Graefe's Archive
Graefe's Arch Clin Exp Ophthalmol (1990)228:73-77
lot Clinical and Experimental
Ophthalmology (c) Springer-Verlag 1990
Metabolic analysis of reepithelializing rabbit cornea using phosphorus-31 nuclear magnetic resonance spectroscopy* Ken Hayashi 2'3'4, Hong-Ming Cheng
1,2, Masayuki Iwasaki 4, Hua Xiong 1,2, and Kenneth R. Kenyon 2,3
1 Howe Laboratory of Ophthalmology, Harvard Medical School 2 Massachusetts Eye and Ear Infirmary, 3 Eye Research Institute of Retina Foundation, Boston, MA 02114, USA 4 Department of Ophthalmology, Faculty of Medicine, Kyushu University, 3-1-1 Maidashi. Higashi-ku, Fukuoka, 812 Japan
Abstract. To investigate metabolic differences between the central and peripheral cornea the latter including the limbal area, corneas were dissected and examined using phosphorus-31 (31p) nuclear magnetic resonance spectroscopy. Since most 31p signals originate from the epithelium, 31p spectra of the cornea primarily represent the metabolic state of the epithelium. The spectra of the peripheral cornea showed all phosphorus resonances detected in the whole cornea; in contrast, the central cornea showed no phosphocreatine and glycerophosrylethanolamine, and only low levels of ATP. These results indicate that there is a higher metabolic activity in the peripheral epithelium, especially in the limbal area, than in the central epithelium. To evaluate the metabolic state of corneal epithelium during regeneration, we also examined corneas reepithelializing after 7 mm of central epithelial tissue had been removed by mechanical scraping. Rabbits were killed 24 and 48 h after scraping. The reepithelializing corneas clearly showed an increase in ATP, phosphocreatine, and sugar phosphates with time, although phosphorylcholine remained depressed. These findings suggest that the reepithelializing cornea has an elevated level of energy production and that it may have reached a higher steady state, thereby indicating accelerated metabolism of the epithelium during regeneration.
study by Kuwabara et al. also showed activation of glycolytic enzyme in the migrating edge of the epithelium [13]. More recently, Ebato has reported that the mitotic rate of epithelial cells from the peripheral cornea is much higher than that of cells from the central cornea, suggesting predominance of metabolic state of peripheral epithelium [3]. Our work and that of others has shown that limbal epithelial cells exhibited high activity of metabolic enzymes during reepithelialization and normal maintenance [9, 21]. These studies seem to agree with the a forementioned hypothesis that corneal epithelial cells first proliferate in the limbus and then the cells move centripetally. Glycolysis is the major energy source of corneal epithelium [12, 22-24] and it has been studied extensively [2, 1 I, 15]. On the other hand, there have been only a few reports on phosphorus metabolite levels of reepithelializing cornea [10]. Recently, phosphorus-31 nuclear magnetic resonance (3~p NMR) spectroscopy has been used for the detection and measurement of phosphorus metabolites of the cornea [5-7]. Since most 3Lp signals originate from the epithelium [6], 31p N M R is ideally suited for the study of corneal epithelium. In the present study, we investigated the 31p metabolites in different zones of the cornea and examined the metabolic changes of reepithelializing cornea using 31p NMR.
Introduction
Materials and methods
The regeneration of corneal epithelium is remarkably rapid and occurs with no remodeling of the cornea [16]. Such active and effective epithelial regeneration is unique among biological tissues. Thoft and Friend hypothesized that corneal epithelial cells move centripetally from the peripheral region during epithelial wound healing and normal maintenance [25]. Recently, Schermer et al. found immature keratin expression in the timbal area and proposed that limbat epithelial cells are the source of epithelial renewal [20]. A number of morphologic studies have provided indirect evidence showing that regeneration of corneal epithelium involves elevated metabolic activity. Matsuda et al. demonstrated an increase in number and size of mitochondria in the regenerating epithelium [17]. A histochemical * Supported in part by NIH grants EY07620 (HMC) and EY05799 (KRK), and a Grant-in-Aid from Bausch and Lomb (KH) Offprint requests to: H.-M. Cheng
Tissue preparation
Thirty-one New Zealand white rabbits weighing 2 to 3 kg were used. For corneal epithelial scraping, the rabbits were anesthesized with ketamine chloride intramuscularly (50 mg/kg weight). The center of each cornea was marked with a 7-mm diameter surgical trephine, and the epithelium within the marked zone was completely removed by mechanical scraping with a No. 15 Bard-Parker blade. Special care was taken not to injure the stroma. The size of the epithelial defect was estimated under the operating microscope and using fluorescein staining immediately after epithelial removal and again when the rabbits were killed. All rabbits were killed by air embolism after sedation with ketamine chloride (50 mg/kg weight). Twelve rabbits were used for the examination of the distribution of phosphorus metabolites in various layers of the cornea (Exp. 1). Four rabbits served as control. The
74
other eight rabbits underwent corneal epithelial removal according to the method mentioned above. Four rabbits each were killed either immediately or 48 h after scraping. Eyes were enucleated immediately after killing and rapidly frozen in liquid nitrogen. Frozen central corneas were then separated using a surgical trephine 7 m m in diameter. Four rabbits were used for the study of the distribution of phosphorus metabolites in the central and peripheralcorneas, the latter including the limbal area (Exp. 2). Both eyes were enucleated immediately after killing and rapidly frozen in liquid nitrogen. The center (7 mm in diameter) of frozen corneas (n = 8) was forcefully trephinated and separated from the eyeballs. The peripheral cornea was then cut circumferentially at 0.5 mm posterior to the limbus with a No. 15 Bard-Parker blade. Twelve rabbits were used for the measurement of the phosphorus metabolite levels o f reepitheliaIizing cornea (Exp. 3). Whole cornea (central +peripheral) was used in this experiment. Four rabbits served as control. Corneal epithelia (7 m m in diameter) of the other eight rabbits were removed by scraping. Four rabbits each were killed at 24 h and 48 h after scraping. Eyes were enucleated immediateIy after killing and rapidly frozen in liquid nitrogen. The frozen corneas including a 0.5 mm scleral rim were then excised using a No. 15 Bard-Parker blade. All manipulations for dissecting tissues were performed while the materials were still frozen. Three rabbits were used for histological examination to observe the reepithelializing process. Following epithelial removal, the rabbits were killed immediately, and 24 h, and 48 h after scraping and eyeballs were enucleated.
Pi
Central Cornea Pi
Pi
i
t~P j
C.reepithelializedil~]~,~
GPC
Fig. 1. Phosphorus-31 nuclear magnetic resonance spectra of central rabbit corneas. A Central control cornea; B deepithelialized central cornea; and C reepithelialized central cornea. SP, sugar phosphates; PCh, phosphorytcholine; GPC, glycerophosphorylcholine; Pi, inorganic phosphate
Perchloric acid extraction Histological examination of reepitheBalizing cornea Frozen corneas were stored at - 8 0 ° C in preweighed Eppendorf centrifuge tubes. Total weight (corneas + tube) was quickly determined with careful attention to avoid thawing and condensation. Corneal wet weight was calculated by substracting tube weight from the total weight. The corneas were then placed in a precooled m o r t o r for perchloric acid (PCA) extraction. The corneas were ground to a fine powder in a liquid-nitrogen bath and mixed with equal weights of 10% PCA. The mixture was allowed to thaw to 0 ° - 4 ° C and immediately centrifuged at 43500 g for 15 min at 4 ° C. The supernatant was then neutralized with concentrated K O H and the pH adjusted to 10. The sample was centrifuged again to remove precipitates. The supernatant was then lyophilized and kept at - 8 0 ° C until use.
Eyeballs with reepithelializing corneas were immediately prefixated in 4% glutaraldehyde buffered at pH 7.2 with 0.1 M cacodilate for 30 min. The corneas were dissected with 0.5 mm scleral rim and fixed overnight. The fixed corneas were cut sagitally through their center with a sharp razor blade and the half pieces o f the corneas were postfixed by 1% OsO4. After dehydration in a graded series ethanol and clearing with prophylene oxide, they were embedded in Epon 812. Semithin sections were cut on a Sorvall MT2B ultramicrotome (Du Pont; Newtown, Conn) and stained for 1 min in 0.1% Azur-II with 1% borax at 90 ° C, and photographed under the light microscope.
Results Phosphorus-31 nuclear magnetic resonance spectroscopy
Phosphorus metabolites of deepithelialized central cornea (Exp. 1)
Lyophilizates of P C A extracts from the corneas were reconstituted at a concentration of 0.4 g wet weight/ml with an N M R solution of 20 mmol/1EDTA, and 20% D 2 0 , pH 9.0. 31p N M R spectroscopy was conducted at 109.3 M H Z using a Bruker HX270 spectrometer. N M R parameters included a 45 ° pulse angle, 4-K data points, 5000-Hz spectral width, 0.5-s interpulse delay, and proton decoupling. Samples were run at room temperature; 160000 accumulations were averaged per spectrum. An exponential filter resulting in 2 Hz line-broadening was used.
Figure 1 shows the 3 ~p spectra o f the central corneas (7-ram diameter) from the control (A) and from the corneas obtained immediately (B) and 48 h (C) after scraping. Only the phosphomonoester and -diester regions are shown. In the normal control sugar phosphates (SP), phosphorylcholine (PCh), glycerophosphorylcholine (GPC), and inorganic phosphate (Pi) are seen. When the corneas were completely deepithelialized, only Pi was detected. However, in the corneas taken at 48 h after scraping, at which the corneas had been already reepithelialized, SP, Pi, and G P C recovered
75 ATP
SP
Peripheral Cornea including Limbal Area
Central
Pi
I I
]/
GPC
|1~
I
|
Cornea
sP ~
f---'A°P7 ~
PCr
GPc
i
l
~ ATP
[
~
~
.8
Fig. 2A, B. Phosphorus-31 nuclear magnetic resonance spectra of central and peripheral corneas. A Central cornea and B peripheral cornea (including limbal area). GPE, glycerophosphorylethanolamine
Reepithelializing
Cornea Pi ATP
C.48 h
SP \
C( G.c GPE ~
:
I
I
I
PCr
ATP
B.24h
A. Control
I
ATP
r
to virtually the same levels as normal controls (SP 96%, Pi 98%, G P C 85%).
Phosphorus metabolites of central and peripheral cornea (Exp. 2) Figure 2 shows the 3~p profiles o f the central (A) and peripheral corneas, the latter including the limbal area (B). The peripheral cornea (Fig. 2A) contained all resonances
(3
Fig. 3A-C. Phosphorus-31 nuclear magnetic resonance spectra of reepithelializing whole (central + peripheral) rabbit corneas. A Normal control cornea; B corneas taken 24 h after scraping; and C cornea taken 48 h after scraping
detected in the whole cornea (Fig. 3 A). The identity of these resonances agrees with that o f previous studies [5-7]. In contrast, the central cornea showed only SP, PCh, GPC, Pi, and low levels of A T P (Fig. l B). Both phosphocreatine (PCr) and glycerophosphorylethanolamine (GPE) were not detected. The percentage o f metabolic distribution in the peripheral cornea relative to the whole cornea is estimated to be SP 90%, PCh 96%, G P E 100%, G P C 80%, P C r 100%, and A T P 88%. The experiment was carried out three times
76
k
[[
-,m
i
Fig, 4A-D. Morphology of reepithelializing corneas. A-C Original magnification, x 100; D, x 80. A Central cornea just after scraping, epithelium is completely removed and the underlying stroma remains intact; St, stroma. B Central cornea 24 h after scraping, monolayer of regenerating epithelium resurfaces the denuded region; Ep, epithelium. C Central cornea 48 h after scraping, whole cornea is resurfaced with a multilayer of regenerated epithelium. D Limbal region 48 h after scraping, reaction of limbal vasculature is minimal ; En, endothelial cells of limbal vessels
under the same conditions and the results were identical. W h e n cornea is dissected with 7-mm diameter, the ratio of peripheral area to the central area is calculated to be a b o u t 3/1 according to the formula o f K w o k [14]. W h e n the difference o f the area is taken into calculation, the postulated percentage of metabolic distribution in peripheral cornea relative to the whole cornea is estimated to be SP 76%, PCh 89%, G P E 100%, G P C 57 %, PC r 100%, and A T P 71%.
Phosphorus metabolites of reepithelializmg cornea (Exp. 3) Figure 3 shows the 31p spectra o f normal control cornea (A) and reepithelializing corneas at 24 h (B) and 48 h (C) after scraping. Fluorescein staining of the cornea showed that the diameter o f the epithelial defect zone was approximately 2 m m after 24 h. N o epithelial defect could be seen after 48 h in all corneas. The reproducibility of the 31p N M R m e t h o d was evident. There was no change in signal intensity o f G P C and G P E t h r o u g h o u t the 48-h period. The ratio o f G P C / G P E remained constant at 3/1. F o r ease o f comparison, the P C r / G P E ratios were calculated; they increased from t.06 + 0.20 at 0 time to 2.33 _+0.09 after 48 h. A similar increase o f SP and A T P , both at 20% and 100% after 24 h and 48 h respectively, was also observed. On the other hand, PCh decreased 50% after 24 h. Since the epithelial defect zone after 24 h was estimated to be less than 5% of total corneal epithelial area and reepithelialization
was complete by 48 h, the increase o f ATP, PCr, and SP is independent of the reepithelialized area; in other words, there a p p e a r e d to be an actual increase o f p h o s p h o r u s metabolite levels.
Histological examination of reepithelializing cornea Right after scraping, the central 7-ram zone was completely deepithelialized, and the stroma underneath remained intact (Fig. 4A). A t 24 h after scraping, m o n o - and multiple layers o f regenerating epithelium covered the peripheral region o f previously denuded area (Fig. 4 B). After 48 h, the whole cornea was resurfaced by a multilayer o f regenerating epithelium that appeared the same as n o r m a l corneal epithelial layer (Fig. 4C). T h r o u g h o u t this process, vascular reaction o f limbal vasculature was not seen (Fig. 4D).
Discussion In evaluating 31p spectra o f the cornea, it is i m p o r t a n t to understand in which layer o f the cornea the metabolites are distributed. When the completely deepithelialized central corneas were examined (Fig. 1 B), only Pi could be observed; this indicates that most resonances detected in our experiment were located in the epithelium. Indeed, 48 h after scraping (Fig. IC), all resonances recovered to the same level as the normal control (Fig. 1 A).
77 Figure 2 clearly demonstrates the different distribution of phosphorus metabolites in the central and peripheral corneas, the latter including the limbal area: both PCr and G P E are not detected in the central cornea. Moreover, the percentage of metabolic distribution of other signals in the peripheral cornea are evidently more than that in the central cornea. Since PCr is a parameter of mitochondrial activity, this result seems to suggest that the peripheral cornea predominates metabolically. Limbal epithelium has been assumed to be the source of epithelial cell renewal [20] and has elevated metabolic activity [9, 21]. It is likely that limbal epithelium is the principal site of 3~p resonances and changes seen in Exp. 3 may indicate an acceleration of epithelial cell metabolism in this region. As shown in Fig. 3, both PCr and A T P increased with time during reepithelialization. After 24 h (Fig. 3 B), the levels of PCr and A T P increased 33% and 20%, respectively, relative to the normal control (Fig. 3 A). More interestingly, at 48 h when epithelial regeneration had been completed (Fig. 3 C), these high energy phosphates increased further. This increase may be due to an elevated production because of increased glucose and glycogen utilization. A decrease in A T P / P C r consumption is unlikely because of the high energy demand during cell replication. Alternatively, the regenerating epithelium may have reached a new steady state, since the elevated levels of A T P and PCr continued even at 96 h, when the corneas had already reepithelialized (data not shown). This is in agreement with the cardiac muscle in which high energy phosphates " o v e r s h o o t " during recovery from ischemia [1, 4]. The increase in ATE and PCr in reepithelializing cornea indicates that corneal epithelial cells are metabolically activated during regeneration. Moreover, since most signals are from the peripheral cornea, metabolic acceleration occurs principally in the peripheral cornea, especially in the limbal area. Previous studies on reepithelializing corneas reported either no significant change or a decrease in A T P levels [18, 19]. This may be due to differences in assay sensitivity or sample preparation. Our 31p N M R results clearly show a prominent rise in high energy phosphorus metabolites, suggesting an acceleration of cellular metabolism during reepithelialization.
Acknowledgements. We wish to thank Prof. Hajime Inomata of Kyushu University for providing the equipment for histological study and Dr. L. Stephen Kwok for his expert advice for the calculation of ocular surface area. References
1. Brooks WM, Willis RJ (1983) 31p nuclear magnetic resonance study of the recovery characteristics of high energy phosphate compounds and intracellular pH after global ischemia in the perfused guinea-pig heart. J Mol Cell Cardiol 15:495-502 2. Burns RP, Roberts H (1969) Effect of wounding on the corneal epithelial glycogen and related enzyme. Invest Ophthalmol 6:541-547 3. Ebato B, Friend J, Thoft RA (1987) Comparison of central and peripheral corneal epithelium in culture. Invest Ophthalmol Vis Sci 28:1450-1463
4. I:lahcrt 5 JT, Wcisfcldt M L, Bulkley BH, Gardncr T J, Gott VL, Jacobs WE (1982) Mechanisms of ischemic myocardial cell damage assessed by phosphorus-31 nuclear magnetic resonance. Circulation 65:561-570 5. Greiner JV, Kopp SJ, Gillette TE, Glonek T (1983) Phosphatic metabolites of the intact cornea by phosphorus-31 nuclear resonance. Invest Ophthalmol Vis Sci 24:535-542 6. Greiner JV, Kopp SJ, Glonek T (1984) Nondestructive metabolic analysis of cornea using phosphorus nuclear magnetic resonance. Arch Ophthalmol 102 : 770-771 7. Greiner JV, Lass JH, Glonek T (1984) Ex vivo metabolic analysis of eye bank corneas using phosphorus nuclear magnetic resonance. Arch Ophthalmol 102:1171-1173 8. Greiner JV, Braude LS, Glonek T (1985) Distribution of phosphatic metabolites in the porcine cornea using phosphorus-31 nuclear magnetic resonance. Exp Eye Res 40:335-342 9. Hayashi K, Kenyon KR (1988) Increased cytochrome oxidase activity in alkali-burned corneas. Curt Eye Res 7:131-138 10. Henninghausen U, Schmidt-Martens FW, Reim M (1972) Metabolitspiegel und Enzymaktivitaten des Energie Iiefernden Stoffwechsels im regenerierenden Corneaepithel. Ber Dtsch Ophthalmol Ges 71:95-99 11. Herrmann H, Hickman FH (1948) Exploratory studies on corneal metabolism. Bull Johns Hopkins Hosp 82:225-250 12. Kinoshita JH (1962) Some aspects of the carbohydrate metabolism of the cornea. Invest Ophthalmol 1:178-186 13. Kuwabara T, Perkins DG, Cogan DG (1976) Sliding of the epithelium in experimental corneal wounds. Invest Ophthalmol 15:4-14 14. Kwok LS (1986) Kinetics of epithelial wound healing in the rabbit cornea. J Infer Deduc Biol 1 : 1-I 5 15. Langham ME (1954) Glycotysis in the cornea of the rabbit. J Physiol 126:396M03 16. Lemp MA (1976) Cornea and sclera. Arch Ophthalmol 94: 473-490 17. Matsuda H, Smelser GK (1973) Electron microscopy of corneal wound healing. Exp Eye Res 16:427-442 18. Reim M, Schmidt-Martens FW (1967) Biochemische VerS.nderungen bei der Vereisung der Hornhaut in vivo. Klin Monatsbl Augenheilkd 150: 96-103 19. Reim M, Conze A, Kaszuba HJ (1982) Adenosine phosphate and glutathione levels in the regenerated corneal epithelium after abrasion and mild alkali burns. Graefe's Arch Clin Exp Ophthalmol 218:42-45 20. Schermer A, Galvin S, Sun T-T (1986) Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggest limbat location of corneal stem cells. J Celt Biol 103 : 49-62 21. Steuhl K-P, Thiel TJ (1987) Histochemical and morphological study of the regenerating corneal epithelium after limbus-tolimbus denudation. Graefe's Arch Clin Exp Ophthalmol 225:53-58 22. Thoft RA, Friend J (1972) Corneal epithelial glucose utilization. Arch OphthaImol 88:58-62 23. Thoft RA, Friend J (1975) Biochemical aspects of contact lens wear. Am J Ophthalmol 80:139-145 24. Thoft RA, Friend J (1976) Corneal epithelial changes during midterm storage. Invest Ophthalmol 15:82-88 25. Thoft RA, Friend J (1983) The X, Y, Z hypothesis of corneal epithelial maintenance. Invest Ophthalmol Vis Sci 24:1442-1443 Received June 5, 1989 / Accepted August 24, 1989
Graefe's Arch Clin Exp Ophthalmol (1990)228:73-77
lot Clinical and Experimental
Ophthalmology (c) Springer-Verlag 1990
Metabolic analysis of reepithelializing rabbit cornea using phosphorus-31 nuclear magnetic resonance spectroscopy* Ken Hayashi 2'3'4, Hong-Ming Cheng
1,2, Masayuki Iwasaki 4, Hua Xiong 1,2, and Kenneth R. Kenyon 2,3
1 Howe Laboratory of Ophthalmology, Harvard Medical School 2 Massachusetts Eye and Ear Infirmary, 3 Eye Research Institute of Retina Foundation, Boston, MA 02114, USA 4 Department of Ophthalmology, Faculty of Medicine, Kyushu University, 3-1-1 Maidashi. Higashi-ku, Fukuoka, 812 Japan
Abstract. To investigate metabolic differences between the central and peripheral cornea the latter including the limbal area, corneas were dissected and examined using phosphorus-31 (31p) nuclear magnetic resonance spectroscopy. Since most 31p signals originate from the epithelium, 31p spectra of the cornea primarily represent the metabolic state of the epithelium. The spectra of the peripheral cornea showed all phosphorus resonances detected in the whole cornea; in contrast, the central cornea showed no phosphocreatine and glycerophosrylethanolamine, and only low levels of ATP. These results indicate that there is a higher metabolic activity in the peripheral epithelium, especially in the limbal area, than in the central epithelium. To evaluate the metabolic state of corneal epithelium during regeneration, we also examined corneas reepithelializing after 7 mm of central epithelial tissue had been removed by mechanical scraping. Rabbits were killed 24 and 48 h after scraping. The reepithelializing corneas clearly showed an increase in ATP, phosphocreatine, and sugar phosphates with time, although phosphorylcholine remained depressed. These findings suggest that the reepithelializing cornea has an elevated level of energy production and that it may have reached a higher steady state, thereby indicating accelerated metabolism of the epithelium during regeneration.
study by Kuwabara et al. also showed activation of glycolytic enzyme in the migrating edge of the epithelium [13]. More recently, Ebato has reported that the mitotic rate of epithelial cells from the peripheral cornea is much higher than that of cells from the central cornea, suggesting predominance of metabolic state of peripheral epithelium [3]. Our work and that of others has shown that limbal epithelial cells exhibited high activity of metabolic enzymes during reepithelialization and normal maintenance [9, 21]. These studies seem to agree with the a forementioned hypothesis that corneal epithelial cells first proliferate in the limbus and then the cells move centripetally. Glycolysis is the major energy source of corneal epithelium [12, 22-24] and it has been studied extensively [2, 1 I, 15]. On the other hand, there have been only a few reports on phosphorus metabolite levels of reepithelializing cornea [10]. Recently, phosphorus-31 nuclear magnetic resonance (3~p NMR) spectroscopy has been used for the detection and measurement of phosphorus metabolites of the cornea [5-7]. Since most 3Lp signals originate from the epithelium [6], 31p N M R is ideally suited for the study of corneal epithelium. In the present study, we investigated the 31p metabolites in different zones of the cornea and examined the metabolic changes of reepithelializing cornea using 31p NMR.
Introduction
Materials and methods
The regeneration of corneal epithelium is remarkably rapid and occurs with no remodeling of the cornea [16]. Such active and effective epithelial regeneration is unique among biological tissues. Thoft and Friend hypothesized that corneal epithelial cells move centripetally from the peripheral region during epithelial wound healing and normal maintenance [25]. Recently, Schermer et al. found immature keratin expression in the timbal area and proposed that limbat epithelial cells are the source of epithelial renewal [20]. A number of morphologic studies have provided indirect evidence showing that regeneration of corneal epithelium involves elevated metabolic activity. Matsuda et al. demonstrated an increase in number and size of mitochondria in the regenerating epithelium [17]. A histochemical * Supported in part by NIH grants EY07620 (HMC) and EY05799 (KRK), and a Grant-in-Aid from Bausch and Lomb (KH) Offprint requests to: H.-M. Cheng
Tissue preparation
Thirty-one New Zealand white rabbits weighing 2 to 3 kg were used. For corneal epithelial scraping, the rabbits were anesthesized with ketamine chloride intramuscularly (50 mg/kg weight). The center of each cornea was marked with a 7-mm diameter surgical trephine, and the epithelium within the marked zone was completely removed by mechanical scraping with a No. 15 Bard-Parker blade. Special care was taken not to injure the stroma. The size of the epithelial defect was estimated under the operating microscope and using fluorescein staining immediately after epithelial removal and again when the rabbits were killed. All rabbits were killed by air embolism after sedation with ketamine chloride (50 mg/kg weight). Twelve rabbits were used for the examination of the distribution of phosphorus metabolites in various layers of the cornea (Exp. 1). Four rabbits served as control. The
74
other eight rabbits underwent corneal epithelial removal according to the method mentioned above. Four rabbits each were killed either immediately or 48 h after scraping. Eyes were enucleated immediately after killing and rapidly frozen in liquid nitrogen. Frozen central corneas were then separated using a surgical trephine 7 m m in diameter. Four rabbits were used for the study of the distribution of phosphorus metabolites in the central and peripheralcorneas, the latter including the limbal area (Exp. 2). Both eyes were enucleated immediately after killing and rapidly frozen in liquid nitrogen. The center (7 mm in diameter) of frozen corneas (n = 8) was forcefully trephinated and separated from the eyeballs. The peripheral cornea was then cut circumferentially at 0.5 mm posterior to the limbus with a No. 15 Bard-Parker blade. Twelve rabbits were used for the measurement of the phosphorus metabolite levels o f reepitheliaIizing cornea (Exp. 3). Whole cornea (central +peripheral) was used in this experiment. Four rabbits served as control. Corneal epithelia (7 m m in diameter) of the other eight rabbits were removed by scraping. Four rabbits each were killed at 24 h and 48 h after scraping. Eyes were enucleated immediateIy after killing and rapidly frozen in liquid nitrogen. The frozen corneas including a 0.5 mm scleral rim were then excised using a No. 15 Bard-Parker blade. All manipulations for dissecting tissues were performed while the materials were still frozen. Three rabbits were used for histological examination to observe the reepithelializing process. Following epithelial removal, the rabbits were killed immediately, and 24 h, and 48 h after scraping and eyeballs were enucleated.
Pi
Central Cornea Pi
Pi
i
t~P j
C.reepithelializedil~]~,~
GPC
Fig. 1. Phosphorus-31 nuclear magnetic resonance spectra of central rabbit corneas. A Central control cornea; B deepithelialized central cornea; and C reepithelialized central cornea. SP, sugar phosphates; PCh, phosphorytcholine; GPC, glycerophosphorylcholine; Pi, inorganic phosphate
Perchloric acid extraction Histological examination of reepitheBalizing cornea Frozen corneas were stored at - 8 0 ° C in preweighed Eppendorf centrifuge tubes. Total weight (corneas + tube) was quickly determined with careful attention to avoid thawing and condensation. Corneal wet weight was calculated by substracting tube weight from the total weight. The corneas were then placed in a precooled m o r t o r for perchloric acid (PCA) extraction. The corneas were ground to a fine powder in a liquid-nitrogen bath and mixed with equal weights of 10% PCA. The mixture was allowed to thaw to 0 ° - 4 ° C and immediately centrifuged at 43500 g for 15 min at 4 ° C. The supernatant was then neutralized with concentrated K O H and the pH adjusted to 10. The sample was centrifuged again to remove precipitates. The supernatant was then lyophilized and kept at - 8 0 ° C until use.
Eyeballs with reepithelializing corneas were immediately prefixated in 4% glutaraldehyde buffered at pH 7.2 with 0.1 M cacodilate for 30 min. The corneas were dissected with 0.5 mm scleral rim and fixed overnight. The fixed corneas were cut sagitally through their center with a sharp razor blade and the half pieces o f the corneas were postfixed by 1% OsO4. After dehydration in a graded series ethanol and clearing with prophylene oxide, they were embedded in Epon 812. Semithin sections were cut on a Sorvall MT2B ultramicrotome (Du Pont; Newtown, Conn) and stained for 1 min in 0.1% Azur-II with 1% borax at 90 ° C, and photographed under the light microscope.
Results Phosphorus-31 nuclear magnetic resonance spectroscopy
Phosphorus metabolites of deepithelialized central cornea (Exp. 1)
Lyophilizates of P C A extracts from the corneas were reconstituted at a concentration of 0.4 g wet weight/ml with an N M R solution of 20 mmol/1EDTA, and 20% D 2 0 , pH 9.0. 31p N M R spectroscopy was conducted at 109.3 M H Z using a Bruker HX270 spectrometer. N M R parameters included a 45 ° pulse angle, 4-K data points, 5000-Hz spectral width, 0.5-s interpulse delay, and proton decoupling. Samples were run at room temperature; 160000 accumulations were averaged per spectrum. An exponential filter resulting in 2 Hz line-broadening was used.
Figure 1 shows the 3 ~p spectra o f the central corneas (7-ram diameter) from the control (A) and from the corneas obtained immediately (B) and 48 h (C) after scraping. Only the phosphomonoester and -diester regions are shown. In the normal control sugar phosphates (SP), phosphorylcholine (PCh), glycerophosphorylcholine (GPC), and inorganic phosphate (Pi) are seen. When the corneas were completely deepithelialized, only Pi was detected. However, in the corneas taken at 48 h after scraping, at which the corneas had been already reepithelialized, SP, Pi, and G P C recovered
75 ATP
SP
Peripheral Cornea including Limbal Area
Central
Pi
I I
]/
GPC
|1~
I
|
Cornea
sP ~
f---'A°P7 ~
PCr
GPc
i
l
~ ATP
[
~
~
.8
Fig. 2A, B. Phosphorus-31 nuclear magnetic resonance spectra of central and peripheral corneas. A Central cornea and B peripheral cornea (including limbal area). GPE, glycerophosphorylethanolamine
Reepithelializing
Cornea Pi ATP
C.48 h
SP \
C( G.c GPE ~
:
I
I
I
PCr
ATP
B.24h
A. Control
I
ATP
r
to virtually the same levels as normal controls (SP 96%, Pi 98%, G P C 85%).
Phosphorus metabolites of central and peripheral cornea (Exp. 2) Figure 2 shows the 3~p profiles o f the central (A) and peripheral corneas, the latter including the limbal area (B). The peripheral cornea (Fig. 2A) contained all resonances
(3
Fig. 3A-C. Phosphorus-31 nuclear magnetic resonance spectra of reepithelializing whole (central + peripheral) rabbit corneas. A Normal control cornea; B corneas taken 24 h after scraping; and C cornea taken 48 h after scraping
detected in the whole cornea (Fig. 3 A). The identity of these resonances agrees with that o f previous studies [5-7]. In contrast, the central cornea showed only SP, PCh, GPC, Pi, and low levels of A T P (Fig. l B). Both phosphocreatine (PCr) and glycerophosphorylethanolamine (GPE) were not detected. The percentage o f metabolic distribution in the peripheral cornea relative to the whole cornea is estimated to be SP 90%, PCh 96%, G P E 100%, G P C 80%, P C r 100%, and A T P 88%. The experiment was carried out three times
76
k
[[
-,m
i
Fig, 4A-D. Morphology of reepithelializing corneas. A-C Original magnification, x 100; D, x 80. A Central cornea just after scraping, epithelium is completely removed and the underlying stroma remains intact; St, stroma. B Central cornea 24 h after scraping, monolayer of regenerating epithelium resurfaces the denuded region; Ep, epithelium. C Central cornea 48 h after scraping, whole cornea is resurfaced with a multilayer of regenerated epithelium. D Limbal region 48 h after scraping, reaction of limbal vasculature is minimal ; En, endothelial cells of limbal vessels
under the same conditions and the results were identical. W h e n cornea is dissected with 7-mm diameter, the ratio of peripheral area to the central area is calculated to be a b o u t 3/1 according to the formula o f K w o k [14]. W h e n the difference o f the area is taken into calculation, the postulated percentage of metabolic distribution in peripheral cornea relative to the whole cornea is estimated to be SP 76%, PCh 89%, G P E 100%, G P C 57 %, PC r 100%, and A T P 71%.
Phosphorus metabolites of reepithelializmg cornea (Exp. 3) Figure 3 shows the 31p spectra o f normal control cornea (A) and reepithelializing corneas at 24 h (B) and 48 h (C) after scraping. Fluorescein staining of the cornea showed that the diameter o f the epithelial defect zone was approximately 2 m m after 24 h. N o epithelial defect could be seen after 48 h in all corneas. The reproducibility of the 31p N M R m e t h o d was evident. There was no change in signal intensity o f G P C and G P E t h r o u g h o u t the 48-h period. The ratio o f G P C / G P E remained constant at 3/1. F o r ease o f comparison, the P C r / G P E ratios were calculated; they increased from t.06 + 0.20 at 0 time to 2.33 _+0.09 after 48 h. A similar increase o f SP and A T P , both at 20% and 100% after 24 h and 48 h respectively, was also observed. On the other hand, PCh decreased 50% after 24 h. Since the epithelial defect zone after 24 h was estimated to be less than 5% of total corneal epithelial area and reepithelialization
was complete by 48 h, the increase o f ATP, PCr, and SP is independent of the reepithelialized area; in other words, there a p p e a r e d to be an actual increase o f p h o s p h o r u s metabolite levels.
Histological examination of reepithelializing cornea Right after scraping, the central 7-ram zone was completely deepithelialized, and the stroma underneath remained intact (Fig. 4A). A t 24 h after scraping, m o n o - and multiple layers o f regenerating epithelium covered the peripheral region o f previously denuded area (Fig. 4 B). After 48 h, the whole cornea was resurfaced by a multilayer o f regenerating epithelium that appeared the same as n o r m a l corneal epithelial layer (Fig. 4C). T h r o u g h o u t this process, vascular reaction o f limbal vasculature was not seen (Fig. 4D).
Discussion In evaluating 31p spectra o f the cornea, it is i m p o r t a n t to understand in which layer o f the cornea the metabolites are distributed. When the completely deepithelialized central corneas were examined (Fig. 1 B), only Pi could be observed; this indicates that most resonances detected in our experiment were located in the epithelium. Indeed, 48 h after scraping (Fig. IC), all resonances recovered to the same level as the normal control (Fig. 1 A).
77 Figure 2 clearly demonstrates the different distribution of phosphorus metabolites in the central and peripheral corneas, the latter including the limbal area: both PCr and G P E are not detected in the central cornea. Moreover, the percentage of metabolic distribution of other signals in the peripheral cornea are evidently more than that in the central cornea. Since PCr is a parameter of mitochondrial activity, this result seems to suggest that the peripheral cornea predominates metabolically. Limbal epithelium has been assumed to be the source of epithelial cell renewal [20] and has elevated metabolic activity [9, 21]. It is likely that limbal epithelium is the principal site of 3~p resonances and changes seen in Exp. 3 may indicate an acceleration of epithelial cell metabolism in this region. As shown in Fig. 3, both PCr and A T P increased with time during reepithelialization. After 24 h (Fig. 3 B), the levels of PCr and A T P increased 33% and 20%, respectively, relative to the normal control (Fig. 3 A). More interestingly, at 48 h when epithelial regeneration had been completed (Fig. 3 C), these high energy phosphates increased further. This increase may be due to an elevated production because of increased glucose and glycogen utilization. A decrease in A T P / P C r consumption is unlikely because of the high energy demand during cell replication. Alternatively, the regenerating epithelium may have reached a new steady state, since the elevated levels of A T P and PCr continued even at 96 h, when the corneas had already reepithelialized (data not shown). This is in agreement with the cardiac muscle in which high energy phosphates " o v e r s h o o t " during recovery from ischemia [1, 4]. The increase in ATE and PCr in reepithelializing cornea indicates that corneal epithelial cells are metabolically activated during regeneration. Moreover, since most signals are from the peripheral cornea, metabolic acceleration occurs principally in the peripheral cornea, especially in the limbal area. Previous studies on reepithelializing corneas reported either no significant change or a decrease in A T P levels [18, 19]. This may be due to differences in assay sensitivity or sample preparation. Our 31p N M R results clearly show a prominent rise in high energy phosphorus metabolites, suggesting an acceleration of cellular metabolism during reepithelialization.
Acknowledgements. We wish to thank Prof. Hajime Inomata of Kyushu University for providing the equipment for histological study and Dr. L. Stephen Kwok for his expert advice for the calculation of ocular surface area. References
1. Brooks WM, Willis RJ (1983) 31p nuclear magnetic resonance study of the recovery characteristics of high energy phosphate compounds and intracellular pH after global ischemia in the perfused guinea-pig heart. J Mol Cell Cardiol 15:495-502 2. Burns RP, Roberts H (1969) Effect of wounding on the corneal epithelial glycogen and related enzyme. Invest Ophthalmol 6:541-547 3. Ebato B, Friend J, Thoft RA (1987) Comparison of central and peripheral corneal epithelium in culture. Invest Ophthalmol Vis Sci 28:1450-1463
4. I:lahcrt 5 JT, Wcisfcldt M L, Bulkley BH, Gardncr T J, Gott VL, Jacobs WE (1982) Mechanisms of ischemic myocardial cell damage assessed by phosphorus-31 nuclear magnetic resonance. Circulation 65:561-570 5. Greiner JV, Kopp SJ, Gillette TE, Glonek T (1983) Phosphatic metabolites of the intact cornea by phosphorus-31 nuclear resonance. Invest Ophthalmol Vis Sci 24:535-542 6. Greiner JV, Kopp SJ, Glonek T (1984) Nondestructive metabolic analysis of cornea using phosphorus nuclear magnetic resonance. Arch Ophthalmol 102 : 770-771 7. Greiner JV, Lass JH, Glonek T (1984) Ex vivo metabolic analysis of eye bank corneas using phosphorus nuclear magnetic resonance. Arch Ophthalmol 102:1171-1173 8. Greiner JV, Braude LS, Glonek T (1985) Distribution of phosphatic metabolites in the porcine cornea using phosphorus-31 nuclear magnetic resonance. Exp Eye Res 40:335-342 9. Hayashi K, Kenyon KR (1988) Increased cytochrome oxidase activity in alkali-burned corneas. Curt Eye Res 7:131-138 10. Henninghausen U, Schmidt-Martens FW, Reim M (1972) Metabolitspiegel und Enzymaktivitaten des Energie Iiefernden Stoffwechsels im regenerierenden Corneaepithel. Ber Dtsch Ophthalmol Ges 71:95-99 11. Herrmann H, Hickman FH (1948) Exploratory studies on corneal metabolism. Bull Johns Hopkins Hosp 82:225-250 12. Kinoshita JH (1962) Some aspects of the carbohydrate metabolism of the cornea. Invest Ophthalmol 1:178-186 13. Kuwabara T, Perkins DG, Cogan DG (1976) Sliding of the epithelium in experimental corneal wounds. Invest Ophthalmol 15:4-14 14. Kwok LS (1986) Kinetics of epithelial wound healing in the rabbit cornea. J Infer Deduc Biol 1 : 1-I 5 15. Langham ME (1954) Glycotysis in the cornea of the rabbit. J Physiol 126:396M03 16. Lemp MA (1976) Cornea and sclera. Arch Ophthalmol 94: 473-490 17. Matsuda H, Smelser GK (1973) Electron microscopy of corneal wound healing. Exp Eye Res 16:427-442 18. Reim M, Schmidt-Martens FW (1967) Biochemische VerS.nderungen bei der Vereisung der Hornhaut in vivo. Klin Monatsbl Augenheilkd 150: 96-103 19. Reim M, Conze A, Kaszuba HJ (1982) Adenosine phosphate and glutathione levels in the regenerated corneal epithelium after abrasion and mild alkali burns. Graefe's Arch Clin Exp Ophthalmol 218:42-45 20. Schermer A, Galvin S, Sun T-T (1986) Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggest limbat location of corneal stem cells. J Celt Biol 103 : 49-62 21. Steuhl K-P, Thiel TJ (1987) Histochemical and morphological study of the regenerating corneal epithelium after limbus-tolimbus denudation. Graefe's Arch Clin Exp Ophthalmol 225:53-58 22. Thoft RA, Friend J (1972) Corneal epithelial glucose utilization. Arch OphthaImol 88:58-62 23. Thoft RA, Friend J (1975) Biochemical aspects of contact lens wear. Am J Ophthalmol 80:139-145 24. Thoft RA, Friend J (1976) Corneal epithelial changes during midterm storage. Invest Ophthalmol 15:82-88 25. Thoft RA, Friend J (1983) The X, Y, Z hypothesis of corneal epithelial maintenance. Invest Ophthalmol Vis Sci 24:1442-1443 Received June 5, 1989 / Accepted August 24, 1989
