effect of hyperglycemia duration on rabbit corneal thickness and endothelial atpase activity

- ACTA OPHTHALMOLOGICA SCANDINAVICA 1995

Effect of hyperglycemia duration on rabbit corneal thickness and endothelial ATPase activitv

J

Peter Herse and Linda Adams

Department of Optometry, University of Auckland, New Zealand

ABSTRACT. This study compares corneal thickness and corneal endothelial Na,K ATPase activity in normal and age-matched diabetic rabbits with up to 10 weeks of hyperglycemia. Corneal endothelial Na,K ATPase activity in normal rabbits increased between 10 and 20 weeks of age. Corneal thickness increased after 5 weeks of hyperglycemia. Decreased corneal endothelial homogenate Na,K ATPase activity was first seen after 10 weeks of hyperglycemia, though an earlier onset was possible due to sampling restrictions. Corneal endothelial homogenate Mg + + ATPase activity showed a transient increase after 2 to 5 weeks of hyperglycemia. This study shows that hyperglycemia in rabbit is associated with complex dynamic interactions between corneal thickness and corneal endothelial Mg + + ATPase and Na,K ATPase activities.

Key words: corneal thickness - diabetes - Na,K ATPase

Acta Ophthalrnol. Scand. 1995: 73: 158-161

iabetes has been associated with D alterations in the function of the corneal endothelium as evidenced by changes in its morphology (Schultz et al. 1984), increased overall corneal thickness (Herse 1990a), and a decreased ability to recover from contact lens induced edema (Herse & Hooker 1994). Maintenance of normal corneal hydration and thickness has been linked to corneal endothelial sodium, potassium adenosine triphosphatase (Na,K ATPase) activity (Geroski et al. 1084). Reduced Na,K ATPase activity has been demonstrated in a number of diabetic tissues including retina, lens, renal glomerulus and sciatic nerve (Greene & Lattimer 1983; MacGreor & Matschinsky 1986; Garner & Spec- tor 1986; Cohen et al. 1985). It has been reported that 10 weeks of hy-

per glycemia in rabbit was associated with reduced corneal endothelial Na,K ATPase activity and increased corneal thickness (Herse 1990b). If corneal thickness is directly linked to endothelial Na,K ATPase activity, then this may suggest that increased corneal thickness may occur soon after commencement of hyperglycemia. How- ever, increased corneal thickness was not seen in the alloxan-induced diabetic rabbit model until about 5 weeks after commencement of hyperglycemia (Herse 1990a). Similarly, it has re- cently been reported that 8 weeks of hyperglycemia was required to produce a significant reduction in retinal Na,K ATPase activity in streptozotocin-induced diabetic rat (Ottlecz et al. 1993). At present, the pattern of changes in corneal endothelial Na,K

ATPase activity over this crucial ‘incu- bation’ period is unknown. The pur- pose of this investigation is to extend the previous studies of Herse (1990a,b) by more fully characterizing the time course of the effects of hyperglycemia on corneal thickness and endothelial Na,K ATPase activity in the alloxan-induced diabetic rabbit model.

Methods Corneal thickness and endothelial Na,K ATPase activities were measured in 2 groups of 10 normal male New Zealand white rabbits (10 and 15 weeks of age), and in 2 groups of 10 age-matched diabetic male New Zea- land white rabbits (2 and 5 weeks of hyperglycemia duration). Hyperglyce- mia was induced by injection in the marginal ear vein of a solution of 110 mg/kg alloxan. The ‘diabetic’ threshold was arbitrarily set at 10 mmol/l blood glucose as measured with a Reflolux II/ BM 1-44 tester. Corneal thickness was measured using an Technar ultrasound pachometer set at 1580 m/sec. Cor- neal endothelial homogenate activity was measured using the method of Whikehart & Soppet (1981). Ethics approval had been obtained from the University of Auckland Animal Ethics Committee. Statistical significance was tested using analysis of variance followed when necessary by Fisher’s PLSD post-hoc comparisons (a =

0.05). For further detail regarding the method, the reader is referred to Herse (1990b).

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Results

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1) Blood glucose Blood glucose levels did not appear to vary between the ages of 10 and 20 weeks in the normal group (p = 0.08). Blood glucose levels for the diabetic group were on average 2.3 times greater (15.0 mmol/l) than the normal group (6.7 mmol/l). The level of hyperglycemia was variable between animals and over time. Data obtained from an earlier study (Herse 1990a) from similar 20 week old normal and diabetic (10 weeks hyperglycemia) rabbits are included for comparison.

2) Corneal thickness The corneal thickness data for the rabbits of this study are shown in Fig. 1. Also included in Fig. 1 at the 20 week normal and diabetic (10 weeks hyperglycemia) positions are data taken from an earlier study (Herse 1990a). This inclusion is considered valid due to the close similarity of the corneal thickness data in both studies for normal and diabetic rabbits of similar ages and durations of hyperglycemia. Cor- neal thickness in the normal group did not appear to vary between 10 and 20 weeks of age (p = 0.96); with the corneal thicknesses of the 10, 15 and 20 week old groups being 364k17 pm (sD), 3 6 4 f 2 0 pm, 363+13 pm, re-

350 3mm 10 I2

I

1 Weeks of age

Fig. 1. Variation in corneal thickness with time in normal (filled bars) and diabetic (un- filled bars) rabbits. The error bars represent f 1 SEM (n = 10). The diabetic rabbits were injected with 1 1 0 mg/kg alloxan at 10 weeks of age. Note that the data for the 20 week rabbits (normal and diabetic) are taken from Herse (IYY0a).

Table 1. Effect of the duration of hyperglycemia on the specific activities of corneal endothelial ATPases in normal and alloxan-induced diabetic New Zealand white rabbits. Mean activities (n = 10) are specified as pmoles Pi released/mg protein/30 rnin. Protein is specified as pg/eye. Errors represent f 1 SEM.

Group Age Diabetes Protein Total Mg++ Na,K (weeks) (weeks) ATPase ATPase ATPase

Control 10 0 1 3 1 f l l 1.1Yf0.08 1.11f0.08 0.09f0.04 Diabetic 12 2 124k 12 1.56f0.16 1.54k0.14* 0.02f0.06 Control 15 0 l4Yk 13 1.20f0.09 1.03f0.08 0.17k0.03 Diabetic 15 5 141 f 9 1.48k0.09 1.31f0.09* 0.18f0.03 Control 20 0 172 f 15 1.01 f 0.05 0.65 f 0.05 0.36 f 0.04 Diabetic 20 10 179 f 13 0.80 f 0.06* 0.6Yf 0.07 0.1 1 f 0.03*

* Represents significantly different from age-matched controls at p < 0.05. Note tht the data for the 20 week rabbits (normal and diabetic) are taken from Herse (l99Ob).

spectively. The corneal thickness of the diabetic group was found to remain relatively constant for the first 2 weeks of hyperglycemia. A significant increase in corneal thickness was first seen to occur after 5 weeks of hyperglycemia.

3) Endothelial ATPase activities Corneal endothelial ATPase activity data for this study are shown in Table 1. Also included for comparison in Table 1 at the 20 week normal and diabetic (10 weeks hyperglycemia) positions are data taken from an earlier study (Herse 1990b). The inclusion of this data is considered valid due to the use in both studies of identical assay procedures, identical rabbit species and sex, identical experimental housing and lighting environments, identical means of hyperglycemia induction and identical tissue preparation procedures. Repetition of the previous study was considered too expensive and raised ethical concerns in the University Ani- mal Ethics Committee. It should be noted that the long-term decrease in corneal endothelial Na,K ATPase activity found in the earlier diabetic rabbit study (Herse 1990b) has been re- plicated by other workers using cultured bovine corneal endothelia (Whikehart et al. 1993).

activities for the normal group did not vary between 10 and 20 weeks of age (p = 0.11). However, there were significant variations in the Mg++ (p= 0.0002) and Na,K ATPase (p = 0.0001 activities. It was found that Mg ++ ac-

The endothelial total Na,K ATPase ,

tivity was significantly less and Na,K ATPase activity significantly greater in 20 weeks old animals than in 10 and 15 week old animals. These results suggest that a maturation of ATPase activity occurs between 10 and 20 weeks of age in the normal rabbit corneal endothelium.

Due to the normal age variation in Mg + + and Na,K ATPase corneal endothelial activity described above, measurement of diabetic rabbit corneal endothelial ATPase activity in- volved comparison of diabetic animals against age and sex-matched normal controls. Note that 10-week-old normal rabbits were contrasted against 12-week-old diabetic rabbits (2 weeks of hyperglycemia). Significant increases in Mg++ ATPase activity were found after both 2 and 5 weeks of hyperglycemia (27-39%). This increase in Mg + + ATPase activity was not evident after 10 weeks of hyperglycemia. Conversely Na,K ATPase activity appeared to remain unaffected by hyperglycemia during the first 5 weeks of the study, with the only statis- tically significance change appearing as a reduction (69%) after 10 weeks of hyperglycemia. A large (78%) reduction in Na,K ATPase activity was noted in the 2 weeks hyperglycemia comparison, though the large variance in the diabetic data at this point resulted in a finding of no statistical significance. In summary; alloxan-induced diabetes in rabbits appears to be associated with a short-term increase in corneal endothelial Mg + + ATPase activity after 2 to 5 weeks hyperglycemia. This is fol-

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- ACTA OPHTHALMOLOGICA SCANDINAVICA 1995

lowed by a return to normal of Mgi-t ATPase activity and a reduction in Na,K ATPase activity after about 10 weeks of hyperglycemia.

Regression analysis demonstrated that endothelial protein levels increase in a linear manner with age in both normal (4 f 2 g/week, p = 0.04) and diabetic (7 f 2 g/week, p = 0.002) groups. No significant difference in protein levels were found between normal and diabetic groups following age-matched contrasts.

Discussion 1) Age variation in corneal Na,K ATPase activity Rabbit corneal thickness has been shown to increase with age until about 12 to 18 weeks (approx. 2 to 3 kg body weight) when the adult corneal thickness is attained (Herse 1990a). As corneal thickness has been linked with corneal endothelial Na,K ATPase activity (Geroski et al. 1984), can a significant relationship be found between endothelial Na,K ATPase activity and age? A significant positive linear regression (slope = 0.03, r2 = 0.49, p = 0.0001) was found between age and endothelial Na,K ATPase activity in the normal rabbit data for the period of 10 to 20 weeks of age (Table 1). As the 20 week normal rabbit data (Herse 1990b) reasonably match literature values for ‘adult’ rabbit corneal thickness and endothelial Na,K ATPase activity from a study using a similar spec- trophotometric method (Zam et al. 1980), it is suggested that a maturation period is required for both rabbit corneal thickness and endothelial Na,K ATPase activity, with adult levels reached at about 20 weeks of age. Sup- port for an age-related maturation of corneal endothelial Na,K ATPase comes from reports that corneal endothelial plasma membrane Na,K ATPase activity increases with time in primary cell cultures (Whikehart et al. 1989) and that the number of rabbit corneal endothelium ouabain-binding Na,K ATPase sites increase with age until maturation of normal corneal hydration and thickness occurs at about 12 weeks (Stiemke et al. 1991). It may appear that the results of the present

study are in conflict with this maturation proposal; with afinding that endothelial Na,K ATPase activity increased while corneal thickness remained constant between the ages of 10 and 20 weeks. However, it should be noted that this apparent conflict is an artifact produced by the small time window used in this study. It has been pre- viously established that rabbit corneal thickness increases until about 18 weeks of age (Herse 1990a).

2) Variation in corneal Na,K ATPase activities following hyperglycemia The effect of hyperglycemia on lens epithelial Na,K ATPase activity has been extensively studied (Garner & Spector 1986). It has been shown that a short period of in vitro hyperglycemia (e.g. 2 days) is sufficient to produce significant reductions in lens epithelial Na,K ATPase activity. What about the cornea? Previous studies have demonstrated that 10 weeks of uncontrolled in vivo hyperglycemia in rabbit was associated with a 69% reduction in corneal endothelial homogenate Na,K ATPase activity (Herse 1990b), and in vitro tissue culture of bovine corneal endothelia in hyperglycemic media (25 mmol/l) for 4 to 5 weeks was associated with a 76% reduction in endothelial plasma membrane Na,K ATPase activity (Whike- hart et al. 1993). Thus, raised glucose levels would appear to be associated with a reduction in endothelial Na,K ATPase activity.

The question remains as to what duration of hyperglycemia is required to produce the activity reduction. A recent study has quantified alterations in retinal ATPase activities in streptozotocin-induced diabetic rats over a period of 6 months (Ottlecz et al. 1993). It was shown that retinal Na,K ATPase activities were significantly reduced (8-14%) only after 4 months of in vivo hyperglycemia. Thus the data suggest that the rapid Na,K ATPase inhibition seen in the in vitro lens situ- ation does not occur in the in vivo retina. This phenomenon was also found in the present study. Reduction in corneal endothelial Na,K ATPase activity was found to occur after an in vivo hyperglycemia duration of between 5 and 10 weeks.

What possible mechanisms could

explain the apparent delay in the inhibition of the corneal endothelial Na,K ATPase activity? For hyperglycemia to have an impact on corneal endothelial enzyme activity, it is necessary to increase the glucose levels in the aqueous humor. It has been shown using fluorophotometry that a breakdown of the blood-retina barrier occurs in human diabetic subjects soon after the onset of hyperglycemia (Cunha-Vaz et al. 1975). Assuming that the blood-aqueous barrier is af- fected by hyperglycemia in a similar manner to the blood-retina barrier, then this would suggest that some short delay should occur between the onset of increased blood glucose levels and increased glucose levels in the aqueous humor and at the corneal endothelium. A further delay may be attributable to the corneal ‘functional reserve’. It has been shown in Fuch’s endothelial dys- trophy that the number of ouabain- binding Na,K ATPase sites increases with the duration of the condition, thus providing a ‘functional reserve’ of Na,K ATPase sites. During the course of the disease this ‘reserve’ is event- ually exhausted, fluid control lost and corneal edema results (Edelhauser et al. 1986). It may be possible that the graudal loss of the blood-aqueous barrier and the corneal endothelial ‘functional reserve’ may help explain the de- layed onset of corneal edema in the diabetic condition. A further possible mechanism has also been proposed. It has been reported (Whikehart et al. 1993) that inhibition of corneal endothelial Na,K ATPase activity occurs only after an extended period of direct contact of living tissue with hyperglycemic media (e.g. 4 to 5 weeks). Short-term in vitro exposure of puri- fied corneal endothelial plasma membranes to hyperglycemic media (e.g. 1 h) was shown to have no significant effect on corneal endothelial Na,K AT- Pase activity. It was also reported in this study that the amount of double stranded DNA in the cultured endothelial cells exposed to long-term hyperglycemia was reduced by about 15%. The interpretation was that long- term hyperglycemia resulted in dam- age to the endothelial DNA and ulti- mately to a decrease in the synthesis of new, active Na,K ATPase (Whikehart et al. 1993). Further research is indi-

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cated to test whether any of these hy- potheses provide useful explanations of the experimental data.

The data of the current study de- monstrate a transient increase in corneal Mg++ ATPase activity after 2 and 5 weeks of hyperglycemia. No significant difference in Mg + + ATPase activity was found after 10 weeks of hyperglycemia. This result support a similar recent finding of an 8 to 13% increase in retinal Mg + + ATPase activity after a 4 to 6 month period of hyperglycemia in in vivo diabetic rat (Ottlecz et al. 1993). These data em- phasize the difficulties in comparing in vivo and in vitro experiments. While Mg + + ATPase activity was found to increase in the in vivo experiments, a recent in vitro tissue culture study has shown a 33% reduction in Mg + + AT- Pase after 4 to 5 weeks exposure to hyperglycemic media (Whikehart et al. 1993). It has also been suggested that Mg + + ATPase and Na,K ATPase activities are linked in a biofeedback mechanism (Whikehart et al. 1989). Decreasing Na,K ATPase activities may down-regulate Mg t + ATPase activity to increase available cellular ATP, and thus up-regulate Na,K AT- Pase activity (Whikehart et al. 1989). If this is so, an early increase in diabetes in retinal (Ottlecz et al. 1993) and corneal endothelial Mg + + ATPase activities would suggest a decrease in cellular ATP and thus a decrease in Na,K ATPase activity. Could an early increase in Mg + + ATPase activity be an important link in the systemic inhibition of Na,K ATPase activity seen in diabetes?

3) Linear regression analysis Linear regression analysis was per- formed on the combined data from the normal and diabetic rabbit groups. No significant relationships were found between corneal thickness and blood glucose level (slope = - 0.35, p = 0.19, r2 = 0.03). The agreement of the current rabbit data with literature reports of no significant relationship between blood glucose and corneal thickness in Type I diabetic human subjects (Busted et al. 1981; Pierro et al. 1993), offers further support for the alloxan- induced diabetic rabbit as a useful model for the human diabetic condition.

Summary The conclusion of this study were:

1. Normal rabbit corneal endothelial Na,K ATPase activity increases between 10 (approx 1.5 kg body weight) and 20 (approx. 3 kg body weight) weeks of age. As the majority of studies in literature use rabbits of these ages, care should be taken when performing longitudinal studies.

2 . Alloxan-induced diabetes in rabbit is associated with a transient increase in corneal endothelial Mg + + ATPase activity after about 2 to 5 weeks of hyperglycemia, followed by a decrease in Na,K ATPase activity after about 5 to 10 weeks of hyperglycemia.

Acknowledgments Supported by grants from the New Zealand Optometric Vision Research Foundation and the New Zealand Lotteries Com- mission.

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Received on June 10th. 1994.

Corresponding author: Peter Herse School of Optometry University of New South Wales Sydney 2052 Australia.

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effect of hyperglycemia duration on rabbit corneal thickness and endothelial atpase activity

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