Hypotony and aqueous humor dynamics in myotonic dystrophy. Stanley D. Walker, Richard F. Brubaker, and Shigetoshi Nagataki

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1 Hypotony and aqueous humor dynamics in myotonic dystrophy Stanley D. Walker, Richard. Brubaker, and Shigetoshi Nagataki A group of 26 subjects with myotonic dystrophy were studied with fluorophotometry to evaluate the relationship between aqueous humor dynamics and, the hypotony found in this disorder. All 26 received topical fluorescein to determine the anterior chamber elimination coefficient; five received systemic fluorescein to evaluate the integrity of the bloodaqueous barrier. The group had a mean intraocular pressure of 7. mm Hg. The rate of clearance of fluorescein from the anterior chamber and the corneatoanterior chamber transfer coefficient for fluorescein were normal. An abnormally high level of fluorescence, three times normal, was observed in the anterior chambers of the myotonic subjects after oral administration of fluorescein. This finding could, not he attributed to abnormal absorption and. elimination of fluorescein or to abnormal plasma binding. This finding indicates that there is a defect in the bloodocular barrier to fluorescein in myotonic subjects. Thus conclusions regarding aqueous humor flow cannot be made from the rate of clearance of topically applied fluorescein in myotonic subjects, since the clearance due to diffusion may represent a significantly large fraction of the total clearance. (INVEST OPHTHALOL VIS SCI 22:74475, 982.) Key words: myotonic dystrophy, bloodaqueous barrier, ocular hypotony, aqueous humor formation, fluorescein clearance, anterior chamber, human eye, intraocular pressure, aqueous humor dynamics yotonic dystrophy is a progressive hereditary disease. It is characterized by myotonia, muscular wasting, frontal baldness, testicular atrophy, and ocular abnormalities. The eye signs include subcapsular cataract, hypotony, acquired ptosis, exposure keratitis, pigmentary abnormalities, an abnormal electroretinogram, abnormal dark adaptation, and weakness of the orbicularis oculi rom the Department of Ophthalmology, ayo Clinic & oundation and ayo edical School, Rochester, inn. Supported by NIH grant EY00634, The Rowland oundation, and the ayo oundation. Dr. Nagataki is a Research to Prevent Blindness International Research Scholar. Submitted for publication Oct. 6, 980. Reprint requests: Richard. Brubaker,.D., Department of Ophthalmology, ayo Clinic, 90 Guggenheim, Rochester, inn muscle. Rarely, extremely low intraocular pressure may be the only sign present. Intraocular pressures in some myotonic persons are lower than generally accepted normal values of episcleral venous pressure. The pathophysiologic mechanism responsible for causing these low intraocular pressures remains unclear. Previous studies have suggested that the hypotony in myotonic dystrophy is due to increased facility of outflow or to decreased aqueous formation. 2 " 8 The facility of outflow has been studied by using tonography, and the rate of flow has been calculated from the measured outflow resistance. However, tonographic data are of questionable value in eyes with very low intraocular pressure. In this study, we attempted to measure aqueous humor flow in myotonic dystrophy using fluorophotometry /82/ /0 982 Assoc. for Res. in Vis. and Ophthal., Inc. Downloaded rom: on 0/22/208

2 Volume 22 Number 6 Hypotony and aqueous dynamics in myotonic dystrophy 745 ethods Two groups of adult subjects were studied. The first group consisted of 26 persons with the typical signs and symptoms of myotonic dystrophy. The diagnosis was based on family history, electromyography, neurologic signs, and ocular manifestations. In addition, we selected for this group only those myotonic subjects whose intraocular pressures were mm Hg or lower. The second group were normal controls from previous studies."' l() The ages of the myotonic persons ranged from 8 to 63 years, with a median age of 37; the ages of the controls ranged from 2 to 78 years, with a median age of 39. On the morning of the study an eye examination was performed. All subjects had biomicroscopically clear anterior chambers. Intraocular pressure was determined with a Goldmann tonometer. The volume of each anterior chamber was determined photogrammetrically. " Iontophoresis was used to deposit fluorescein into the central 4 mm of both corneas. 2 A current of 200 /xa was passed through the cornea for 7 sec, depositing 0.5 to 0.6 fjig of fluorescein beneath the epithelium. The conjunctival sac was irrigated with balanced salt solution to remove any fluorescein from the surface of the eye. A fiuorophotometer was used to measure the mass and concentration of fluorescein in the cornea and anterior chamber at 0.5, 2, and 7 hr after iontophoresis. These data were used to calculate the anterior chamber fluorescein elimination coefficient of fluorescein, k 0. The method described by Coakes and Brubaker was used. 3 luorescein is eliminated from the anterior chamber either by flow of aqueous humor or by diffusion into surrounding tissues. To assess the permeability of the iris vessels to fluorescein, we asked those myotonic persons who lived near Rochester, inn., to return for additional studies. ive subjects were willing to return for the additional study. We advertised for normal volunteers to study in the same way, and six normal subjects were studied. The myotonic subjects received an oral dose of 500 mg of fluorescein (which was very near 7 mg/kg for all but one subject). The apparent concentration of fluorescence was measured in the anterior chamber. The total fluorescence was measured in the combined central 0 mm of the cornea and anterior chamber at, 2, and 3 hr after administration. A blood sample was drawn 2 hr after oral administration. The normal subjects received 7 mg/kg oral fluorescein (except one subject who received 5 mg/kg, 500 mg). In the normals, anterior chamber measurements were made and blood samples were drawn at V4, Vi,, 2, and 3 hr. Heparinized blood samples were centrifuged, and the plasma was analyzed with a fluorometer to determine the total concentration of fluorescein. The ratio of bound to unbound fluorescein in the plasma was determined by the method of fluorescence polarization. 4 " 6 The KinseyPalm model 7 was used to compare the anterior chamber fluorescence data after oral administration to model behavior, using assumed values of k dpa and Ch: dc a =, C a ) () C a is the concentration of fluorescein in the anterior chamber, Q, the concentration in the posterior chamber, C p the concentration of unbound fluorescein in the plasma, k (lpa the transfer coefficient due to diffusion, and k fa the transfer coefficient due to flow. The anterior chamber loss coefficient is the sum of the coefficients due to diffusion and flow., _, i, (o) K o ~~ K clpa ' K fa W The measured concentrations of unbound fluorescein in the plasma of the normal subjects was fit by the method of least squares to an equation of the following form: C p = A (e CTt e (3) Results Table I outlines the results of fluorophotometry after topical fluorescein. The mean intraocular pressure of the myotonic group was 7. ± 2.2 mm Hg. Compared to a group of 3,000 asymptomatic patients tested at the ayo Clinic, this figure corresponds to the 0.2 percentile. Three eyes in the myotonic group were observed to have pressures of 3 mm Hg. uch to our surprise, we found that the anterior chamber elimination coefficient, k 0, was normal in the myotonic group, notwithstanding the observed ocular hypotony. The myotonic dystrophy subjects were found to have a mean k 0 of.5 ± 0.37 x 0~ 2 min~' compared to the mean of.5 ± 0.44 found among the normals. There was no statistically significant correlation between k 0 and the intraocular pressure in the myotonic Downloaded rom: on 0/22/208

3 746 Walker et al. Invest. Ophthalmol. Vis. Sci. June 982 Table I. Topical fluorescein Patient No. Sex Age (years) Intraocular pressure (mm Hg) OD OS Anterior chamber volume (/xl) OD OS Anterior chamber elimination coefficient (k 0 x 0 2 tnin~ l ) OD OS Group (myotonic dystrophy) ean ± S.D. Group 2 (normal)t ean ± S.D ± 2 39 ± * ± ± * ± ± i : i: 0.44 This person also had unilateral Horner's syndrome and was not included in analysis. tgroup 2: n = 37; 7 males, 20 females. subjects, as illustrated in ig.. ig. 2 shows the relation between the age of the subject and the k 0 value for each eye. The apparent correlation is not quite statistically significant (twotailed linear regression analysis of the mean of the two eyes vs. age, p > 0.05). The corneatoanterior chamber transfer coefficient, k a, was found to be 3.0 ±.0 0" 3 min in the myotonic subjects. This value is identical to the mean, 3.0 ±.4, of a group of normal subjects who were studied by us using the same technique. Thus the entry of fluorescein into the anterior chamber after topical administration and the disappearance thereafter appear to be normal in myotonic dystrophy. In the five myotonic subjects who received oral fluorescein, fluorescein concentrations were measured in the plasma 2 hr after administration (Table II). The total fluorescein concentration at this time (for a dose of 7 mg/kg) was 230 ± 50 ng/ml. The unbound concentration was 328 ± 8 ng/ml, and the unbound fraction was 0.4 ± In the normal group, the total fluorescein concentration at 2 hr was 3650 ± 970 ng/ml. The unbound concentration was 478 ± 70 ng/ ml, and the unbound fraction was 0.3 ± There was no statistically significant difference (unpaired t test, p > 0.05) between the bound concentrations, the unbound concentrations, or the unbound fractions of the Downloaded rom: on 0/22/208

4 Volume 22 Number 6 Hypotony and aqueous dynamics in myotonic dystrophy 747 normal subjects and the myotonic subjects. The plasma concentrations of fluorescein of the normal subjects were also measured at other times between 5 min and 3 hr. The ratio of free to bound fluorescein was constant over this time interval. The best fit parameters of equation 3 were A = 360 ng/ ml, a = " 3 min" and (3 = " 2 min". The intensity of fluorescence in the anterior chamber was higher in the five myotonic subjects after oral administration than in the six normal subjects, approximately three times higher. These data are presented in Table III. In addition, the combined fluorescence in the central 0 mm of the cornea and the anterior chamber was measured, from which the initial fluorescence of the same field was subtracted. These data are presented in Table IV. It was estimated from the anterior chamber photographs used to calculate anterior chamber volume that 75% of the anterior chamber is included in the 0 mm diameter excitation field of our fluorophotometer. Using this figure, we calculated the fluorescence which was present in the central 0 mm of the anterior chamber as a = C a (0.75 anterior chamber volume). The apparent corneal mass of fluorescein in the central 0 mm can be estimated from the difference between the total fluorescence and the calculated anterior chamber fluorescence. These data are presented in Table IV. The calculated corneal fluorescence is higher in the myotonics than in the normals, but the differences in anterior chamber fluorescence are more striking than the differences in corneal fluorescence. Discussion It was our expectation that people with myotonic dystrophy would be found to have a low rate of aqueous formation. Thus it was surprising to us to discover that the clearance of fluorescein in the eyes of myotonic subjects was the same as the clearance from normal eyes. This finding, however, does not exclude the possibility that the rate of aqueous humor Ko (x0 2 mirr ).2 O.6 IOP vs Ko 8 x 8 I I r I! ' I ' I Intraocular pressure (mmhg) ig.. Relation between intraocular pressure and the coefficient of fluorescein elimination from the anterior chamber in myotonic dystrophy. Ko (x0 2 min Ko vs Age x x * xx V( x x * * *x * «x * Age (yrs) S ig. 2. Relation between age and the coefficient of fluorescein elimination from the anterior chamber in myotonic dystrophy. flow through the anterior chamber is abnormally low in myotonic dystrophy. luorescein is lost from the anterior chamber by flow and by diffusion. In the normal eye, the former mechanism has been shown to dominate. 8 ' 9 Such, however, may not be the case in myotonic subjects. Downloaded rom: on 0/22/208

5 748 Walker et al. Invest. Ophthalmol. Vis. Sci. June 982 Table II. Oral fluorescein Plasma concentration (2 hr) Patient No. Total ( fjtg/ml) Unbound( /xglml) Unbound fraction Group 3 (myotonic dystrophy) * ± ± 0.04 ean ± S.D. Group 4 (normals)t ean ± S.D ± ± ± ± 0.05 All subjects were male. *This subject received a dose of 4 mg/kg. His total and unbound levels were multiplied by 7/4 for inclusion in the Table. One normal subject received 5.6 mg/kg and his data were multiplied by 7/5.6. t Group 4: n = 6; mean age = 24. Table III. Apparent anterior chamber concentrations after oral fluorescein (7 mg/kg)* Concentration in anterior chamber (ng/ml) Time (hr) 2 3 Normal group (n = 6) yotonic group (n 20.5 ± ± ± ± ± ± 29.4 Normal Values are mean ± S.E. *The data of one normal subject who received 5.6 mg/kg and one myotonic subject who received 4 mg/kg were multiplied by factors of 7/5.6 and 7/4, respectively. The concern that diffusional losses might interfere with measurement of aqueous humor flow in myotonic subjects was bolstered by published reports that iris permeability is abnormal in this disease. 20 " 22 Therefore we restudied a small group of our subjects. luorescein was given systemically to determine whether the bloodaqueous barrier was normal. The results in this group indicated that the bloodaqueous barrier in myotonic dystrophy is abnormally leaky to fluorescein. An attempt was made to determine whether the observed abnormality in bloodaqueous barrier permeability to fluorescein would eliminate the possibility of estimating the rate of aqueous humor flow from the observed clearance of topically applied fluorescein. We reasoned that an abnormality of the bloodaqueous barrier posterior to the iris need not be considered; such an abnormality would not affect the clearance of topically applied fluorescein. Thus, we must consider only the possibilities that either () iridal or corneal exchange might be abnormal or (2) absorption of orally administered fluorescein or plasma binding of fluorescein might be abnormal in the myotonic group. No abnormality of the cornea was expected, and the observed corneatoanterior chamber transfer rate of fluorescein in the myotonic eyes indicated that the exchange between the cornea and the anterior chamber was normal in these eyes. After oral fluorescein, the myotonic subjects had higher intensities of fluorescence in the cornea, but the differences between the myotonic subjects and the normals could be explained by the higher anterior chamber levels in the myotonic eyes as shown in Table IV. Araie et al. 23 have shown that orally administered fluorescein is rapidly and completely absorbed in normal subjects. In our normal Downloaded rom: on 0/22/208

6 Volume 22 Number 6 Hypotony and aqueous dynamics in myotonic dystrophy i mean ± SE, myotonics Apparent luorescein in Anterior Chamber, ng/ml mean ± SE, normals Hours S8949B ig. 3. easured levels of fluorescein in the anterior chamber of normal subjects (n = 6) and myotonic dystrophy subjects (n = 5) after oral dosage of fluorescein. Curve illustrates levels predicted by the simplified version of the KinseyPalm Equation, equation 4. or the normal curve, the value of k () was.5 0~ 2 min", and the value of k fa was.35 0~ 2 min". or the myotonic curve, the value of k 0 was.5 x 0~ 2 min" and the value of k fa was. x 0~ 2 min". The values of C p for both curves were computed from equation 3. Table IV. Apparent mass of fluorescein in central 0 mm of cornea and anterior chamber after oral fluorescein administration (ean ± S.E.) Time (hr) Combined mass (ng) Anterior* chamber mass (ng) Corneal mass t (ng) Normals (n = 6) 2 3 yotonics (n = 5) ± ± ± ± ± ± ± ± ± ± ± ± " Calculated from concentration x (anterior chamber volume x 0.75). t Column 2 column 3. subjects, peak concentrations were reached in less than hr after oral ingestion. Only one sample was tested in our myotonic subjects, drawn 2 hr after administration. The concentration was lower in the myotonic subjects but was not significantly different from the normal. The ratio of bound to free fluorescein was the same in the two groups. It would have been better to take frequent blood samples from our myotonic subjects. It seems unlikely, however, that the higher levels of anterior chamber fluorescence after oral administration in the myotonic subjects could have been explained by abnormal systemic pharmacokinetics of fluorescein. We cannot exclude the possibility that the vessels of the iris are abnormally permeable to fluorescein in myotonic dystrophy. 20 The problem of abnormal iris permeability can be put into perspective by using the Kinsey Palm equation to estimate the extent of iris abnormality that must exist to account solely Downloaded rom: on 0/22/208

7 750 Walker et al. Invest. Ophthalmol. Vis. Sci. June 982 for the elevated anterior chamber fluorescein levels in the myotonic subjects. or this purpose, we have necessarily made some simplifying assumptions about the entry of fluorescein from the pupil, since the concentration of fluorescein in the pupillary aqueous humor, Cf,, was not measured. Estimates by Goldmann 8 and by Nagataki 9 would place the normal level of Ch at approximately 7% of the unbound concentration of fluorescein in the plasma. If this simplification is allowed, equations and 2 combine as follows: dc, dt = k k fa C p C a (4) or our normals, we assumed that k fa was 90% of the measured ^. The values of C p were calculated from equation 3. Equation 4 was integrated numerically to produce the curve shown in ig. 3. or the myotonic subjects the same C p curve was used, and the value of k fa that produced the leastsquares deviation between the C a curve and the observed data points was calculated. The value of k fa thus determined was found to be 0.0 min", 20% smaller than the value used for the normals. It is evident from ig. 3 that the curves produced by the simplified form of the KinseyPalm equations do not fit the time course of the anterior chamber data. The disparity between the curves and the data could be explained partly by the true value of k 0 being lower than the measured value or by lack of a constant relation between C h and C p. In the latter case, it would seem rather likely that diffusional exchange between the posterior chamber and the vitreous would blunt early levels of fluorescein concentration in the posterior chamber and augment later levels. Such a change would alter the time course of the model curves toward that of the observed data. However, the true cause of the disparity cannot be determined in the absence of measurements of the time course of the fluorescein concentration in the pupil. In addition, the KinseyPalm equation itself is a simplification because it makes no allowance for the depot effect that the peripheral cornea is likely to have as fluorescence enters the limbus from the blood and diffuses centrally. We had originally hoped to be able to make an estimate of the probable range of aqueous humor flow in myotonic dystrophy, but within the limits of available data, there are too many unknowns to do so. The data of this study indicate that the exchange of fluorescein between the cornea and the anterior chamber and the clearance of fluorescein from the anterior chamber are normal in myotonic dystrophy but that the bloodaqueous barrier to fluorescein is abnormal. The profound ocular hypotony remains unexplained. Because of the abnormality of the ocular barrier to fluorescein, it would seem preferable to study the dynamics of aqueous flow by the method proposed by Araie et al. 23 combined with careful measurements of fluorescein concentrations in the pupillary aqueous and fluorescein concentrations in the peripheral cornea. REERENCES. Junge J: Ocular changes in dystrophia myotonica, paramyotonia and myotonia congenita. Doc Ophthalmol 2:, Burian H and Burns CA: Ocular changes in myotonic dystrophy. Am J Ophthalmol 63:22, Burns CA: Ocular histopathology of myotonic dystrophy. Am J Ophthalmol 68:46, Ginsberg J, Hamblet J, and enetee : Ocular abnormality in myotonic dystrophy. Ann Ophthalmol 0:02, anschot WA: Histological findings in a case of dystrophia myotonica. Presented at Netherlands Ophthalmological Society, 57 eeting, Groningen, 966. Ophthalmologica 55:294, Nappi G: Tonographic evaluation of intraocular pressure in myotonic dystrophy. Boll Soc Ital Biol Sper 54:80, Vos TA: 25 years dystrophia myotonica (D..) Presented at Netherlands Ophthalmological Society, 43 eeting, Rotterdam, 959. Ophthalmologica 4:37, Houber JP and Babel J: Les lesions uveoretiennes de la dystrophie myotonique. Ann Oculist 203:067, Townsend DJ and Brubaker R: Immediate effect of epinephrine on aqueous formation in the normal human eye as measured by fluorophotometry. IN VEST OPHTHALOL VIS SCI 9:256, Burns RR, Bourne W, and Brubaker R: Endothelial function in patients with cornea guttata. IN VEST OPHTHALOL VIS SCI 20:77, 98.. Johnson SB, Coakes RL, and Brubaker R: A simple Downloaded rom: on 0/22/208

8 Volume 22 Hypotony and aqueous dynamics in myotonic dystrophy 75 Number 6 photogrammetric method of measuring anterior chamber volume. Am J Ophthalmol 85:469, Jones R and aurice D: New methods of measuring the rate of aqueous flow in man with fluorescein. Exp Eye Res 5:208, Coakes RL and Brubaker R: ethod of measuring aqueous humor flow and corneal endothelial permeability using a fluorophotometry nomogram. IN VEST OPHTHALOL VIS SCI 8:288, Penniston JT: luorescence polarization measurement of binding of fluorescein to albumin, Exp Eye Res (in press). 5. Brubaker R, Penniston JT, Grotte DA, and Nagataki S: easurement of fluorescein binding in human plasma using fluorescence polarization. Arch Ophthalmol (in press). 6. Dandliker WB, Kelly RJ, Dandliker J, arquhar J, and Levin J: luorescence polarization immunoassay. Theory and experimental method. Immunochemistry 0:29, Kinsey VE and Palm E: Posterior and anterior chamber aqueous humor formation. Arch Ophthalmol 53:330, Goldmann H: Uber luorescein in der menschlichen Vorderkammer. Das Kammerwasserinutenvolumen des enschen. Ophthalmologica 20:65, Nagataki S: Aqueous humor dynamics of human eyes as studied using fluorescein. Jpn J Ophthalmol 9:235, Stern LZ, Cross HE, and Crebo AR: Abnormal iris vasculature in myotonic dystrophy. Arch Neurol 35:224, Cobb S, Schilling JS, and Chisholm B: Vascular tufts at the pupillary margin in myotonic dystrophy. Am J Ophthalmol 69:573, ason GI: Iris neovascular tufts: relationship to rubeosis, insulin and hypotony. Arch Ophthalmol 97:2346, Araie, Sawa, Nagataki S, and ishima S: Aqueous humor dynamics in man as studied by oral fluorescein. Jpn J Ophthalmol 24:346, 980. Downloaded rom: on 0/22/208