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Endothelial Function and Aqueous Humor Flow Rate in Patients With Fuchs’ Dystrophy
Endothelial Function and Aqueous Humor Flow Rate in Patients With Fuchs' Dystrophy Steven E. W i l s o n , M . D . , William M. Bourne, M . D . , Peter C. O'Brien, P h . D . , and Richard F. Brubaker, M . D . Using a two-dimensional scanning fluorophotometer, we studied 50 subjects with sym metric ocular involvement of Fuchs' dystrophy without epithelial edema. Twenty-six subjects with confluent or nearly confluent cornea guttata with increased corneal thickness and 24 subjects with mild to moderate cornea guttata with normal corneal thickness were compared to normal control subjects. There were no statistically significant differences in endothe lial permeability between the three groups. Corneal thickness was significantly increased in the subjects with confluent to nearly conflu ent guttae, however. These results suggest that endothelial pump function may be affected in subjects with advanced cornea guttata with stromal edema. FUCHS' DYSTROPHY is a progressive disorder of the corneal endothelium characterized by a decrease in the number and function of endo thelial cells. 1 Clinically, the disease is charac terized by the appearance of small drop-like excrescences (guttae) in Descemet's mem brane, a condition termed cornea guttata. Ulti mately, stromal and epithelial edema may occur, resulting in severe visual loss. The ori gin of the disease is unknown. We undertook the present study to assess endothelial function in Fuchs' dystrophy using the two-dimensional scanning ocular fluoro-
Accepted for publication June 23, 1988. From the Department of Ophthalmology (Drs. Wil son, Bourne, and Brubaker) and the Section of Biostatistics, Department of Health Sciences Research (Dr. O'Brien), Mayo Clinic, Rochester, Minnesota. This study was supported in part by National Institutes of Health grants EY 02037 and EY 00634, Research to Prevent Blindness, Inc., and the Mayo Clinic and Foun dation. Reprint requests to William M. Bourne, M.D., Depart ment of Ophthalmology, Mayo Clinic, Rochester, MN 55905.
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photometer. We also investigated the rate of flow of aqueous humor in Fuchs' dystrophy.
Patients and Methods We recruited 50 patients with stage 1 (no epithelial edema) Fuchs' dystrophy and rela tively symmetric ocular involvement. Twentysix of the patients had confluent or nearly confluent guttae (21 confluent, five nearly con fluent) and 24 had mild to moderate guttae (Fig. 1). We also recruited 41 control subjects without ocular disease except for cataract. We obtained a complete ocular history from all participants and performed a physical exami nation, including biomicroscopy with a slit lamp and a Hruby lens, indirect ophthalmoscopy, and Goldmann tonometry. We measured background fluorescence of the cornea and
Fig. 1 (Wilson and associates). Representative en dothelial specular micrographs of patients with Fuchs' dystrophy with confluent or nearly confluent guttae (top) and mild to moderate guttae (bottom).
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anterior chamber with a two-dimensional scan ning fluorophotometer. 2 All subjects had clear corneal stroma and no epithelial edema. All subjects had an initial intraocular pressure in both eyes of less than or equal to 22 mm Hg. No subject had extraocular or intraocular inflam mation (no conjunctival injection, no inflam matory cells in the cornea on slit-lamp exami nation, and less than one cell per 1 x 2-mm slit-lamp beam in the anterior chamber). No subject was taking ocular medications such as beta-receptor blockers or agonists, antiinflammatory medications, or oral carbonic anhydrase inhibitors for at least two weeks before the start of the study. None of the subjects had previous ocular surgery or ocular abnormalities other than cataract. We obtained detailed in formed consent from each participant. At 4 A.M. on the first day of the study, each subject administered one drop of 2% fluorescein in each eye, followed by a second drop five minutes later. The subject was instructed to remove excess fluorescein from the eyelids with a moist cottonball and return to sleep. We measured fluorescein concentration in the cor neal stroma and anterior chamber with the two-dimensional scanning fluorophotometer at 10 A.M., 11 A.M., 12 noon, 1 P.M., and 2 P.M. Between measurements, the subjects were per mitted to participate in their normal activities. The subjects were instructed to refrain from drinking alcohol or large volumes of water, using any drugs, or eating large meals, any of which might alter anterior chamber aqueous humor dynamics. We instilled another drop of 2% fluorescein in both eyes in most patients from all three groups after the 2 P.M. measurement, when corneal and anterior chamber fluorescein concentra tions had decayed to lower levels. After ten minutes, we repeatfd measurements of fluores cein concentration in! the corrieal stroma arid anterior chamber. The ratio of the change in anterior chamber fluorescein concentration to the change in corneal stroma fluorescein con centration over the ten-minute period was de termined for each eye; this was referred to as the optical boundary function. 3 Ten minutes after this application of fluorescein, little if any of the applied fluorescein would have reached the anterior chamber. Increased optical bound ary function in the patients with Fuchs' dystro phy compared with normal controls would in dicate a false increase in the measured anterior chamber fluorescein concentration caused by fluorescein in the thicker stroma. Polarization
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of fluorescence measurements of the cornea and anterior chamber were made for all sub jects. 4 We then measured corneal thickness with a Heyer-Schulte specular microscope. We calculated endothelial permeability and aqueous humor flow rate using previously pub lished methods. 5,6 Endothelial permeability to fluorescein was determined from the rate of disappearance of fluorescein from the cornea and the gradient between the stroma and the anterior chamber. The cornea-to-anterior chamber mass transfer coefficient (kcca) was calculated using equations derived from those of Jones and Maurice, 7 assuming the stroma-toanterior chamber distribution ratio of fluoresce in at equilibrium (rca) in humans is 1.6.8 We calculated endothelial permeability to fluores cein in centimeters per minute as follows9: permeability = kcca x CT x rca, where kcca is the mean of the values calculated for each of the four hourly intervals and CT is the central corneal thickness in millimeters as measured with the specular microscope. The volume of corneal stroma was assumed to be 70 |xl for corneas with a central thickness of 0.55 mm, the average thickness found in nor mal subjects measured with the same specular microscope. 6 We then calculated corneal vol ume for each subject using the following equa tion to adjust for corneal thickness: corneal volume in \xl = CT/0.55 x 70 (Jil. Anterior chamber volume was determined by using a photogrammetric method. 10 Since subjects with relatively symmetric cor neal involvement were studied, values from the two eyes for endothelial permeability, aqueous humor flow rate, polarization of fluorescence in the stroma, polarization of fluorescence in the anterior chamber, and optical boundary func tion were averaged to give a single value for each parameter for each subject. In seven patients with Fuchs' dystrophy and six normal controls, endothelial permeability and aqueous humor flow rate were measured on five separate occasions, a minimum of five days apart. We selected subjects for multiple measurements so that individuals with both normal and relatively low aqueous humor flow rates on the first measurements were present in each group. Statistical comparisons were performed by using a standard Student's (-test for unpaired
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data. P :£ .05 was considered significant. Addi tionally, data on aqueous humor flow rate and endothelial permeability for the Fuchs' dystro phy and control subjects were compared by means of an extension of the (-test for situa tions in which patients respond differentially to disease, 11 for example, the effect of flow rate on the disease varies from patient to patient. The standard test assumes that if patients with Fuchs' dystrophy have a diminution in flow rate, the magnitude of the diminution is the same in every patient. The mathematical impli cation of this assumption is as follows. Let Y equal the log odds of the probability that a person has Fuchs' dystrophy. If one were to graph Y against flow rate (X), the standard t-test assumes that the plot would be a straight line (which would have zero slope if there were no association between flow rate and disease and a nonzero slope otherwise). The observed relationship in this study was significantly nonlinear (P = .007). Therefore, we used the generalized f-test, which allows for the possi bility that the relationship may be nonlinear.
Results Table 1 contains means and standard devia tions for all of t h e . measured and calculated values for the normal subjects, subjects with mild to moderate guttae, and subjects with confluent to nearly confluent guttae. In one control subject and one subject with mild to moderate guttae, calculations for endothelial permeability and aqueous humor flow rate were not possible for one eye because of inad
vertent transfer of fluorescein into the eye from the eyelids during fluorophotometry. There fore, data for these subjects were not used for statistical computations involving these varia bles. Corneal thickness (Fig. 2) was higher in subjects with advanced cornea guttata com pared with subjects with early cornea guttata or control subjects. Mean endothelial permeabili ty (Fig. 3) was similar in the three groups. The range of values tended to be greater, however, in subjects with cornea guttata. Mean aqueous humor flow rates were also similar for the three groups (Fig. 4). Again, however, the range of aqueous humor flow rates was greater for sub jects with either advanced or early cornea guttata compared with control subjects. In a comparison between the normal subjects and subjects with mild to moderate guttae or confluent to nearly confluent guttae, there was no statistically significant difference between the normal subjects and subjects with mild to moderate cornea guttata for any of the mea sured variables (Table 2). There was also no statistically significant difference between the normal subjects and subjects with confluent to nearly confluent guttae in endothelial permea bility, aqueous humor flow rate, fluorescence polarization of the corneal stroma, or optical boundary function. Statistically significant dif ferences were noted between controls and sub jects with confluent to nearly confluent guttae in corneal thickness and fluorescence polariza tion in the aqueous humor. Corneal thickness and fluorescence polarization in the aqueous humor were increased in subjects with conflu ent to nearly confluent guttae compared with controls. Mean and range of intraocular pres sure were not statistically significantly different
TABLE 1 MEANS OF MEASURED VARIABLES*
AQUEOUS HUMOR FLOW RATE
CORNEAL
NO. OF
AGE
CORNEAL THICKNESS
SUBJECTS
(YHS)
(MM)
( X 1 0 ~ 4 CM/MIN)
(HL'MIN)
Normal
41
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24
63.2 (12.7) 69.2 (10.2) 61.6 (14.4)
0.57 (0.03) 0.56 (0.02) 0.59 (0.03)
4.27 (0.74) 4.32 (1.04) 4.45 (1.14)
2.52 (0.47) 2.35 (0.58) 2.52 (0.83)
GROUP
26
•Numbers in parentheses are standard deviations.
FLUORESCENCE
ENDOTHELIAL PERMEABILITY TO FLUORESCEIN
POLARIZATION
OPTICAL
STROMA
AQUEOUS HUMOR
BOUNDARY FUNCTION
0.1655 (0.0247) 0.1700 (0.0254) 0.1661 (0.0249)
0.0341 (0.0139) 0.0364 (0.0152) 0.0469 (0.0237)
0.023 (0.011) 0.026 (0.007) 0.025 (0.011)
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standard deviation used was that of normal subject group). Since fluorescence polarization in the aque ous humor was statistically significantly in creased in subjects with confluent to nearly confluent guttae compared with normal con trols, correlation coefficients were calculated between fluorescence polarization in the aque ous humor and the other variables in Table 1 for the subjects with confluent to nearly confluent guttae. There were no statistically significant correlations. Endothelial permeability and aqueous humor flow rates for an individual eye appeared to remain stable over time (Figs. 5 and 6). For an individual subject, endothelial permeability and aqueous humor flow rate tended to be similar in the right and left eye. The clustering of either measurement for an individual eye also confirms the precision of the measurement of endothelial permeability and aqueous humor flow rate obtained with the two-dimensional scanning ocular fluorophotometer.
Discussion In the present study, performed with the two-dimensional scanning fluorophotometer, we found no difference in endothelial permea bility between normal subjects and subjects with Fuchs' dystrophy with increased central
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subjects (Fig. 3). In neither of these previous studies was the difference in endothelial per meability between patients with cornea guttata and normal subjects evaluated statistically. Burns, Bourne, and Brubaker, 15 using the slit-lamp fluorophotometer, compared 21 pa tients with prominent cornea guttata without epithelial edema to 17 age- and sex-matched normal controls. A statistically significant in crease in the mean endothelial permeability was found in the patients with Fuchs' dystro phy. The calculated mean endothelial pump rate did not differ between the two groups. These last results suggested that a breakdown in endothelial barrier function was the earliest defect that occurred in Fuchs' dystrophy. Patients with confluent to nearly confluent guttae in the present study were comparable in the extent of disease to subjects in the Burns, Bourne, and Brubaker study. The present study is larger, and subjects with mild to mod erate cornea guttata were also studied. The present study was also performed with the more precise two-dimensional scanning fluorophotometer, and five measurements of stromal and anterior chamber fluorescein con centration were used to calculate the endotheli al permeability for a subject on a particular day instead of three. Figures 5 and 6 demonstrate the precision of the measurements with the two-dimensional scanning fluorophotometer and the relative consistency of these measure ments over time for a particular normal subject or a subject with Fuchs' dystrophy. It must be emphasized that absolute values of endothelial permeability obtained using the slit-lamp fluorophotometer and the twodimensional scanning fluorophotometer are not directly comparable. Mean endothelial per meability to fluorescein determined for control subjects by using the slit-lamp fluoro photometer in the Burns, Bourne, and Brubaker study 15 (2.89 x 10"4 cm/min) was com parable to that found using the same instru ment in a study of 112 normal subjects 16 (2.4 x 10~4 cm/min). Similarly, mean endothelial per meability to fluorescein for control subjects in the present study determined with the twodimensional scanning fluorophotometer (4.27 x 10"4 cm/min) is similar to that found with the same instrument in a study of 80 normal sub jects 6 (4.03 x 10"4 cm/min). Any conclusions regarding changes in endothelial permeability must, therefore, be based on concurrent con trol subjects, regardless of which technique is used.
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Explanations for finding no difference in en dothelial permeability by using a more precise measurement when a difference has been pre viously found with a less precise method in clude the possibility of a type I error in the Burns, Bourne, and Brubaker study. 15 This would be expected to occur 5% of the time. Alternatively, differences between the studies may reflect variability between Fuchs' dystro phy patients in the different studies. Whatever the correct explanation, the present study sug gests that increased endothelial permeability is not an invariable finding in patients with cor nea guttata and increased corneal thickness. Data in the present study suggesting that endothelial permeability is normal in patients with Fuchs' dystrophy" without epithelial edema and that pump function, therefore, di minishes with progression of the disease must, however, be viewed with caution. Several as sumptions that may not be valid are inherent in the methods used to reach this conclusion. 5 We assumed that the cornea-to-anterior chamber distribution ratio (rca), used in the calculation of endothelial permeability, is the same in normal subjects and patients with Fuchs' dystrophy. Whether this is true or not is unknown. The fluorescence of fluorescein in the corneal stroma is known to be quenched to a certain extent compared to fluorescence in water, 1718 although the mechanism for the phenomenon is un known. Whether this quenching effect is differ ent in normal controls and subjects with Fuchs' dystrophy is unknown. If a difference were present, calculations of endothelial permeabili ty would be affected. We have assumed that the baseline central corneal thickness for patients with Fuchs' dystrophy, before developing the disease, does not differ from the normal popu lation. Whether this is true or not is unknown, although the central corneal thickness for the subjects with mild to moderate cornea guttata in this study did not differ statistically from the normal subjects. We also assumed that the rate of transfer of fluorescein across the endothelium is proportional to that of water and that the proportion is the same in the different groups studied. Finally, we used methods that are based on the work of Hedbys and Dohlman 19 and Ytteborg and Dohlman 20 and the assump tions made in reaching their conclusions. Even though endothelial permeability may be normal early in the course of the disease, it may be increased in patients with more ad vanced Fuchs' dystrophy with epithelial edema, since endothelial permeability cannot
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be measured in such corneas by this method. Recent studies have found alterations in ouabain binding sites, adenosine triphosphatase activity, and cytochrome oxidase activity in the endothelium of patients with Fuchs' dystro phy. Geroski and associates, 21 using specific binding of tritiated ouabain, reported a signifi cant increase in Na/K adenosine triphosphatase-associated ouabain binding site densi ty in cadaver eyes with moderate cornea guttata compared with normal cadaver eyes. Na/K adenosine triphosphatase activity was not measured in this study. Conversely, Mc Cartney and colleagues, 22 in a more recent study using tritiated ouabain and autoradiographic techniques, found a decrease in adeno sine triphosphatase-associated ouabain bind ing sites in the endothelium on both a per unit basis and a per cell basis in eyes with advanced Fuchs' dystrophy compared with age-matched eye bank eyes. They also noted a reduction in adenosine triphosphatase activity in the endo thelium in Fuchs' dystrophy. Tuberville, Wood, and McLaughlin 23 recently described a decrease in cytochrome oxidase activity in the central corneal endothelium in Fuchs' dystrophy. Since endothelial pump function is an energyrequiring process, a decrease in p u m p function could be expected in cells in which oxidative phosphorylation activity is markedly de creased. The results of the present study are consistent with these recent experiments The fluorescence polarization of the aqueous humor was statistically significantly higher in the subjects with confluent to nearly confluent guttae compared with normal subjects. This difference could be caused by the difference in corneal thickness, or possibly, a difference in the aqueous humor colloid concentration be tween the two groups. 4 The actual difference was relatively small, and there was no correla tion between this variable and the other varia bles measured in the subjects with confluent to nearly confluent cornea guttata. Data collected for aqueous humor flow rate suggest that there could be a difference in flow between subjects with guttae and normal con trols. The difference is significant using a gen eralized t-test assuming a nonlinear (quadratic) model. 11 Two explanations for the apparent dif ference in variability of aqueous humor flow in these two groups are possible. The difference may be caused by a reduction in the optical uniformity of the cornea in subjects with cor nea guttata. Alternatively, a real difference in rate of aqueous humor flow could be present in
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at least a subgroup of patients with Fuchs' dystrophy. We cannot discern, based on these techniques, which explanation is correct. At least we know that the rate of aqueous humor flow is approximately the same in groups of persons with cornea guttata as in normals, suggesting that guttae do not result from a reduction in the rate of aqueous humor flow in most patients with Fuchs' dystrophy. An alter ation in the composition of aqueous humor, however, cannot be excluded as a contributing factor.
References 1. Wilson, S. E., and Bourne, W. M.: Fuchs' dys trophy. Cornea 7:2, 1988. 2. McLaren, J. W.,'and Brubaker, R. F.: A twodimensional scanning ocular fluorophotometer. In vest. Ophthalmol. Vis. Sci. 26:144, 1985. 3. Brubaker, R. F.: The flow of aqueous humor in the human eye. Trans. Am. Ophthalmol. Soc. 80:391, 1982. 4. Herman, D. C , McLaren, J. W., and Brubaker, R. F.: A method of determining concentration of albumin in the living eye. Invest. Ophthalmol. Vis. Sci. 29:133, 1988. 5. Wilson, S. E., Bourne, W. M., and Brubaker, R. F.: Effect of dexamethasone on corneal endotheli al function in Fuchs' dystrophy. Invest. Ophthalmol. Vis. Sci. 29:357, 1988. 6. Carlson, K. H., Bourne, W. M., and Brubaker, R. F.: Variations in endothelial cell morphology and permeability to fluorescein with age. Exp. Eye Res. In press. 7. Jones, R. F., and Maurice, D. M.: New methods of measuring the rate of aqueous flow in man with fluorescein. Exp. Eye Res. 5:208, 1966. 8. Ota, Y., Mishima, S., and Maurice, D. M.: En dothelial permeability of the living cornea to fluores cein. Invest. Ophthalmol. 13:945, 1974. 9. Coakes, R. L., and Brubaker, R. F.: Methods of measuring aqueous humor flow and corneal endo thelial permeability using a fluorophotometer nomogram. Invest. Ophthalmol. Vis. Sci. 18:288, 1979. 10. Johnson, S. B., Coakes, R. L., and Brubaker, R. F.: A simple photogrammetric method for measur ing anterior chamber volume. Am. J. Ophthalmol. 85:469, 1978. 11. O'Brien, P. C : Comparing two samples. Exten sions of the T, rank sum, and log rank tests. J. Am. Stat. Assoc. 83:52, 1988. 12. Lachin, J. M.: Introduction to sample size de termination and power analysis for clinical trials. Controlled Clin. Trials 2:93, 1981. 13. Stanley, J. A.: Water permeability of the human cornea. Arch. Ophthalmol. 87:568, 1972.
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14. Waltman, S. R., and Kaufman, H. E.: In vivo studies of human corneal endothelial permeability. Am. J. Ophthalmol. 70:45, 1970. 15. Burns, R. R., Bourne, W. M., and Brubaker, R. F.: Endothelial function in patients with cornea guttata. Invest. Ophthalmol. Vis. Sci. 20:77, 1981. 16. Bourne, W. M., Nagataki, S., and Brubaker, R. F.: The permeability of the corneal endothelium to fluorescein in the normal human eye. Curr. Eye Res. 3:509, 1984. 17. Araie, M., and Maurice, D. M.: A reevaluation of corneal endothelial permeability to fluorescein. Exp. Eye Res. 41:383, 1985. 18. Araie, M.: Carboxyfluorescein. A dye for eval uating the corneal endothelial barrier function in vivo. Exp. Eye Res. 42:141, 1986. 19. Hedbys, B. P., and Dohlman, C. H.: A new
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method for the determination of the swelling pres sure of the corneal stroma in vitro. Exp. Eye Res. 2:122, 1963. 20. Ytteborg, J., and Dohlman, C. H.: Corneal edema and intraocular pressure. II. Clinical results. Arch. Ophthalmol. 74:477, 1965. 21. Geroski, D. H., Matsuda, M , Yee, R. W., and Edelhauser, H. F.: Pump function of the human corneal endothelium. Ophthalmology 92:759, 1985. 22. McCartney, M. D., Robertson, D. P., Wood, T. O., and McLaughlin, B. J.: ATPase pump site density in human dysfunctional corneal endotheli um. Invest. Ophthalmol. Vis. Sci. 28:1955, 1987. 23. Tuberville, A. W., Wood, T. O., and McLaughlin, B. J.: Cytochrome oxidase activity of Fuchs' endothelial dystrophy. Curr. Eye Res. 5:939, 1986.
O P H T H A L M I C MINIATURE
"There are people gifted by nature with powers of observation. Without effort they form a sharp impression of whatever is going on around them, in themselves, and in others. Also they know how to cull out of these observations whatever is most significant, typical, or colourful. W h e n you hear such people talk you are struck by the a m o u n t that an unobservant person misses. " O t h e r people are unable to develop this power of observation even sufficiently to preserve their own simplest interests. How much less able, then, are they to do it for the sake of studying life itself." Constantin Stanislavski, An Actor Prepares N e w York, Theatre Arts Books, 1981, p . 86
Accepted for publication June 23, 1988. From the Department of Ophthalmology (Drs. Wil son, Bourne, and Brubaker) and the Section of Biostatistics, Department of Health Sciences Research (Dr. O'Brien), Mayo Clinic, Rochester, Minnesota. This study was supported in part by National Institutes of Health grants EY 02037 and EY 00634, Research to Prevent Blindness, Inc., and the Mayo Clinic and Foun dation. Reprint requests to William M. Bourne, M.D., Depart ment of Ophthalmology, Mayo Clinic, Rochester, MN 55905.
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photometer. We also investigated the rate of flow of aqueous humor in Fuchs' dystrophy.
Patients and Methods We recruited 50 patients with stage 1 (no epithelial edema) Fuchs' dystrophy and rela tively symmetric ocular involvement. Twentysix of the patients had confluent or nearly confluent guttae (21 confluent, five nearly con fluent) and 24 had mild to moderate guttae (Fig. 1). We also recruited 41 control subjects without ocular disease except for cataract. We obtained a complete ocular history from all participants and performed a physical exami nation, including biomicroscopy with a slit lamp and a Hruby lens, indirect ophthalmoscopy, and Goldmann tonometry. We measured background fluorescence of the cornea and
Fig. 1 (Wilson and associates). Representative en dothelial specular micrographs of patients with Fuchs' dystrophy with confluent or nearly confluent guttae (top) and mild to moderate guttae (bottom).
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anterior chamber with a two-dimensional scan ning fluorophotometer. 2 All subjects had clear corneal stroma and no epithelial edema. All subjects had an initial intraocular pressure in both eyes of less than or equal to 22 mm Hg. No subject had extraocular or intraocular inflam mation (no conjunctival injection, no inflam matory cells in the cornea on slit-lamp exami nation, and less than one cell per 1 x 2-mm slit-lamp beam in the anterior chamber). No subject was taking ocular medications such as beta-receptor blockers or agonists, antiinflammatory medications, or oral carbonic anhydrase inhibitors for at least two weeks before the start of the study. None of the subjects had previous ocular surgery or ocular abnormalities other than cataract. We obtained detailed in formed consent from each participant. At 4 A.M. on the first day of the study, each subject administered one drop of 2% fluorescein in each eye, followed by a second drop five minutes later. The subject was instructed to remove excess fluorescein from the eyelids with a moist cottonball and return to sleep. We measured fluorescein concentration in the cor neal stroma and anterior chamber with the two-dimensional scanning fluorophotometer at 10 A.M., 11 A.M., 12 noon, 1 P.M., and 2 P.M. Between measurements, the subjects were per mitted to participate in their normal activities. The subjects were instructed to refrain from drinking alcohol or large volumes of water, using any drugs, or eating large meals, any of which might alter anterior chamber aqueous humor dynamics. We instilled another drop of 2% fluorescein in both eyes in most patients from all three groups after the 2 P.M. measurement, when corneal and anterior chamber fluorescein concentra tions had decayed to lower levels. After ten minutes, we repeatfd measurements of fluores cein concentration in! the corrieal stroma arid anterior chamber. The ratio of the change in anterior chamber fluorescein concentration to the change in corneal stroma fluorescein con centration over the ten-minute period was de termined for each eye; this was referred to as the optical boundary function. 3 Ten minutes after this application of fluorescein, little if any of the applied fluorescein would have reached the anterior chamber. Increased optical bound ary function in the patients with Fuchs' dystro phy compared with normal controls would in dicate a false increase in the measured anterior chamber fluorescein concentration caused by fluorescein in the thicker stroma. Polarization
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of fluorescence measurements of the cornea and anterior chamber were made for all sub jects. 4 We then measured corneal thickness with a Heyer-Schulte specular microscope. We calculated endothelial permeability and aqueous humor flow rate using previously pub lished methods. 5,6 Endothelial permeability to fluorescein was determined from the rate of disappearance of fluorescein from the cornea and the gradient between the stroma and the anterior chamber. The cornea-to-anterior chamber mass transfer coefficient (kcca) was calculated using equations derived from those of Jones and Maurice, 7 assuming the stroma-toanterior chamber distribution ratio of fluoresce in at equilibrium (rca) in humans is 1.6.8 We calculated endothelial permeability to fluores cein in centimeters per minute as follows9: permeability = kcca x CT x rca, where kcca is the mean of the values calculated for each of the four hourly intervals and CT is the central corneal thickness in millimeters as measured with the specular microscope. The volume of corneal stroma was assumed to be 70 |xl for corneas with a central thickness of 0.55 mm, the average thickness found in nor mal subjects measured with the same specular microscope. 6 We then calculated corneal vol ume for each subject using the following equa tion to adjust for corneal thickness: corneal volume in \xl = CT/0.55 x 70 (Jil. Anterior chamber volume was determined by using a photogrammetric method. 10 Since subjects with relatively symmetric cor neal involvement were studied, values from the two eyes for endothelial permeability, aqueous humor flow rate, polarization of fluorescence in the stroma, polarization of fluorescence in the anterior chamber, and optical boundary func tion were averaged to give a single value for each parameter for each subject. In seven patients with Fuchs' dystrophy and six normal controls, endothelial permeability and aqueous humor flow rate were measured on five separate occasions, a minimum of five days apart. We selected subjects for multiple measurements so that individuals with both normal and relatively low aqueous humor flow rates on the first measurements were present in each group. Statistical comparisons were performed by using a standard Student's (-test for unpaired
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data. P :£ .05 was considered significant. Addi tionally, data on aqueous humor flow rate and endothelial permeability for the Fuchs' dystro phy and control subjects were compared by means of an extension of the (-test for situa tions in which patients respond differentially to disease, 11 for example, the effect of flow rate on the disease varies from patient to patient. The standard test assumes that if patients with Fuchs' dystrophy have a diminution in flow rate, the magnitude of the diminution is the same in every patient. The mathematical impli cation of this assumption is as follows. Let Y equal the log odds of the probability that a person has Fuchs' dystrophy. If one were to graph Y against flow rate (X), the standard t-test assumes that the plot would be a straight line (which would have zero slope if there were no association between flow rate and disease and a nonzero slope otherwise). The observed relationship in this study was significantly nonlinear (P = .007). Therefore, we used the generalized f-test, which allows for the possi bility that the relationship may be nonlinear.
Results Table 1 contains means and standard devia tions for all of t h e . measured and calculated values for the normal subjects, subjects with mild to moderate guttae, and subjects with confluent to nearly confluent guttae. In one control subject and one subject with mild to moderate guttae, calculations for endothelial permeability and aqueous humor flow rate were not possible for one eye because of inad
vertent transfer of fluorescein into the eye from the eyelids during fluorophotometry. There fore, data for these subjects were not used for statistical computations involving these varia bles. Corneal thickness (Fig. 2) was higher in subjects with advanced cornea guttata com pared with subjects with early cornea guttata or control subjects. Mean endothelial permeabili ty (Fig. 3) was similar in the three groups. The range of values tended to be greater, however, in subjects with cornea guttata. Mean aqueous humor flow rates were also similar for the three groups (Fig. 4). Again, however, the range of aqueous humor flow rates was greater for sub jects with either advanced or early cornea guttata compared with control subjects. In a comparison between the normal subjects and subjects with mild to moderate guttae or confluent to nearly confluent guttae, there was no statistically significant difference between the normal subjects and subjects with mild to moderate cornea guttata for any of the mea sured variables (Table 2). There was also no statistically significant difference between the normal subjects and subjects with confluent to nearly confluent guttae in endothelial permea bility, aqueous humor flow rate, fluorescence polarization of the corneal stroma, or optical boundary function. Statistically significant dif ferences were noted between controls and sub jects with confluent to nearly confluent guttae in corneal thickness and fluorescence polariza tion in the aqueous humor. Corneal thickness and fluorescence polarization in the aqueous humor were increased in subjects with conflu ent to nearly confluent guttae compared with controls. Mean and range of intraocular pres sure were not statistically significantly different
TABLE 1 MEANS OF MEASURED VARIABLES*
AQUEOUS HUMOR FLOW RATE
CORNEAL
NO. OF
AGE
CORNEAL THICKNESS
SUBJECTS
(YHS)
(MM)
( X 1 0 ~ 4 CM/MIN)
(HL'MIN)
Normal
41
Mild to moderate cornea guttata Confluent to nearly confluent cornea guttata
24
63.2 (12.7) 69.2 (10.2) 61.6 (14.4)
0.57 (0.03) 0.56 (0.02) 0.59 (0.03)
4.27 (0.74) 4.32 (1.04) 4.45 (1.14)
2.52 (0.47) 2.35 (0.58) 2.52 (0.83)
GROUP
26
•Numbers in parentheses are standard deviations.
FLUORESCENCE
ENDOTHELIAL PERMEABILITY TO FLUORESCEIN
POLARIZATION
OPTICAL
STROMA
AQUEOUS HUMOR
BOUNDARY FUNCTION
0.1655 (0.0247) 0.1700 (0.0254) 0.1661 (0.0249)
0.0341 (0.0139) 0.0364 (0.0152) 0.0469 (0.0237)
0.023 (0.011) 0.026 (0.007) 0.025 (0.011)
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standard deviation used was that of normal subject group). Since fluorescence polarization in the aque ous humor was statistically significantly in creased in subjects with confluent to nearly confluent guttae compared with normal con trols, correlation coefficients were calculated between fluorescence polarization in the aque ous humor and the other variables in Table 1 for the subjects with confluent to nearly confluent guttae. There were no statistically significant correlations. Endothelial permeability and aqueous humor flow rates for an individual eye appeared to remain stable over time (Figs. 5 and 6). For an individual subject, endothelial permeability and aqueous humor flow rate tended to be similar in the right and left eye. The clustering of either measurement for an individual eye also confirms the precision of the measurement of endothelial permeability and aqueous humor flow rate obtained with the two-dimensional scanning ocular fluorophotometer.
Discussion In the present study, performed with the two-dimensional scanning fluorophotometer, we found no difference in endothelial permea bility between normal subjects and subjects with Fuchs' dystrophy with increased central
Vol. 106, No. 3
Endothelial Function in Fuchs' Dystrophy
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subjects (Fig. 3). In neither of these previous studies was the difference in endothelial per meability between patients with cornea guttata and normal subjects evaluated statistically. Burns, Bourne, and Brubaker, 15 using the slit-lamp fluorophotometer, compared 21 pa tients with prominent cornea guttata without epithelial edema to 17 age- and sex-matched normal controls. A statistically significant in crease in the mean endothelial permeability was found in the patients with Fuchs' dystro phy. The calculated mean endothelial pump rate did not differ between the two groups. These last results suggested that a breakdown in endothelial barrier function was the earliest defect that occurred in Fuchs' dystrophy. Patients with confluent to nearly confluent guttae in the present study were comparable in the extent of disease to subjects in the Burns, Bourne, and Brubaker study. The present study is larger, and subjects with mild to mod erate cornea guttata were also studied. The present study was also performed with the more precise two-dimensional scanning fluorophotometer, and five measurements of stromal and anterior chamber fluorescein con centration were used to calculate the endotheli al permeability for a subject on a particular day instead of three. Figures 5 and 6 demonstrate the precision of the measurements with the two-dimensional scanning fluorophotometer and the relative consistency of these measure ments over time for a particular normal subject or a subject with Fuchs' dystrophy. It must be emphasized that absolute values of endothelial permeability obtained using the slit-lamp fluorophotometer and the twodimensional scanning fluorophotometer are not directly comparable. Mean endothelial per meability to fluorescein determined for control subjects by using the slit-lamp fluoro photometer in the Burns, Bourne, and Brubaker study 15 (2.89 x 10"4 cm/min) was com parable to that found using the same instru ment in a study of 112 normal subjects 16 (2.4 x 10~4 cm/min). Similarly, mean endothelial per meability to fluorescein for control subjects in the present study determined with the twodimensional scanning fluorophotometer (4.27 x 10"4 cm/min) is similar to that found with the same instrument in a study of 80 normal sub jects 6 (4.03 x 10"4 cm/min). Any conclusions regarding changes in endothelial permeability must, therefore, be based on concurrent con trol subjects, regardless of which technique is used.
September, 1988
Explanations for finding no difference in en dothelial permeability by using a more precise measurement when a difference has been pre viously found with a less precise method in clude the possibility of a type I error in the Burns, Bourne, and Brubaker study. 15 This would be expected to occur 5% of the time. Alternatively, differences between the studies may reflect variability between Fuchs' dystro phy patients in the different studies. Whatever the correct explanation, the present study sug gests that increased endothelial permeability is not an invariable finding in patients with cor nea guttata and increased corneal thickness. Data in the present study suggesting that endothelial permeability is normal in patients with Fuchs' dystrophy" without epithelial edema and that pump function, therefore, di minishes with progression of the disease must, however, be viewed with caution. Several as sumptions that may not be valid are inherent in the methods used to reach this conclusion. 5 We assumed that the cornea-to-anterior chamber distribution ratio (rca), used in the calculation of endothelial permeability, is the same in normal subjects and patients with Fuchs' dystrophy. Whether this is true or not is unknown. The fluorescence of fluorescein in the corneal stroma is known to be quenched to a certain extent compared to fluorescence in water, 1718 although the mechanism for the phenomenon is un known. Whether this quenching effect is differ ent in normal controls and subjects with Fuchs' dystrophy is unknown. If a difference were present, calculations of endothelial permeabili ty would be affected. We have assumed that the baseline central corneal thickness for patients with Fuchs' dystrophy, before developing the disease, does not differ from the normal popu lation. Whether this is true or not is unknown, although the central corneal thickness for the subjects with mild to moderate cornea guttata in this study did not differ statistically from the normal subjects. We also assumed that the rate of transfer of fluorescein across the endothelium is proportional to that of water and that the proportion is the same in the different groups studied. Finally, we used methods that are based on the work of Hedbys and Dohlman 19 and Ytteborg and Dohlman 20 and the assump tions made in reaching their conclusions. Even though endothelial permeability may be normal early in the course of the disease, it may be increased in patients with more ad vanced Fuchs' dystrophy with epithelial edema, since endothelial permeability cannot
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Endothelial Function in Fuchs' Dystrophy
be measured in such corneas by this method. Recent studies have found alterations in ouabain binding sites, adenosine triphosphatase activity, and cytochrome oxidase activity in the endothelium of patients with Fuchs' dystro phy. Geroski and associates, 21 using specific binding of tritiated ouabain, reported a signifi cant increase in Na/K adenosine triphosphatase-associated ouabain binding site densi ty in cadaver eyes with moderate cornea guttata compared with normal cadaver eyes. Na/K adenosine triphosphatase activity was not measured in this study. Conversely, Mc Cartney and colleagues, 22 in a more recent study using tritiated ouabain and autoradiographic techniques, found a decrease in adeno sine triphosphatase-associated ouabain bind ing sites in the endothelium on both a per unit basis and a per cell basis in eyes with advanced Fuchs' dystrophy compared with age-matched eye bank eyes. They also noted a reduction in adenosine triphosphatase activity in the endo thelium in Fuchs' dystrophy. Tuberville, Wood, and McLaughlin 23 recently described a decrease in cytochrome oxidase activity in the central corneal endothelium in Fuchs' dystrophy. Since endothelial pump function is an energyrequiring process, a decrease in p u m p function could be expected in cells in which oxidative phosphorylation activity is markedly de creased. The results of the present study are consistent with these recent experiments The fluorescence polarization of the aqueous humor was statistically significantly higher in the subjects with confluent to nearly confluent guttae compared with normal subjects. This difference could be caused by the difference in corneal thickness, or possibly, a difference in the aqueous humor colloid concentration be tween the two groups. 4 The actual difference was relatively small, and there was no correla tion between this variable and the other varia bles measured in the subjects with confluent to nearly confluent cornea guttata. Data collected for aqueous humor flow rate suggest that there could be a difference in flow between subjects with guttae and normal con trols. The difference is significant using a gen eralized t-test assuming a nonlinear (quadratic) model. 11 Two explanations for the apparent dif ference in variability of aqueous humor flow in these two groups are possible. The difference may be caused by a reduction in the optical uniformity of the cornea in subjects with cor nea guttata. Alternatively, a real difference in rate of aqueous humor flow could be present in
277
at least a subgroup of patients with Fuchs' dystrophy. We cannot discern, based on these techniques, which explanation is correct. At least we know that the rate of aqueous humor flow is approximately the same in groups of persons with cornea guttata as in normals, suggesting that guttae do not result from a reduction in the rate of aqueous humor flow in most patients with Fuchs' dystrophy. An alter ation in the composition of aqueous humor, however, cannot be excluded as a contributing factor.
References 1. Wilson, S. E., and Bourne, W. M.: Fuchs' dys trophy. Cornea 7:2, 1988. 2. McLaren, J. W.,'and Brubaker, R. F.: A twodimensional scanning ocular fluorophotometer. In vest. Ophthalmol. Vis. Sci. 26:144, 1985. 3. Brubaker, R. F.: The flow of aqueous humor in the human eye. Trans. Am. Ophthalmol. Soc. 80:391, 1982. 4. Herman, D. C , McLaren, J. W., and Brubaker, R. F.: A method of determining concentration of albumin in the living eye. Invest. Ophthalmol. Vis. Sci. 29:133, 1988. 5. Wilson, S. E., Bourne, W. M., and Brubaker, R. F.: Effect of dexamethasone on corneal endotheli al function in Fuchs' dystrophy. Invest. Ophthalmol. Vis. Sci. 29:357, 1988. 6. Carlson, K. H., Bourne, W. M., and Brubaker, R. F.: Variations in endothelial cell morphology and permeability to fluorescein with age. Exp. Eye Res. In press. 7. Jones, R. F., and Maurice, D. M.: New methods of measuring the rate of aqueous flow in man with fluorescein. Exp. Eye Res. 5:208, 1966. 8. Ota, Y., Mishima, S., and Maurice, D. M.: En dothelial permeability of the living cornea to fluores cein. Invest. Ophthalmol. 13:945, 1974. 9. Coakes, R. L., and Brubaker, R. F.: Methods of measuring aqueous humor flow and corneal endo thelial permeability using a fluorophotometer nomogram. Invest. Ophthalmol. Vis. Sci. 18:288, 1979. 10. Johnson, S. B., Coakes, R. L., and Brubaker, R. F.: A simple photogrammetric method for measur ing anterior chamber volume. Am. J. Ophthalmol. 85:469, 1978. 11. O'Brien, P. C : Comparing two samples. Exten sions of the T, rank sum, and log rank tests. J. Am. Stat. Assoc. 83:52, 1988. 12. Lachin, J. M.: Introduction to sample size de termination and power analysis for clinical trials. Controlled Clin. Trials 2:93, 1981. 13. Stanley, J. A.: Water permeability of the human cornea. Arch. Ophthalmol. 87:568, 1972.
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14. Waltman, S. R., and Kaufman, H. E.: In vivo studies of human corneal endothelial permeability. Am. J. Ophthalmol. 70:45, 1970. 15. Burns, R. R., Bourne, W. M., and Brubaker, R. F.: Endothelial function in patients with cornea guttata. Invest. Ophthalmol. Vis. Sci. 20:77, 1981. 16. Bourne, W. M., Nagataki, S., and Brubaker, R. F.: The permeability of the corneal endothelium to fluorescein in the normal human eye. Curr. Eye Res. 3:509, 1984. 17. Araie, M., and Maurice, D. M.: A reevaluation of corneal endothelial permeability to fluorescein. Exp. Eye Res. 41:383, 1985. 18. Araie, M.: Carboxyfluorescein. A dye for eval uating the corneal endothelial barrier function in vivo. Exp. Eye Res. 42:141, 1986. 19. Hedbys, B. P., and Dohlman, C. H.: A new
September, 1988
method for the determination of the swelling pres sure of the corneal stroma in vitro. Exp. Eye Res. 2:122, 1963. 20. Ytteborg, J., and Dohlman, C. H.: Corneal edema and intraocular pressure. II. Clinical results. Arch. Ophthalmol. 74:477, 1965. 21. Geroski, D. H., Matsuda, M , Yee, R. W., and Edelhauser, H. F.: Pump function of the human corneal endothelium. Ophthalmology 92:759, 1985. 22. McCartney, M. D., Robertson, D. P., Wood, T. O., and McLaughlin, B. J.: ATPase pump site density in human dysfunctional corneal endotheli um. Invest. Ophthalmol. Vis. Sci. 28:1955, 1987. 23. Tuberville, A. W., Wood, T. O., and McLaughlin, B. J.: Cytochrome oxidase activity of Fuchs' endothelial dystrophy. Curr. Eye Res. 5:939, 1986.
O P H T H A L M I C MINIATURE
"There are people gifted by nature with powers of observation. Without effort they form a sharp impression of whatever is going on around them, in themselves, and in others. Also they know how to cull out of these observations whatever is most significant, typical, or colourful. W h e n you hear such people talk you are struck by the a m o u n t that an unobservant person misses. " O t h e r people are unable to develop this power of observation even sufficiently to preserve their own simplest interests. How much less able, then, are they to do it for the sake of studying life itself." Constantin Stanislavski, An Actor Prepares N e w York, Theatre Arts Books, 1981, p . 86