Solutions containing miotic agents: Effects on corneal transendothelial electrical potential difference

Graefe's Arch Clin Exp Ophthalmol (1997) 235:379-383 © Springer-Verlag 1997

Ritsuko Akiyama Kunyan Kuang Jan E Koniarek Pablo A. Chiaradia Calvin W. Roberts Jorge Fischbarg

Solutions containing miotic agents: effects on corneal transendothelial electrical potential difference

Received: 14 September 1995 Revised version received: 14 January 1997 Accepted: 15 January 1997

Abstract • Background: Anterior

R. A k i y a m a ~ - K . K u a n g • J . R K o n i a r e k

RA. Chiaradia 2, J. Fischbarg (C5~) Department of Ophthalmology Columbia University, College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032, USA C.W. Roberts Department of Ophthalmology, Cornell University Medical Center, New York, USA J. Fischbarg Department of Physiology and Cellular Biophysics, Columbia University, New York, USA Present address:

l Department of Ophthalmology, School of Medicine, Kyorin University, Tokyo, Japan Permanent address:

2 Department of Ophthalmology, School of Medicine, University of Buenos Aires, Argentina

chamber miotic solutions are widely used during anterior chamber surgery. We examined the effects of solutions containing miotic agents such as carbachol and/or acetylcholine on corneal endothelial pumping activity. • Methods: We monitored, in vitro, the transendothelial electrical potential difference of isolated rabbit corneal endothelial preparations. As controls, we used solutions without miotics. • Results: We found that a solution containing 55 mM acetylcholine and minimal amounts of salts (Miochol E) maintains transendothelial electrical potential difference some 30% above control levels for up to 4 h. Two other solutions, one including balanced salts and 0.55 mM carbachol (Miostat), the other a mixture of 0.19 mM carbachol and 55 mM acetylcholine plus minimal salts, are adequate to

Introduction

maintain the potential difference at control levels. Lastly, a solution with acetylcholine but without any salts (Miochol) greatly decreases the potential difference, to 30% of the control level, in 100 min. • Conclusion: Our results indicate that: (1) 55 mM (1%) acetylcholine stimulates the endothelial electrical potential difference; (2) addition of 0.19 mM (0.003%) carbachol negates the stimulatory effect of acetylcholine; and (3) absence of electrolytes severely depresses the endothelial electrical activity.

endothelial layer, it would be useful to determine whether they may have adverse effects on the endothelium.

Anterior chamber miotic solutions are used in cataract surgery, intraocular lens implantation, corneal transplantation, glaucoma surgery, and vitrectomy. Their purpose is to constrict the pupil and thus secure the placement of the posterior chamber intraocular lens, to protect the crystalline lens during anterior chamber surgery, to keep the peripheral iris out of the surgical incision, and to function as an ocular hypotensive agent. Since during use these solutions come into contact with the corneal

The tolerance of the corneal endothelium to commercially prepared solutions containing acetylcholine chloride and carbachol has been investigated by Vaughn et al. [14], Yee and Edelhauser [15], and Birnbaum et al. [3], who assessed their effect in vitro in rabbit and human corneas by measuring changes in corneal thickness and in electron microscopic (EM) morphology. A summary of their results is shown in Table 1. Their results were not always in agreement, but their respective experimental

380

Table 1 Effects of intraocular

miotics in previous reports and the present study (EM electron microscopy, TEPD transendothelial electrical potential difference)

Study

Miochol

Old Miostat

Vaughn et al. 1978 [18] Yee and Edelhauser 1986 [15] Birnbaum et al. 1987 [3] Akiyama et al. (this paper)

No swelling; no EM damage Increased swelling; EM damage

Increased swelling; EM damage?

Materials and methods

Miochol E

No swelling; no EM damage Increased swelling; no EM damage TEPD normal TEPD slightly above normal for 4 h for4h

TEPD abolished in 2 h

c o n d i t i o n s were different. We therefore d e c i d e d to c o m pare such solutions u n d e r our own conditions. We m o n i tored the t r a n s e n d o t h e l i a l e l e c t r i c a l p o t e n t i a l d i f f e r e n c e ( T E P D ) i n s t e a d of the rate of swelling. T h e T E P D has b e e n c o r r e l a t e d with the rate of the e n d o t h e l i a l fluid p u m p [2, 5, 6, 8] that m a i n t a i n s the c o r n e a at the low level of h y d r a t i o n r e q u i r e d for t r a n s p a r e n c y [7, 12], so we took T E P D as a more sensitive and i n s t a n t a n e o u s index of tissue function. We found that the p r e s e n c e of electrolytes was b e n e f i c i a l to m a i n t a i n T E P D , and that a c e t y l c h o l i n e r e s u l t e d in slight s t i m u l a t i o n of e n d o t h e lial f u n c t i o n . A c o m p a r i s o n of our results with previous f i n d i n g s is given in Table 1.

Miostat

PHM82 Standard pH Meter (Radiometer, Copenhagen). The pH values listed in Table 2 are those at room temperature and without gassing. However, for pH control, oxygenation, and stirring, the solutions on both experimental hemichambers were continuously bubbled with a mixture of 5%, COa and 95% air. For comparison, the pH values of the solutions thus gassed were: (A) 6.1, (B) 4.8, (C) 4.4, (D) 4.4 (E) 7.7, and (F) 4.0. Solution exchanges and TEPD recording At the beginning of each experiment, a control TEPD reading was taken with solution E (balanced salts solutions, BS) bathing both the stromal and the endothelial sides of the preparation. The TEPD values were read as a continuous trace in the chart recorder, except for short interruptions while electrodes were being zeroed. The TEPD took about 20 rain to stabilize at a baseline value. After such stabilization, the BS solution on the endothelial side was replaced by the appropriate test solution, while the stromal side was left undisturbed. Since the test solutions had salt concentrations widely different from BS, the resulting electrolyte gradients across the

Animals Male New Zealand white rabbits weighing 3 kg were killed with an overdose of sodium pentobarbital injected into the marginal ear vein and their eyes were enucleated. The epithelium was scraped from the cornea, and the cornea was removed from the globe using the procedure of Dikstein and Maurice [4] and mounted in the chamber. Transendothelial electrical potential difference measurements The technique and experimental protocol utilized for measuring TEPD have been described previously [5, 6, 11]. The cornea was clamped between two hemichambers (Fig. 1). A calomel electrode was connected by a saline bridge to each hemichamber (Fig. 1). The saline bridges were checked against each other approximately every 5 rain, and any electrode drift was corrected with an adjustable series battery. The entire experimental setup was enclosed in a steel Faraday cage to decrease electromagnetic interference. All experiments were performed at 37 °C. Experimental solutions The solutions used were: (A) Miostat, Alcon Laboratories, Ft. Worth, Tex; (B) Miochol and (C) Miochol E, both from Iolab Pharmaceuticals, Claremont, Calif.); (D) a solution containing carbachol 0.01% and acetylcholine 1%; (E) a solution of balanced salts; and (F) the electrolytes used in the acetylcholine solutions (C) and (D). Their compositions are given in Table 2. Osmolarities were determined with a Micro-Osmette osmometer (Precision Systems, Natick, Mass.) using 50 gl samples, pH was determined with a

solution

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Chart recorder Fig. 1 Schematic diagram of the chamber and associated recording equipment for measuring transendothelial electrical potential difference, a Upper (aqueous side) hemiehamber and solution in it, b endothelial cell layer, c deepithelialized stroma, d lower (stromal side) hemi-chamber and solution, e aeration funnel. Details not shown: the stroma is supported by a stainless steel net, and the chamber and aeration funnel are jacketed for temperature control. Both the upper and lower solutions are continuously bubbled with a mixture of 5% CO 2 and 95% air. Arrows in the lower hemichambet denote bubbling-induced fluid circulation between chamber and aeration funnel. Voltmeter: Keithley 610C Electrometer

381

Table 2 Ionic composition (mM) of the solutions used (BS balanced salts)

a Gassed with air plus 5% CO 2

A Miostat

B Miochol

D mixed

NaC1 NaHCO 3 KC1 KHCO~ MgC1 KH2PO4 MgSO4 CaC12 Sucrose Acetylcholine C1 Mannitol Carbachol Sodium acetate Sodium citrate

109.6

5.1

5.1

10.0

5.4

5.4

Osmolarity (mOsm; --SD) pH

303.5+ 1.5

E BS

F low NaC1

112.9 39.2

5.1 5.4

3.8 1.5

1.5

3.3

1.5

6.7

1.5 1 0.8 1.7

0.7

0.7

55.1 164.7

55.1 153.7

55.1 153.7 0.2

305.5+0.9

308.0+1.4

308.0+1. 4 5

0.6 28.7 5.8

5.2

endothelium gave rise to liquid junction electrical potentials of some 2 mV across the layer, the polarity of which was reversed with respect to the normal TEPD. Such artifactual junction potentials were monitored (approximately every 20 min) only to ensure that they were stable and within normal bounds. After a 1-h period of exposure to the test solution, the test solution was replaced with BS so as to be able to measure TEPD correctly and thus determine the effect of such exposure. After this return to BS, the TEPD always returned to the correct polarity and magnitude, showing that TEPD reversals per se had no lasting effect on the endotheliurn. Once more, approximately 20 rain was required for the TEPD to stabilize, at which point its value was noted. This cycle was repeated for as long as the TEPD, upon washing and return to BS, was more than 5% of its initial value; if it was less, the experiment was terminated.

Fig. 2 Transendothelial electrical potential difference values (aqueous side negative) as a function of time. Each curve represents one of the six solutions tested. Symbols denote the means (n=number of experiments); deviations are SEM. Values were grouped by time; SEMs in the time axis were typically only 2-3%, and were thus neglected

C Miochol E

5.2

0.7 112 153.7

289.8+1. 5 7.7 a

303.8+1. 6 5

Results

F i g u r e 2 depicts the e x p e r i m e n t a l results. W h e n the end o t h e l i u m was e x p o s e d to solution B, the T E P D d e c l i n e d rapidly, to nearly zero after s o m e 4 h. In c o n t r a d i s t i n c tion, all the other solutions tested were adequate to m a i n tain the T E P D at or above c o n t r o l levels. O f these, solutions A, D, E, and F gave a p p r o x i m a t e l y the same results, with the PD i n c r e a s i n g b y some 25% by the second hour, and d e c r e a s i n g slowly after that, r e a c h i n g some 10% of the i n i t i a l PD b y the fifth hour. S o l u t i o n C i n d u c e d the highest T E P D values, r e a c h i n g a level of 110% of the i n i t i a l value vs 75% for that of BS at the 3 h mark. This s t i m u l a t i o n , w h i c h we a t t r i b u t e to

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acetylcholine, is evident by the end of the first hour, and lasts for nearly 4 h. At the 180-min time point, comparison of the TEPD values obtained with the different solutions gave the following results (Student's t-test): C>A, P-E, P D , C < > F , P-