s i o n T h e m e t a b o l i c c h a n g e s i n P M M A - C L c o r n e a s a n d in e y e c l o s u r e p r o b a b l y r e s u l t e d f r o m h y p o x i a a n d a c t i v a t i o n o f a n a e r o b i c g l y c o l y s i s , as s h o w n b y T h o f t a n d F r i e n d (1975). T h e a p p e a r a n c e o f P C r i n t h e r e c o v e r i n g c o r n e a is i n t e r e s t i n g , as i t i n d i c a t e s a c t i v a t i o n o f m i t o c h o n d r i a l a c t i v i t y . T h i s p r o c e s s m a y b e s i m i l a r t o t h a t in m u s c l e , i n w h i c h r e c o v e r y is v i a t h e o x i d a t i o n o f l a c t a t e a n d p y r u v a t e , l e a d i n g t o t h e r e g e n e r a t i o n o f P C r , A T P , a n d g l y c o g e n . C o r n e a s m a y h a v e a s i m i l a r c a p a c i t y , so t h a t deleterious metabolic consequence can be avoided. Clinically, CL patients are i n s t r u c t e d t o w e a r t h e i r P M M A l e n s e s for less t h a n 12 h r p e r d a y . D u r i n g t h e p e r i o d when the lenses were removed, the corneas can again be exposed to atmospheric o x y g e n . H o w e v e r , a l t h o u g h 31p m e t a b o l i s m r e t u r n e d t o n o r m a l [Fig. 1(C)1, t h e g l y c o g e n r e s e r v e w a s o n l y p a r t i a l l y r e s t o r e d , as i n d i c a t e d b y t h e r e c o v e r y e x p e r i m e n t [Fig. 2 (E)]. F u r t h e r , i f t h e r e c o v e r y p e r i o d c o i n c i d e s w i t h e y e c l o s u r e (i.e. sleep), t h e metabolic recovery may not be total. T h e a p p e a r a n c e o f a n u n k n o w n (in t h e p h o s p h o m o n o e s t e r r e g i o n ) in c o r n e a s f i t t e d w i t h o x y g e n - p e r m e a b l e C L s is u n e x p e c t e d (Fig. 1). C h a n g e in m e m b r a n e m e t a b o l i s m
FIG. 2. Scanning and electron micrographs of rabbit corneas. Left: scanning electron micrograph; center: transmission electron micrograph ; right : periodic acid-Schiff (PAS) staining. (A) Control cornea; (B) : after 24 hr of PMMA CL wear; (C) after 48 hr of PMMA CL wear ; (D) after 48 hr of silicone lens (Dk = 12"5) wear ; (E) recovery for 24 hr after PMMA lens wear for 48 hr; and (F) lid closure for 24 hr. The nearly normal features in (A), (D), (E) and (F) should be noted. PAS staining intensity decreased with increasing duration of PMMA lens wear (0-, 24-, and 48 hr), suggesting a depletion of glycogen. Only minor changes were observed in the corneas fitted with oxygen-permeable silicone lenses.
HARD
CONTACT
LENS INDUCED
CORNEAL
METABOLIC
CHANGES
773
A
B
C
D
E
F
FIG. 2. For legend see facing page.
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K. TSUBOTA ET AL.
in PMMA-CL fitted corneas, i.e. fluctuation of PC, GPE, and GPC, is a novel finding. The decrease in PC can be attributed to the decrease in ATP, required for the phosphorylation of choline. The significance of the change in GPE and GPC is not clear. Burt and Ribolow (1984) have proposed that both phosphodiesters may be metabolic messengers between the cytoplasm and the membrane and can regulate membrane order. As PC, GPE and GPC are cytosolic metabolites, as are other 31p metabolites shown in Fig. 1, their selective loss cannot be explained by traumainduced cell disintegration alone. Disturbance apparently occurred to corneal membrane metabolite pattern under CL-induced hypoxia. The deterioration of corneal epithelial integrity appears to be associated with a decrease in membrane metabolites PC, GPC, and GPE (Table I and Fig. 2), but not with ATP levels. In sum, these data show multifaceted change in corneal metabolism induced by hard CL wear. These reversible changes include energy metabolism and previously unreported disturbance to membrane metabolites. Further studies on the effect of CL parameters such as the base curve, thickness, diameter and materials are currently under way. ACKNOWLEDGMENTS This project was supported by grants EY07620 (to H.M.C.) from the National Eye Institute, Bethesda, MD. NM1% experiments were conducted at Francis Bitter National Magnet Laboratory, Massachusetts Institute of Technology (supported by 1%R0095from the National Institutes of Health). Contact lenses used in this study were donated by Hoya Corporation, Tokyo. REFERENCES Alfonso, E., Mandelbaum, S., Fox, M. J. and Forster, R.K. (1986). Ulcerative keratitis associated with contact lens wear. Am. J. Ophthalmol. 101,429-33. Barr, J. T. and Schoessler, J. P. (1980). Corneal endothelial response to rigid contact lenses. Am. J. Optom. Physiol. Opt. 57, 267-74. Begmanson, J. P. G. and Chu, L. W-F. (1982). Contact lens-induced corneal epithelial injury. Am. J. Optom. Physiol. Opt. 59, 500-6. Burns, R. P., Roberts, H. and Rich, L. F. (1971). Effect of silicone contact lenses on corneal epithelial metabolism. Am. J. Ophthalmol. 71,486-9. Burt, C. T. and Ribolow, H. J. (1984). A hypothesis: noncyclic phosphodiesters may play a role in membrane control. Biochem. Med. 31, 21-30. Cheng, H.-M. and Gonzdlez, 1%. G. (1986). The effect of high glucose and oxidative stress on lens metabolism, aldose reductase and senile cataractogenesis. Metabolism 35 (Suppl. l), 10-14. Davis, B. D., Dulbecco, R., Eisen, H. N. and Grisherg, H. S. (1984). In Microbiology. Pp. 831-832. Harper & Row: Philadelphia. Greiner, J. V., Braude, L. S. and Glonek, T. (1985). Distribution of phosphatic metabolites in the porcine cornea using phosphorus-31 nuclear magnetic resonance. Exp. Eye Res. 40, 335-42. Honan, P. R. (1979). Complications associated with hard contact lenses. Ophthalmology 86, 1102-3.
Klyce, S. D. (1981). Stromal lactate accumulation can account for corneal edema osmotically following epithelial hypoxia in the rabbit. J. Physiol. (London) 321, 49 64. MacRae, S. M., Matsuda, M. and Yee, R. (1985). The effect of long-term hard contact lens wear on the corneal endothelium. CLAO J. 11,322-6. Matsuda, M., MacRae, S. M., Inaba, M. and Manabe, R. (1989). The effect of hard contact lens wear on the keratoconic corneal endothelium after penetrating keratoplasty. Am. J. Ophthalmol. 107, 246-51.
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Olson, L. E., Hoffert, J. R. and Fromm, P. O. (1970). Glucose metabolism and maintenance of transparancy in ocular tissues of rainbow trout. Exp. Eye Res. 9, 260-7. Riley, M. V. (1969a). Glucose and oxygen utilization by the rabbit cornea. Exp. Eye Res. 8, 193-200. Riley, M. V. (1969b). Glycolysis in the ox cornea. Exp. Eye Res. 8, 201~4. Sherrard, E. S. (1978). Characterization of changes observed in the corneal endothelium with the specular microscope. Invest. Ophthalmol. Vis. Sci. 17, 322 6. Thoft, R. A. and Friend, J. (1972). Corneal epithelial glucose utilization. Arch. Ophthalmol. ~ , 58-62. Thoft, R . A . and Friend, J. (1975). Biochemical aspects of contact lens wear. Am. J. Ophthalmol. 80, 139-45. Zantos, S. G. and Holden, B. A. (1977). Transient endothelial changes soon after wearing contact lenses. Am. J. Optom. Physiol. Opt. 54, 856-8.
Hard Contact L e n s - I n d u c e d M e t a b o l i c C h a n g e s In Rabbit Corneas KAZUO TSUBOTA* t , K E N N E T H R. KENYON t AND H o N G - M I N G
CHENG*~
* Howe Laboratory of Ophthalmology, Harvard Medical School and the Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, MA 02114, U.S.A. and ?Eye Research Institute, 20 Staniford Street, Boston, MA 02114, U.S.A. (Received 14 September 1988 and accepted in revised form 20 June 1989) The biochemistry of contact lens-cornea interaction is not well understood, although previous studies have suggested that corneal metabolic changes may be the underlying factor in morphological alterations. Using a rabbit model, this interaction has been examined with 3~p nuclear magnetic resonance (NMR) spectroscopy, which detects signals principally from the epithelium. The examination was supplemented with electron microscopy and histochemistry. Polymethylmethacrylate lenses caused reversible changes, including activation of anaerobic glycolysis and disturbance of membrane metabolite levels. These changes were far more severe than those occurring during prolonged eye closure. There appears to be an association between cellular deterioration and loss of membrane metabolites. On the other hand, oxygen-permeable silicone lenses allowed maintenance of nearly normal metabolic patterns. These results show multifaceted corneal response to hard contact lens wear. Key words: contact lens ; cornea ; Dk ; NMR ; glycolysis; membrane nmtabolism.
1. I n t r o d u c t i o n Most studies on c o r n e a - c o n t a c t lens (CL) i n t e r a c t i o n h a v e e m p h a s i z e d m o r p h o l o g i c a l aspects. I t is well k n o w n t h a t h a r d c o n t a c t lenses can alter corneal thickness a n d e n d o t h e l i a l cell s h a p e / d e n s i t y , a n d cause epithelial i n j u r y (see, e.g. Z a n t o s a n d H o l d e n , 1977 ; S h e r r a r d , 1978 ; B a r r a n d Schoessler, 1980; B e g m a n s o n a n d Chu, 1982 ; M a c R a e , M a t s u d a a n d Yee, 1985; M a t s u d a , M a c R a e , I n a b a a n d Manabe, 1989). These changes are m o s t l y t r a n s i e n t ; nevertheless, l o n g - t e r m h a r d CL wear can lead to corneal complications, p a r t i c u l a r l y u l c e r a t i v e k e r a t i t i s (Honan, 1979; Alfonso, M a n d e l b a u m , F o x a n d F o r s t e r , 1986). The b i o c h e m i c a l basis of C L - i n d u c e d changes, on t h e o t h e r hand, is n o t as well d o c u m e n t e d . Burns, R o b e r t s a n d Rich (1971) found an increase of A T P a n d l a c t a t e a n d a decrease of glycogen a n d glucose following p o l y m e t h y l m e t h a c r y l a t e (PMMA) lens wear. T h o f t a n d F r i e n d (1975) r e p o r t e d similar results, e x c e p t t h a t A T P decreased with a c o n c o m i t a n t increase of A D P . The reason for this d i s c r e p a n c y is unclear, a l t h o u g h a loss in A T P can b e t t e r e x p l a i n declined corneal functions such as t h e ionic p u m p s . K l y c e (1981) p r o p o s e d t h a t a c c u m u l a t i o n of l a c t a t e m a y be the osmotic basis of corneal edema, whereas B a r r a n d Schoessler (1980) suggested t h a t h a r d CLs m a y a d v e r s e l y affect the e n d o t h e l i a l b i c a r b o n a t e p u m p a n d therefore corneal deturgescence. Conflicting d a t a a n d differing v i e w p o i n t s n o t w i t h s t a n d i n g , it is conceivable t h a t C L - i n d u c e d change in corneal m e t a b o l i s m m a y be t h e basis of m o r p h o l o g i c a l complications. B o t h aerobic a n d a n a e r o b i c glycolysis are a c t i v e in the cornea (Riley, 1969a,b; Olson, Hoffert a n d F r o m m , 1970; Thoft a n d F r i e n d , 1972). The cornea r e s p o n d s to t r a u m a a n d h y p o x i a b y v a r y i n g the p r o p o r t i o n of these two modes (Thoft a n d F r i e n d , $* To whom correspondence should be addressed, at the Howe Laboratory of Ophthalmology. 0014-4835/89/110769+07 $03.00/0 26
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K. TSUBOTA ET AL.
1975). T r a u m a causes an initial decrease b u t e v e n t u a l r e c o v e r y of A T P a n d glycogen levels. H y p o x i a , on t h e o t h e r hand, results in decreased A T P a n d glycogen, increased l a c t a t e , a n d d i m i n i s h e d o x y g e n u p t a k e . T h e e x t e n t of t r a u m a t i c i n j u r y a n d h y p o x i a therefore d e t e r m i n e t h e final corneal t o l e r a n c e to CLs. I n this p a p e r , t h e i n t e r a c t i o n b e t w e e n corneal e p i t h e l i u m a n d the h a r d CL is e x a m i n e d . As m o s t ( > 9 5 % ) a l p signals, e x c e p t t h a t of inorganic p h o s p h a t e Pi, originate from t h e e p i t h e l i u m (Greiner, B r a u d e a n d Glonek, 1985), the all) n u c l e a r m a g n e t i c resonance (NMR) a p p r o a c h is ideal for this purpose. A d d i t i o n a l l y , electron m i c r o s c o p y was used to e x a m i n e the a s s o c i a t e d m o r p h o l o g y , a n d periodic a c i d - S c h i f f (PAS) staining was used to e s t i m a t e t h e glycogen content. The N M R results a n d t h e i r m o r p h o l o g i c a l correlations are r e p o r t e d here.
2. M a t e r i a l s a n d M e t h o d s New Zealand albino rabbits, weighing 2-5 kg, were fitted with hard contact lenses in one eye, and the contralateral eye served as the control. The lenses were made of either polymethylmethacrylate (PMMA) or oxygen-permeable silicone lenses (Dk -- 12-5 or 55-5, manufacturer's specification). They were the same in design, each having a diameter of 13 mm and a base curve of 7"8 ram. This design permitted total coverage of the cornea (diameter l0 ram). The lens curvatures were steeper than those of the corneas as determined with fluorescein stain. After 12-, 24-, and 49 hr of continuous wear, the lenses were removed, the animals (eight in each group) were killed, and the eyes were enucleated and immediately frozen in liquid nitrogen. The corneas were excised and extracted with perchloric acid. In the eye closure experiment, designed to provide information on corneal metabolism under baseline hypoxia, the closure period was either 12- or 24 hr. The eyes/corneas were processed as described above. Extreme care had been taken to ensure that the final samples contained the same corneal wet weight per unit volume, at 2'5 g m1-1 NMR solution (20 % DsO + 20 mM EDTA), so the signal intensities from different samples (e.g. Fig. 1) could be compared quantitatively. F o r all samples, the alP-NMR spectroscopy was conducted at 109"3 MHz, using a Bruker HX270 spectrometer in the Fourier-transform mode. The NMR parameters were pulse angle 60 ~ (14 #see pulse duration), sweep width +_2500 Hz, recycle delay 0"5 see, line broadening 2 Hz, and 160000 acquisitions per spectrum. The resonances were identified according to previously described procedures (Cheng and Gonzglez, 1986). Electron microscopy was performed on corneas fixed in 4 % glutaraldehyde in 0"1 M eaeodylate (pH 7"2) and post-fixed in 1% OsO 4. Sectioning was achieved using a Sorvall MT2B mierotome on samples embedded in Epon 812. PAS staining for polysaccharides (Davis, Dulbeceo, Eisen and Grisherg, 1984) was performed to estimate glycogen content in the corneas.
3. R e s u l t s a l P - N M R s p e c t r a of the perchloric acid (PCA) e x t r a c t s of r a b b i t corneas are shown in Fig. 1, which includes the control corneas (A), corneas fitted w i t h P M M A lenses for 48 hr (B), corneas allowed to recover for 24 hr following 24 hr of P M M A lens wear (C), a n d corneas fitted w i t h o x y g e n - p e r m e a b l e silicone lenses (D/c---55-5) (D). These e x p e r i m e n t s were r e p e a t e d twice with t h e s a m e results. A c o m p a r i s o n between Fig. I(A) a n d (B) clearly shows t h a t w i t h P M M A lenses (1) A T P a n d glycerop h o s p h o r y l c h o l i n e (GPC) levels b o t h decreased b y 50 %, (2) p h o s p h o r y l c h o l i n e (PC) a n d g l y c e r o p h o s p h o r y l e t h a n o l a m i n e (GPE) d i s a p p e a r e d , a n d (3) a - g l y c e r o p h o s p h a t e (GP) doubled. A l t h o u g h cell loss occurred d u r i n g CL wear, this w o u l d n o t in itself affect t h e r e l a t i v e p r o p o r t i o n of the cellular m e t a b o l i t e s . These changes progressed after 12 hr of lens wear (Table I).
HARD CONTACT LENS-INDUCED
CORNEAL
METABOLIC
I
CHANGES
771
~ \l\
I GPE GPC
C
A
~ A T P ~
o
-Io
-;s
-5o
6(ppm) FIG. 1. atp NMR spectra of the PCA extracts of rabbit corneas. (A) Normal rabbit corneas, resonances include ccPOEO-glycerophosphate (GP), sugar phosphates (SP), phosphorylcholine (PC), inorganic phosphate (the most intense resonance), glycerophosphorylethanolamine (GPE), glycerophosphorylcholine (GPC), dinucleotides (N), ADP and ATP. (B) After 48 hr of PMMA CL wear. (C) After 24 hr of PMMA CL wear, followed by 24 hr of recovery; the appearance of the PCr resonance is noteworthy. (D) After 48 hr of silicone CL (Dk = 55"5) wear ; the appearance of an unknown resonance (X) is noteworthy. E v e n t h o u g h corneas fitted with PMMA lenses showed abnormal 31p-profiles after 12 hr, t h e y were able to recover. A 24-hr period appeared adequate [see Fig. 1 (C) and Table I]; however, the level of metabolites, except PC, was twice as high as in the control. This ' o v e r s h o o t ' phenomenon was also observed in corneas recovering from epithelial wounds. I t should be noted t h a t phosphocreatine (PCr) also became a p p a r e n t [Fig. 1 (C)]. The hypoxic effect can be diminished somewhat by increasing oxygen permeability, as with high oxygen-permeable silicone lenses ( D k = 55"5), although an u n k n o w n resonance [{Fig. 1 (D)] in the phosphomonoester region appeared. F u r t h e r study is necessary to investigate the nature of this metabolic change. E y e closure caused changes in the 31P-profile, particularly an increase in G P and decrease in A T P which a p p r o x i m a t e d t h a t of corneas fitted with PMMA lenses for 24 hr (Table I). I t should be noted, however, t h a t PC, G P E and GPC levels stayed relatively constant. The electron micrographs are shown in Fig. 2. PMMA-CL fitted corneas suffered severe d a m a g e after 24 hr, whereas eyes with oxygen-permeable CL or lid closure had minimal change. The morphological abnormalities are accompanied by a decrease in PC, G P E and GPC, suggesting a possible correlation between these compounds and cell structure. PAS staining of PMMA-CL fitted corneas showed m u c h less intense staining t h a n the control, indicating extensive loss of glycogen in the former. 26-2
772
K. T S U B O T A E T AL. TABLE 1
Change of P-31 profiles of rabbit corneas under various conditions. % Phosphorus signal
GP SP PC P1 GPE GPC PCr ATP ADP+N+DS
Normal
PMMAI2
PPMA24
PMMA48
Recov
0p48
2"1 8"4 3"2 15.8 3-2 3"2 0"0 50'5 13-7
5"4 9"7 2"7 12-4 2"7 3"2 0"0 45-4 18"4
5"5 14.8 0"9 22"2 1.9 1-9 0-0 33"3 19.4
3"8 6-4 0"0 38"5 0"0 1"3 0"0 11"5 38"5
2"5 .15"4 2"6 10"3 1"3 3"8 2"6 46"2 15"4
1"7 8-5 1"6 16"1 3.4 3-4 1"7 45"8 17"8
LidC12 LidC24 3"8 9"2 2.3 15"4 2-3 3"8 0'0 39"2 23"8
4"6 11-5 2-2 15"3 2"3 3"8 0"0 36"6 23'7
Phosphorus signal intensity was estimated by integrating the areas under each resonance. Values shown are the mean of duplicate experiments (eight corneas per experiment). PMMA12, 24, 48: PMMA lenses worn for 12-; 24-; and 48 hr, respectively. Recov : Recovery after 24 hr of PMMA lens wear. Op48 : Oxygen-permable lenses worn for 48 hr. LidC12, 24: Lid closure ibr 12- and 24 hr, respectively. Abbreviations : GP : a-glycerophosphate ; SP : sugar phosphates ; PC : phosphoryieholine ; P~ : inorganic phosphate ; GPE : glycerophosphorylethanolamine ; GPC : glycerophosphoryleholine ; PCr : phosphocreatine; ATP: a-, /?-, and y-ATP; ADP: a- and /?-ADP; N: dinucleotids; DS: nueleoside diphosphosugar.
4. D i s c u s s i o n T h e m e t a b o l i c c h a n g e s i n P M M A - C L c o r n e a s a n d in e y e c l o s u r e p r o b a b l y r e s u l t e d f r o m h y p o x i a a n d a c t i v a t i o n o f a n a e r o b i c g l y c o l y s i s , as s h o w n b y T h o f t a n d F r i e n d (1975). T h e a p p e a r a n c e o f P C r i n t h e r e c o v e r i n g c o r n e a is i n t e r e s t i n g , as i t i n d i c a t e s a c t i v a t i o n o f m i t o c h o n d r i a l a c t i v i t y . T h i s p r o c e s s m a y b e s i m i l a r t o t h a t in m u s c l e , i n w h i c h r e c o v e r y is v i a t h e o x i d a t i o n o f l a c t a t e a n d p y r u v a t e , l e a d i n g t o t h e r e g e n e r a t i o n o f P C r , A T P , a n d g l y c o g e n . C o r n e a s m a y h a v e a s i m i l a r c a p a c i t y , so t h a t deleterious metabolic consequence can be avoided. Clinically, CL patients are i n s t r u c t e d t o w e a r t h e i r P M M A l e n s e s for less t h a n 12 h r p e r d a y . D u r i n g t h e p e r i o d when the lenses were removed, the corneas can again be exposed to atmospheric o x y g e n . H o w e v e r , a l t h o u g h 31p m e t a b o l i s m r e t u r n e d t o n o r m a l [Fig. 1(C)1, t h e g l y c o g e n r e s e r v e w a s o n l y p a r t i a l l y r e s t o r e d , as i n d i c a t e d b y t h e r e c o v e r y e x p e r i m e n t [Fig. 2 (E)]. F u r t h e r , i f t h e r e c o v e r y p e r i o d c o i n c i d e s w i t h e y e c l o s u r e (i.e. sleep), t h e metabolic recovery may not be total. T h e a p p e a r a n c e o f a n u n k n o w n (in t h e p h o s p h o m o n o e s t e r r e g i o n ) in c o r n e a s f i t t e d w i t h o x y g e n - p e r m e a b l e C L s is u n e x p e c t e d (Fig. 1). C h a n g e in m e m b r a n e m e t a b o l i s m
FIG. 2. Scanning and electron micrographs of rabbit corneas. Left: scanning electron micrograph; center: transmission electron micrograph ; right : periodic acid-Schiff (PAS) staining. (A) Control cornea; (B) : after 24 hr of PMMA CL wear; (C) after 48 hr of PMMA CL wear ; (D) after 48 hr of silicone lens (Dk = 12"5) wear ; (E) recovery for 24 hr after PMMA lens wear for 48 hr; and (F) lid closure for 24 hr. The nearly normal features in (A), (D), (E) and (F) should be noted. PAS staining intensity decreased with increasing duration of PMMA lens wear (0-, 24-, and 48 hr), suggesting a depletion of glycogen. Only minor changes were observed in the corneas fitted with oxygen-permeable silicone lenses.
HARD
CONTACT
LENS INDUCED
CORNEAL
METABOLIC
CHANGES
773
A
B
C
D
E
F
FIG. 2. For legend see facing page.
774
K. TSUBOTA ET AL.
in PMMA-CL fitted corneas, i.e. fluctuation of PC, GPE, and GPC, is a novel finding. The decrease in PC can be attributed to the decrease in ATP, required for the phosphorylation of choline. The significance of the change in GPE and GPC is not clear. Burt and Ribolow (1984) have proposed that both phosphodiesters may be metabolic messengers between the cytoplasm and the membrane and can regulate membrane order. As PC, GPE and GPC are cytosolic metabolites, as are other 31p metabolites shown in Fig. 1, their selective loss cannot be explained by traumainduced cell disintegration alone. Disturbance apparently occurred to corneal membrane metabolite pattern under CL-induced hypoxia. The deterioration of corneal epithelial integrity appears to be associated with a decrease in membrane metabolites PC, GPC, and GPE (Table I and Fig. 2), but not with ATP levels. In sum, these data show multifaceted change in corneal metabolism induced by hard CL wear. These reversible changes include energy metabolism and previously unreported disturbance to membrane metabolites. Further studies on the effect of CL parameters such as the base curve, thickness, diameter and materials are currently under way. ACKNOWLEDGMENTS This project was supported by grants EY07620 (to H.M.C.) from the National Eye Institute, Bethesda, MD. NM1% experiments were conducted at Francis Bitter National Magnet Laboratory, Massachusetts Institute of Technology (supported by 1%R0095from the National Institutes of Health). Contact lenses used in this study were donated by Hoya Corporation, Tokyo. REFERENCES Alfonso, E., Mandelbaum, S., Fox, M. J. and Forster, R.K. (1986). Ulcerative keratitis associated with contact lens wear. Am. J. Ophthalmol. 101,429-33. Barr, J. T. and Schoessler, J. P. (1980). Corneal endothelial response to rigid contact lenses. Am. J. Optom. Physiol. Opt. 57, 267-74. Begmanson, J. P. G. and Chu, L. W-F. (1982). Contact lens-induced corneal epithelial injury. Am. J. Optom. Physiol. Opt. 59, 500-6. Burns, R. P., Roberts, H. and Rich, L. F. (1971). Effect of silicone contact lenses on corneal epithelial metabolism. Am. J. Ophthalmol. 71,486-9. Burt, C. T. and Ribolow, H. J. (1984). A hypothesis: noncyclic phosphodiesters may play a role in membrane control. Biochem. Med. 31, 21-30. Cheng, H.-M. and Gonzdlez, 1%. G. (1986). The effect of high glucose and oxidative stress on lens metabolism, aldose reductase and senile cataractogenesis. Metabolism 35 (Suppl. l), 10-14. Davis, B. D., Dulbecco, R., Eisen, H. N. and Grisherg, H. S. (1984). In Microbiology. Pp. 831-832. Harper & Row: Philadelphia. Greiner, J. V., Braude, L. S. and Glonek, T. (1985). Distribution of phosphatic metabolites in the porcine cornea using phosphorus-31 nuclear magnetic resonance. Exp. Eye Res. 40, 335-42. Honan, P. R. (1979). Complications associated with hard contact lenses. Ophthalmology 86, 1102-3.
Klyce, S. D. (1981). Stromal lactate accumulation can account for corneal edema osmotically following epithelial hypoxia in the rabbit. J. Physiol. (London) 321, 49 64. MacRae, S. M., Matsuda, M. and Yee, R. (1985). The effect of long-term hard contact lens wear on the corneal endothelium. CLAO J. 11,322-6. Matsuda, M., MacRae, S. M., Inaba, M. and Manabe, R. (1989). The effect of hard contact lens wear on the keratoconic corneal endothelium after penetrating keratoplasty. Am. J. Ophthalmol. 107, 246-51.
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Olson, L. E., Hoffert, J. R. and Fromm, P. O. (1970). Glucose metabolism and maintenance of transparancy in ocular tissues of rainbow trout. Exp. Eye Res. 9, 260-7. Riley, M. V. (1969a). Glucose and oxygen utilization by the rabbit cornea. Exp. Eye Res. 8, 193-200. Riley, M. V. (1969b). Glycolysis in the ox cornea. Exp. Eye Res. 8, 201~4. Sherrard, E. S. (1978). Characterization of changes observed in the corneal endothelium with the specular microscope. Invest. Ophthalmol. Vis. Sci. 17, 322 6. Thoft, R. A. and Friend, J. (1972). Corneal epithelial glucose utilization. Arch. Ophthalmol. ~ , 58-62. Thoft, R . A . and Friend, J. (1975). Biochemical aspects of contact lens wear. Am. J. Ophthalmol. 80, 139-45. Zantos, S. G. and Holden, B. A. (1977). Transient endothelial changes soon after wearing contact lenses. Am. J. Optom. Physiol. Opt. 54, 856-8.