Evaluation of the effects of saline versus bicarbonate-containing mixed salts solutions on rabbit corneal epithelium in vitro

Ophrhal. Physiol. Opt. Vol. 15, No. 6, pp. 585-599, 1995 Copyright 0 1995 Elsevier Science Ltd for British College of Optometrists Printed in Great Britain. All rights reserved 0275-5408195 $10.00 + 0.00

Evaluation of the effects of saline versus bicarbonate-containing mixed salts solutions on rabbit cornea1 epithelium in vitro Michael

J. Doughty

University

of Waterloo,

School

of Optometry,

Waterloo,

Ontario,

N2L 3Gl

Canada

Summary Rabbit corneas were incubated, over 4 h in vitro, with the cornea1 epithelial surface exposed to various solutions to assess their utility as incubation solutions for physiological, pharmaceutical or toxicological studies. The cornea1 endothelium was perfused with a 35m~ bicarbonate-mixed salts solution equilibrated at 36°C. Cornea1 thickness, cornea1 hydration or epithelial cell appearance (as assessed by scanning electron microscopy) were found to be similar to in vivo if a 35 mM bicarbonate, mixed salts solution (equilibrated with 5% CO,-air) hydration and minor cell was used for the epithelium. Some swelling (14p.m h -‘I, increased exfoliation were seen if this 35 mM bicarbonate solution was equilibrated with 5% CO,-95% 0, (hyperoxia). Solutions with only 5m~ bicarbonate (0.5% CO,-air) produced rapid swelling, large increases in hydration and marksed cellular damage. Slightly hypertonic (310mOsm kg-‘) solutions containing 5m~ bicarbonate caused some swelling at 15pm h-‘, small increases in hydration and some cell damage but the swelling and cellular damage were further reduced by making the solution slightly more hypertonic (325 mOsm kg-‘) by addition of NaCl and KCI. Saline (NaCI 0.9% or 0.97%) or phosphate-buffered saline (PBS) (300 in hydration and almost mOsm kg-‘) produced swelling at 21-28pm hh’, 30% increases total destruction of the superficial cell layers. These studies confirm in vivo experiments that saline (and also buffered saline solutions) are rather toxic to the cornea1 epithelium and thus should not be used as epithelial incubation solutions. Even when using mixed salts solutions and even with bicarbonate present, small differences in composition can have marked effects on cornea1 thickness, hydration or cell appearance. Hyperoxic solutions appear to be mildly cytotoxic compared with normoxic solutions. Ophthal.

Physiol.

Opt.

1995,

15,

585-599

Eyedrops of aqueous solutions are commonly used for purposes ranging from rewetting solutions, artificial tears, diagnostic or therapeutic pharmaceutical agents. Such solutions need to be formulated such that they are stable, that they are reasonably comfortable to the patient, are sterile and that all of the ingredients have minimum toxicity to the ocular surface’. Three main methods have been used to assess the compatibility of eyedrops with the eye, especially if the active ingredients of the eyedrops are meant to penetrate the eye. These methods have involved

assessment of potential toxicity using the Draize test or modifications thereof 2-5, the in vivo or in vitro pharmacokinetics for the active ingredients or preservatives6 and the clinically observed tolerance to solutions or eyedrops’.‘. The first method involves assignment of subjective scores for damage to the cornea1 and conjunctival surface, the second method involves measurement of drug concentrations on the endothelial side of the cornea (or in the cornea1 tissue) and the third method utilizes subjective scores provided by a patient or test subject. Despite extensive evaluation of eyedrops and their ingredients from a toxicological or safety perspective, relatively little is known about the structural or physiological condition of the cornea1 tissue, especially the corneal epithelium, after the single or multiple use of eyedrops indicated for use

Received: 26 June 1994 Revised .form: 12 December 1994

Present address: Glasgow-Caledonian University, Sciences, City Campus, Glasgow G4 OBA, UK.

Department of Vision

585

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as artificial tears. The rational design of an artificial tear that might sustain or promote certain structural or physiological characteristics of the superficial epithelial cells requires that methods be available to assess the effects of different solutions on the cornea1 surface (e.g. cell morphology) and its physiological properties (e.g. its barrier function). Assessments can be performed on the living eye in vivo, such as measurements of increases in central cornea1 thickness after irrigation of the ocular surface with saline solutions’, psychophysical assessments of perceived light scatter after irrigation of the ocular surface with saline’ or measurements of the relative absorption of fluorescein into the cornea1 epithelium after exposure of the ocular surface to eyedrops” or salt solutions”,‘2. Recently, both quantitative impression cytology’3 and scanning electron microscopy’4-‘6 have also been used to assess the effects of different artificial tears on the morphology of epithelial cells at the ocular surface after use of eyedrops in vivo. While all of these methods might be considered as ideal since they involve the assessment of the impact of solutions or chemicals on the ocular surface of the living eye in situ, there are some limitations. The methods of presentation of the test solutions to the ocular surface have sometimes required special procedures such as fitting goggles to the eye to permit ocular surface irrigation8,9, the use of eye cups containing fluorescein dye” or using lid retractors and a speculum to form a conjunctival lid cup that can be filled with test solution with or without fluorescein”*‘2. In vivo methods may be most useful or even required for the final assessment of a solution or eyedrops on the surface of an animal or human eye. An equivalent ex vivo technique that could encompass the various in vivo eye tests for the cornea could be most useful for both screening different solutions for toxicity or, more importantly, evaluating the mechanism of action of the various ingredients of artificial tear solutions. The principles of such an in vitro technique” have been well established but very little attention has been given to the status of the cornea1 epithelium after the use of various irrigating or incubation solutions during in vitro (ex vivo) techniques on the excised eye or cornea. The specular microscope system, as detailed by Dikstein and Maurice”, has been used extensively to evaluate the effects of various solutions and chemicals on the cornea1 endothelium’8.‘9. The technique involves perfusion of the endothelium with test solutions while the epithelial surface is usually covered with silicone oil. The perfusion technique can be followed by the application of quantitative scanning electron microscopy of the cornea1 endothelium2’. A number of investigators have demonstrated that the same apparatus can also be used to present test solutions to the epithelial surface while the endothelial aspect of the isolated corneas is perfused with silicone oi12’-24 or an artificial aqueous humour25m29. Using the specular microscope system or a slightly different cornea1 incubation system with half-chambers,

numerous evaluations have been made of the effects of various solutions on cornea1 thickness or its electrophysiological properties. A large number of different solutions containing mixtures of salts (Na+, Kf, Ca2+, Mg2+ along with Cl-, HCO;, phosphate and sulphates) have been used for many years in such physiological studies or in studies involving assessments of pharmaceuticals or their ingredients on the cornea23~25,28~40. Some of these solutions have also contained glucose (up to at least 33.9 mu), sucrose, phosphate or Tris buffers, gluconate salts and buffers, adenosine, glutathione or even y-aminobutyric acid. Despite the extensive numbers of studies and the use of a large number of different solutions (without toxic chemicals), very little data appears to have been published on the structure and morphological state of the cornea1 epithelium after such studies23X27,38. This lack of information is in marked contrast to tests on endothelial solutions where both scanning and transmission electron microscopy have been very widely used to provide images of the endothelium after the tests’8”9. However, with the use of bicarbonatecontaining solutions, it could be reasonably argued that their complex composition should support maintenance of epithelial cell physiology rather than be detrimental to these cells. However, such a logic is limited by the fact that conflicting data has been published concerning the effects of solutions containing fewer salts and lacking the physiological HCO,-CO, buffer system. Furthermore, the micrographs that have been published indicate moderate to marked differences in the appearance of the cornea1 surface after exposure to just the irrigating solution (without test chemicals)23,28.Mixed salts solutions without bicarbonate but buffered with phosphate, citrate or acetate have been used to incubate or irrigate cornea1 epithelia4’X42without marked cellular damage being evident. Phosphate-buffered saline (PBS) so1utions43X44, NaCl/KCl solutions26 or just NaCl solutions21,*5-*‘,45,46 have also been used as epithelial incubation solutions and it is for the latter solution that there are such differences in reported effects. While the use of a 0.9 % NaCl solution on the epithelium for 6 h was not found to cause obvious increases in cornea1 thickness in vitro25, 0.97-l .O% NaCl solutions have been reported to produce marked increases in epithelial thickness”, changes in light scattering properties of the epithelial cell layer26 and substantial desquamation of superficial cells*‘. In vivo studies* indicate that only marginal changes in central cornea1 thickness would be expected after irrigation of the cornea1 surface with 1% NaCl. These rather different results could perhaps be attributed to the fact that different laboratories performed the analyses and used a range of different techniques to assess the impact of NaCl solutions on the cornea1 epithelium. With the general lack of data on the effects of different solutions on the cornea1 epithelium, cornea1 hydration or cornea1 thickness, a comparative study was therefore undertaken of a range of epithelial incubation solutions using the same specular microscope system.

Effects

of salt solutions

The primary reason for the comparisons was to have a standardized set of experimental conditions in which changes in cornea1 thickness and hydration could be compared. A few preparations were also examined by scanning electron microscopy. Methods Rabbits Female. grey Dutch Belt rabbits of 1.9-2.2 kg weight (10 wei-ks of age) were obtained from a registered local breeder and individually caged (63 X 55 cm floor size, 45 cm high) in Canadian Council for Animal Care (CCAC) approved quarters. All procedures were approved by the local animal care committee which is subject to periodic review by the CCAC. The rabbits were held in quarantine to allow for adjustment from road shipment and acclimation to an artificially imposed light: dark cycle of 14 : 10 hours; the light cycle was started at 06.00h and was provided by ceiling-located cool white fluorescent tubes. After being monitored for 6-8 days to verify that they were free of any clinically discernible upper respiratory or gastrointestinal health problems, the corneas of all animals were checked for normality by slit-lamp biomicroscopy as previously detailed4’. In vitro

incubation

of isolated

corneas

Individual rabbits were euthanized between 14.30 and 15.30h with T-61TM solution (Hoetsch, Canada; 0.5 mlkg-‘; via peripheral ear vein). All neck blood vessels were then severed and the eyes were carefully enucleated with the lids lightly closed. Undue pressure on the epithelial surface, even through the lids, can result in significant epithelial compromise. Cornea1 preparations were made for specular microscope perfusion within 10 min of death essentially following previously detailed protoco120,25.48. In all experiments, the cornea1 endothelium was perfused with a 35 mM bicarbonate, mixed salts solution48 equilibrated with 5 % CO,-10% O2 under an applied hydrostatic pressure (posterior to anterior) of 20 cmH,O at 36 i 1“C. The epithelial side of the preparation was usually covered with 0.75 ml of the test solution and cornea1 thickness measured with the specular microscope. These epithelial-side solutions were then carefully aspirated, replaced with fresh solution and the epithelial-side chamber (see Doughty49 for illustration) covered with a glass plate (after Riley25). Every 15 min, the place was removed, the cornea1 thickness remeasured as quickly as possible, the solution replaced with fresh prewarmed (and gas equilibrated, where appropriate) solution and the glass plate replaced. The solution that had been in contact with the epithelium for 15 min was frequently assessed for composition at this stage, i.e. the solution being removed each time was analysed instead of

on rabbit

cornea/

epithelium:

M. J. Doughty

587

being discarded (see below). Every effort was made to avoid touching the cornea1 surface with either the pipette used to remove the solutions or with the specular microscopic objective. This procedure was followed for a total of 4 h for each cornea. A few corneas were evaluated using the same apparatus but with the epithelial surface covered with a layer of medical grade silicone oi125rather than an aqueous solution. Cornea1 thickness measurements were made every 15 min through the silicone oil and corrected for refraction errors by 7 % Assessment of cornea1 hydration for fied

corneas

After in vitro incubation, both the endothelial- and epithelial-side solutions were replaced with a freshly prepared fixative solution. The cornea was perfusion-fixed for 1 h at 36°C and then a 9mm button was trephined out from the endothelial side. As a result of fixation, little change in cornea1 thickness occurred (average 2.7%) with the maximum change being a 4.3 % decrease in thickness. Most of the buttons were then weighed to 1 mg accuracy and dried in a 60°C oven for 3-4 days. The buttons were cooled to room temperature in a closed vial, weighed to 0.1 mg accuracy, further dried for another 24 h and weighed again to verify that they were fully dried. The water content of the glutaraldehyde-fixed cornea1 buttons was then assessed by calculation of the percentage hydration (%H) where: %H = [ 1 -(dry

weight/wet

weight) x 100133

The same procedure for assessment of wet weight and dry weight was also used on a set of freshly excised and unfixed 9 mm cornea1 buttons. Scanning electron microscopy All corneas from perfusion experiments were glutaraldehydefixed so that samples could be randomly taken for scanning electron microscopy. From each set of epithelial incubation experiments, 1 or 2 corneas were prepared for scanning electron microscopy (following previously detailed techniques2’) rather than being dried. Experimental solutions The bicarbonate-containing, mixed salts solution that was used to perfuse the endothelium (Table I; endothelial solution) was prepared as previously detailed4* using Water for Injection (Baxter Hospital Division). This solution was then immediately equilibrated with a medical grade mixture of 5% CO,-10% O,-balance N, at 36°C. The various epithelial incubation solutions were also prepared with Water for Injection; the composition of the solutions is given in Tables 1 and 2. Some of these solutions were

588

Ophthal.

Table

Physiol.

1. Cornea1

Opt.

incubation

1995

solutions*

Endothelial perfusion solutiont NaCl KCI CaCI, MgCI, NaHCO, Glucose

Epithelial solution

115.0 5.5 1.8 0.8 35.0 5.5

Osmolality PH co2 Pco,

295 7.52 34.5 41.3* 34.0 78 1.24

[HCO,l PO2

Ca*+

15: No 6

1

Epithelial solution 2

Epithelial solution 3

Epithelial solution 4

Epithelial solution 5

115.0 5.5 1.8 0.8 35.0 0.5

145.0 5.5 1.8 0.8 5.0 0.5

155.0 5.5 1.8 0.8 5.0 0.5

155.0 20.0 1.8 0.8 5.0 0.5

115.0 5.5 1.8 0.8 35.0 0.5

f 1 5 0.01 +_ 0.5 1.0 * 0.4 k 2 k 0.07

293 i. 7.54 + 34.7 + 41.7 + 34.1 + 153k2 1.25 f

1 0.04 0.4 0.9 0.5 0.06

294 7.51 34.0 44.1 33.7 686 1.27

+ * f + + & k

2 0.04 0.1 0.4 0.1 7 0.01

296 + 7.34 * 3.9 f 7.0 + 11.5 + 153&l 1.48 +

1 0.02 0.1 0.2 0.2

312+2 7.34 * 3.8 f 6.9 f 11.4iO.l 152+ 1.49 &

0.02

325 f 1 7.33 * 0.04 4.1 kO.1 6.8 * 0.1 11.0~0.2 154+1 1.46 + 0.05

0.03 0.2 0.1 1 0.04

*Endothelial perfusion solution equilibrated with 5% CO,- 10% O,-balance N,; epithelial solution 1 equilibrated with 5% CO,-balance air; epithelial solution 2 equilibrated with 5% CO,-95% 0,; epithelial solutions 3, 4 and 5 equilibrated with 0.5% CO,-21 % O,-balance. N,. Concentrations of salts and glucose are given in mM I-‘; gaseous compositions are as mmHg. All solutions were equilibrated and used at 36OC. -Supplemented with 1 mM adenosine and 0.1 mM glutathione (reduced as added).

Table

2. Cornea1

incubation

solutions

NaCl KCI Na, HPO,-NaH,PO, Osmolality PH PO2 *All

solutions

were

equilibrated

6 to 9” Epithelial solution 6

Epithelial solution 7t

Epithelial solution 8t

140 0 10 299 + 4 7.18 f 0.09 154+6

159.0 4.8 0 302 k 3 6.50 f 0.14 157+3

166 0 0 304 f 6.41 + 0.13 156+7

(with

air) and used

Epithelial solution 9 154 0 0 288 + 2 6.81 +-0.10 153*4

at 36°C.

fAfter Bachman and Wilson26.

equilibrated with 5% CO,-air, 5% CO,-9.5% 0, or 0.5 % CO,-21 % 0, as indicated. All solutions were prepared immediately before use and samples were frequently taken to check maintenance of gaseous composition, pH and osmolality as previously detailed49,50. Materials

All electron microscopy supplies were taken from JBEM (Quebec, Canada). The fixative was 2% high purity glutaraldehyde in 80mM cacodylate buffer, pH 7.2-7.4. The solution was freshly prepared, had a bulk solution osmolality of 33-340 mOsm kg-’ and was routinely checked by UVvisible spectroscopy to ensure that the polymer content was minimal*“. All chemicals and biochemicals used for the cornea1 incubation solutions were obtained from Sigma Chemical (St. Louis, MO, USA) and were of the highest purity available.

Results of the endothelial

Evaluation thickness silicone

and hydrations

perfusion

in vitro

solution

(epithelium

on cornea1 covered

with

oil)

The overall experimental approach adopted was to perfuse the endothelium with an artificial aqueous humour and to incubate cornea1 epithelium in various test solutions. For such an experimental approach, it is important that the endothelium perfusion solution was non-toxic and that its use resulted in maintenance of a non-swelling cornea. That this was the case was checked by perfusing the endothelium with this solution (endothelial perfusion solution, Table 1) and covering the epithelium with silicone oil. As illustrated in Figure I, corneas generally maintained their thickness over a 4 h period (Figure la) although up to a 6 % temporary increase in cornea1 thickness occurred during the first

Effects

320L

I 0

I 1

I 2

of salt solutions

I 3

on rabbit

cornea/

epithelium:

M. J. Doughty

589

4

Time (hours)

Figure 1. Assessment of the endothelial perfusion solution on central cornea1 thickness (a) with time in vitro, and (b) by scanning electron microscopy. See text for further details. The cornea1 thickness values are means *SD for seven preparations. The electron micrograph was taken close to the middle of the endothelial surface. Bar indicates 42pm (corrected for tissue shrinkage).

40 min of perfusion. Recovery back to the initial values then occurred and a slight de-swelling then followed; the final cornea1 thickness in these experiments was 344 -t 6p.m. With the use of this mixed salts solution containing 35 rnM bicarbonate, the cornea1 endothelium was always clearly visible in the specular microscope. After the perfusion. all of the corneas were fixed with glutaraldehyde. The fixed cornea1 buttons had an average wet weight of 29.2 & 0.5 mg, the dried buttons weighed an average of 9.4 mg so giving an average hydration of 67.3 % . If freshly excised (9 mm) cornea1 buttons were dried (without fixation), a hydration of 72.2 f 1.1% (n = 5) was obtained. The difference is assumed to be due to both the slightly greater thickness of the fresh corneas and the fact that the perfused corneas were fixed prior to being dried. Evaluation of the effect of the endothelial pe@sion scanning electron microscopy

by

Scanning electron microscopy of the endothelium consistently showed an uninterrupted mosaic of similar sized polygonal cells (Figure lb). This same general appearance for the endothelium was seen both in corneas where the epithelium was covered with silicone oil and where the epithelium was incubated in various aqueous solutions (unpublished data).

Evaluation of epithelial solutions I and 2 (high bicarbonate) on cornea1 thickness and hydration in vitro under normoxic and hyperoxic conditions With the endothelium perfused with a mixed salt solution containing 35 mM bicarbonate (i.e. endothelial perfusion solution, Table I), the first epithelial experiment was to verify that cornea1 thickness could be maintained in vitro when a very similar bicarbonate solution was used on the epithelium as well (instead of silicone oil). Epithelial solution 1 contained 35 mM bicarbonate (in equilibration with 5% CO,-air; pH of 7.54; bulk solution osmolality of 293 mOsm kg-‘) but only 0.5m~ glucose and neither adenosine nor glutathione were added. A constant cornea1 thickness was observed over 4 h when epithelial solution 1 was used to cover the epithelium (Figure 2). Repeated measurements of the composition of this solution after contact with the epithelium confirmed that the frequent changing of the solutions was adequate to maintain the composition within &5 % of the specified values of Pco?, PO, and osmolality (see Doughty49 for detailed evaluation). The final cornea1 thickness after the 4 h incubation was 347 f 4 pm giving fixed cornea1 buttons with an average wet weight of 30.9 mg; a hydration of 70.6% was calculated (Table 3). This value is within the same range as the hydration values found for freshly isolated cornea1 buttons

590

Ophthal.

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Physiol.

L!

0

Opt.

1995

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I

I

I

1

2

3

in epithelial solution 1 showed a remarkably unblemished surface with almost no exfoliating cells evident (Figure 3). The epithelial surface appeared as a heterogeneous mosaic of light, medium and dark cells with good contrast between each type (Figure 3a). This general appearance was evident even at ~25 at-stage magnification and appeared very similar to that observed for freshly isolated corneas without any form of in vitro irrigation or incubation (see Doughty5’ for micrographs at ~25 magnification). At x 1000 at-stage magnification (Figure 3b), it can be seen that the cell-cell borders are clearly evident. In some places, what appears to be adherent material (visible as thin white lines on the micrographs) was present along the cell-cell borders. On some of the cells, especially the dark reflex cells, the cell surfaces are embellished with round features that have been termed ‘epithelial holes’ or ‘epithelial craters’; some of these have a prominent collar or rim to them (Figure 3~). At x 5000 at-stage magnification (Figure 3c), the surfaces of the actual cells can be seen to be composed of a tightly packed array of erect microplicae. Along many of the cell-cell borders, the cell-cell boundary is delineated by a narrow darker strip that can be likened to the appearance of caulking between masonry or woodwork (see arrowheads on Figure 3~). With the use of the same solution equilibrated with hyperbaric oxygen (epithelial solution 2)) scanning electron microscopy revealed that the surface was slightly altered. A sizeable number of exfoliating cells were present as well as cells with bright electron reflexes and coarse surface features. Occasional damaged cells were also present (not shown but see Figure 6a and related text below).

I 4

Time (hours) Figure 2. Changes in central cornea1 thickness in vitro following epithelial surface incubation in: (0 1, epithelial solution 1, or (n), epithelial solution 2. The endothelium was perfused; see text for further details. Results are presented with mean +SD for 6 or 7 preparations.

and those incubated with silicone oil on the epithelium. The use of 5 % CO,-air as the gas mixture to equilibrate these types of solution might be questioned since many of the published epithelial electrophysiology studies have used high bicarbonate, mixed salt solutions that were gassedwith hyperbaric oxygen (95% 0, with 5% C02). Epithelial solution 2 (equilibrated with 95 % O,-5 % CO,) consistently produced slight cornea1 swelling (Figure 2) with a final rate of 14 + 4 pm h-’ The fixed cornea1 buttons were consistently found to have slightly larger wet weights (35.2 f 1.7mg) than those incubated in high bicarbonate but normoxic solutions (30.9 f 0.4 mg; P < 0.01). Dry weights were not obtained for these samples since they were used in other experiments.

Evaluation of epithelial solutions 3, 4 and 5 (low bicarbonate) on cornea1 thickness and hydration in vitro

Evaluation of epithelial solutions I and 2 (high bicarbonate) under normoxic conditions by scanning electron microscopy

In a second series of experiments, the epithelium was incubated in mixed salt solutions that contained only 5 mM bicarbonate in equilibrium with 21% 0, (i.e. equivalent to

Corneas that maintained a stable thickness after incubation Table

3. Summary

Experimental

of cornea1

solution

swelling

and cornea1

Cornea1

swelling

rate (pm h- ‘I Epithelial Epithelial Epithelial Epithelial Epithelial Epithelial Epithelial Epithelial

solution solution solution solution solution solution solution solution

1 3 4 5 6 7 8 9

(PBS) (NaCIIKCI) (saline) (saline)

Zero t 15+3 8*2 21 +3 17*4 23 f 7 28 i 3

hydration

data

Final cornea1 thickness (pm)

347 543 427 388 452 427 475 488+

2 + k + + + +

4 6 4 3 11 5 8 12

*Hydration determined by comparing the value of the wet weight The dry weights were all in the range of 9.1-9.7 mg. iA swelling rate was not determined because of non-linearity. PBS, phosphate-buffered saline.

Wet weight of cornea/ buttons (mg) 30.9 47.7 37.2 34.5 39.5 37.6 42.5 44.5

with

+ f + + + + + *

Hydration of cornea1 buttons f%I

0.4 1.77 2.1 1.3 1.5 0.8 1.6 2.0

the dry weight

71.6+ 1.4 82.2 + 1.6 74.2 f 1.9 73.3 i 2.6 Not determined 74.7 + 0.8 76.8 + 1.9 79.2 + 2.6 where

H = [(W-D)

No. of *

corneas 5 5 5 5 4 5 6 4

/ W I X 100%.

Effects

of salt solutions

on rabbit

cornea/

epithelium:

591

M. J. Doughty

Figure 3. Scanning electron microscope images of the mid-peripheral cornea1 epithelial surface after 4 h incubation in x 1000; vitro with epithelial solution 1 (high bicarbonate): (a) low magnification: x 200; (b) medium magnification: (c) high magnification: x 5000. Arrowheads in (c) indicate ‘caulking’ between cells (see text). Bar indicates: (a) 150pm; (b) 84pm; (c) 15pm (corrected for tissue shrinkage).

the oxygen concentration in air). To provide an equilibrium pH that was similar to that of the 35 mM bicarbonate solution (e.g. epithelium solution l), epithelial solutions 3, 4 and 5 were equilibrated with 0.5 % CO,-21 % 0, rather than 5% CO,-air; the resultant pH was 7.34 f 0.02. With the use of isotonic 5 mM bicarbonate solution (epithelial solution 3), cornea1 swelling developed within 15 min (Figure 4). The initial rate of swelling was close to 15 pm h-’ but, after 2 h, a much faster rate of swelling developed (Figure 4). Central cornea1 thickness values averaged nearly 550 pm after 4 h to give cornea1 hydration values from fixed buttons of 82.2 % (Table 3). None of the five preparations evaluated showed a linear phase of cornea1 swelling. Since swelling was observed with the use of epithelial solution 3, an attempt was made to attenuate this swelling by supplementing this solution with NaCl to raise the osmolality to 312 k 2 mOsm kg-‘; this gave epithelial solution 4 (Table I). With the use of this solution, the overall swelling rate was markedly reduced so that a net change in thickness of only 56 k 4 pm developed over 4 h (Figure 4; Table 3). A linear swelling rate of 15 -t 3 pm h-’ was recorded in the fourth hour. Further supplementation of the low bicarbonate solution was attempted with KC1 by raising the concentration of this salt from 5.5 to 20 mM (Table 1) with or without equivalent reductions in NaCl concentration. If the KC1 concentration was simply increased, the final osmolality of the solution was 325 f. 1 mOsm kg-’ (epithelial solution 5). With the use of this slightly hypertonic solution, swelling was further reduced; a net swelling rate of only 8 f 2 pm h-’ in the fourth hour was measured (Figure 4) and the final buttons

350

300 ! ;

I 1

I 2 Time

I 3

I 4

(hours)

Figure 4. Changes in central cornea1 thickness with time in vitro following epithelial surface incubation with: (O), epithelial solution 3; ( l ), epithelial solution 4; or (0). epitheiial solution 5. The endothelium was perfused; see text for further details. Results are means *SD for five preparations for each solution.

had hydration values close to those for fresh corneas, i.e. 73.3 % vs 72.2 % (Table 3). Several other slightly different formulations were tried but no further improvement was achieved over the results obtained with epithelial solution 5. For example, with the use of a 5 mM bicarbonate solution

592

Ophthal. Physiol. Opt. 1995 15: No 6

in which the 5 or 15 mM KC1 was substituted for an equivalent quantity of NaCl, produced cornea1 swelling rates intermediate between those recorded for epithelial solution 3 and epithelial solution 4 (data not shown); K+ supplementation (and thus increase in osmolality) produced superior results to Na+ substitution with K+. Evaluation of epithelial solutions 3, 4 and 5 (low bicarbonate) by scanning electron microscopy Scanning electron microscopy (SEM) of one or two of those corneas for which the epithelium was incubated in either of the three low bicarbonate solutions (epithelial solutions 3, 4 and 5) revealed similar appearances and so these will be considered as a group. A representative low magnification montage is shown in Figure 5 of a cornea incubated in epithelial solution 5. The cornea1 buttons were deliberately cut in half prior to the final preparation for SEM simply so that the endothelium could be examined as well (see Figure lb). The half-cornea button retains its curvature after fixation and it should be noted that the surface is not compromised by wrinkles or creases. There were also no obvious substantial mechanical abrasions or similar artefacts

on the samples examined. Such montages were made of the cornea1 surface for corneas incubated in all three of the low bicarbonate solutions. All five samples showed the epithelial surface to be largely unblemished with only minor mechanical artefacts (see arrowheads on Figure 5). The epithelial surface, in the low magnification montages, appeared as a slightly mottled surface of lighter and darker shades. One might conclude therefore that the epithelial surface was uncompromised by the 4 h incubation in low bicarbonate, slightly hypertonic solutions. However, just slightly higher magnification imaging revealed the presence of exfoliating cells. The insert to Figure 5 shows a number of exfoliating cells with upturned borders. A similar appearance was seen for the corneas incubated in the three different solutions containing 5 mM bicarbonate in that some exfoliating cells were present across the entire epithelial surface. With the use of epithelial solution 5, examination of the cornea1 surface at four different locations (at the magnification of the insert for Figure 5) revealed 5-15 obviously exfoliating cells at each location. With the use of epithelial solution 4, this number was 20-30 cells and with the use of epithelial solution 3, 30-50 obviously exfoliating cells were evident in such sampled areas of about 0.4 mm*. It is important to

Figure 5. Typical very low magnification ( x 25 at-stage) scanning electron microscope image of the surface of a bissected cornea1 button after 4 h incubation in epithelial solution 5 (low bicarbonate). Arrowheads mark areas of minor damage to superficial cells. Bar represents 1.8 mm (corrected for tissue shrinkage). The inset shows the area outlined by the box at x 125 at-stage magnification.

Effects of salt solutions on rabbit cornea/ epithelium: M. J. Doughty note, however, that while exfoliating cells were present, the epithelial surface did not appear to be substantially damaged after 4 h exposure to the 5 mM bicarbonate solutions. The SEM also reveals numerous subtle characteristics of the slightly altered epithelial surface. After the use of epithelial solution 5, the exfoliating cells were not always very obvious (Figure 6). At ~200 at-stage magnification (Figure 6u), a number of cells can instead be seen to have a darker central region and/or an unusually brighter electron reflex. The same bright reflex cells were also evident after use of epithelial solution 2. Higher magnification imaging (Figure 6b; x 1000) shows these ‘bright’ cells to have edges that are either thickened or slightly upturned suggestive of initial stages of the exfoliation process. Adjacent to the bright cell (Figure 6b) is a cell with a much darker electron reflex (that would be termed a dark cell; Doughty”) and for which one edge has clearly peeled away from the surface to uncover a lighter cell. At very high magnification (Figure 6c; x 5000 at-stage magnification), the surfaces of unblemished cells can be seen to have a dense array of erect microplicae on them. On the dark reflex cell (upper right, Figure 6c), a few microvilli can be seen as well. The surface of medium reflex cells is also decorated with microscopic debris (the white spots). Along the cell-cell borders, some sort OF ‘caulking’ was also evident (see arrowheads in Figure 6~). In the samples analysed, epithelial craters were almost completely absent from the cells after this in vitro exposure to 5 mM bicarbonate-containing, mixed salt solutions.

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Evaluation of epithelial solutions 6, 7, 8 and 9 (salines) on cornea1 thickness and hydration in vitro If the cornea1 epithelium was incubated in an isotonic citrate-acetate buffered mixed salt solution that did not contain bicarbonate, cornea1 swelling developed at a rate of 20-25 ym h-’ (unpublished data, 3 experiments only). A commercial balanced salt solution (BSS)*’ was used for these studies in an attempt to see if complete removal of bicarbonate would further increase the swelling observed with epithelial solution 3. Since a dramatic increase in the rate of swelling was not observed, no further studies were done and a simpler solution was tried. If the epithelium was incubated in PBS (epithelial solution 6; Table 2), some cornea1 swelling was evident after 15 min but the overall rate of swelling (2 1 f 3 pm h-‘) was slightly less than that observed with epithelial solution 3. The PBS solution has an osmolality of 299 + 4 mOsm kg-’ (compared to 296 k 1mOsm kg-’ epithelial solution 3). The pH of the PBS was 7.18 f 0.09 (4 different batches prepared). Dry weights were not obtained for these samples after incubations as they were used in other experiments. However, the wet weight of the fixed buttons averaged 39.5 mg which is consistent with significantly increased hydration. With phosphate-buffered saline (PBS) producing only about 20 pm h -’ initial cornea1 swelling, simpler unbuffered saline solutions were tested even though the pH of these solutions is not predictable. The use of an NaCliKCl mixture (epithelial solution 7, Tub/e 2) resulted in a cornea1 swelling rate averaging only 17 pm h-’ (Figure 7; Table 3) to give cornea1 buttons with a net hydration of 74.7 %

Figure 6. Scanning electron microscope images of part of the mid-peripheral cornea1 epithelial surface after 4 h incubation in vitro with epithelial solution 5 (low bicarbonate): (a) low magnification: x 200; (b) medium magnification: x 1000; (c) high magnification: x 5000. Arrowheads in (a) indicate abnormal ‘bright’ cells; arrowheads in (c) indicate ‘caulking’ between cells (see text). Bar represents: (a) 150 pm; (b) 84 pm and (c) 15 pm (corrected for tissue shrinkage).

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Figure 7. Changes in central cornea1 thickness with time in vitro following epithelial surface incubation with: (A.), epithelial solution 7; (01, epithelial solution 8; and (W), epithelial solution 9. The endothelium was perfused; see text for further details. Results are mean *SD for 4 or 5 preparations for each epithelial solution.

If this NaCliKCl solution was supplemented with 0.5 mM glucose, the same degree of cornea1 swelling occurred (2 experiments, data not shown). A rather similar result was obtained if isotonic saline (epithelial solution 8, Table 2) was used with a rate of swelling of 23 ,um h-’ being recorded (Figure 7; Table 3). The net change in cornea1 thickness was significantly greater than the thickness in increase seen with the use of PBS for the epithelium. Lastly, incubation of the epithelium in slightly hypotonic NaCl (0.9%; epithelial solution 9, Table 2) was found to produce cornea1 swelling that was measurable after 15 min and continued over 4 h (Figure 7). The swelling, like that seen with epithelial solution 3, was not linear. In the fourth hour, the swelling rate was nearly linear with an average value of 28 f 3 pm h-’ (n = 4) to give a final thickness of 488 + 12 pm. The wet weight of the fixed cornea1 buttons averaged 44.5 mg to give a calculated net hydration of 79.2% (Table 3). Evaluation of epithelial solutions 6, 7, 8 and 9 (salines) by scanning electron microscopy One or two of each of the corneas incubated in the saline solutions (epithelial solutions 6, 7, 8 and 9) were examined by scanning electron microscopy; all showed similar substantial alterations to the epithelial surface and so they will also be discussed as a group. A representative montage of one of these corneas (after incubation in epithelial solution 8) is shown in Figure 8. The only image artefacts of note are the few black spots scattered over the surface that are due to loss of the gold-palladium coat (which occurs easily when the cornea1 surface is very irregular, as it was in all

these cases). At x 20 to x 25 at-stage magnification, the cornea1 surface now was observed to have a very irregular electron reflex. This type of appearance was seen after exposure to epithelial solutions 7, 8 or 9. After exposure to PBS (epithelial solution 6), the surfaces were a little more homogeneous but there was still significant irregularity (not shown). With all four solutions, the image consisted of numerous lighter grey irregular patches on a darker grey background. The inset to Figure 8 (at X5 higher magnification) shows the lighter grey irregular patches to be very damaged and/or exfoliating cells; the darker grey reflex of the image is the underlying layer of cells. In this example, 100% of the surface cells are damaged. In Figure 9 is part of the same epithelium illustrated in Figure 8 at X200 (Figure 9a), X 1000 (Figure 9b) and X 5000 (Figure SC). At ~200 and especially at x 1000, the lighter grey irregular patches are clearly severely damaged cells; darker reflex cells have lesser surface damage; most cells showed severe damage after epithelial solution 7 but only about half the cells showed severe damage after exposure to epithelial solution 6 (PBS). Higher magnification imaging reveals the transition between severe damage and earlier stages (Figure SC). The surfaces of cells with early damage were embellished with widely spaced irregular microplicae with occasional nodular (microvillus) projections as well (see left-hand side of Figure SC). Adjacent to this cell is a cell with a wrinkled surface membrane that contains numerous small holes. Above this severely damaged cell is a cell in which the surface membrane has completely disintegrated (arrowhead in Figure SC). Discussion These studies clearly show that the use of different epithelial incubation solutions in vitro can produce very marked differences in cornea1 thickness, cornea1 hydration and the structural state of the superficial cells of the rabbit cornea1 surface. The type of solutions used in the present studies have all been used, with little or no comment, in published physiological, pharmaceutical or toxicological studies. The specular microscope perfusion system can clearly be used to evaluate the effects of various solutions and chemicals on the cornea1 epithelium. The changes in cornea1 thickness that result from the toxic effects of the solutions used in the present study are clearly less than the changes that can be easily demonstrated using the same method and exposing the cornea1 epithelium to preservative-containing solutionsz8. However, some solutions that do not contain test chemicals can clearly produce damage to the surface cells and such changes could interfere with studies designed to explore the mechanisms of toxicant-induced damage or could interfere with determination of threshold damage effects for toxic chemicals (such as preservatives). It is interesting that the maximum rate of cornea1 swelling observed was only around 30 pm even though gross damage to the superficial

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Figure 8. Typical very low magnification ( x 25 at-stage) scanning electron microscope image of the surface of a bissected cornea1 button after 4 h incubation of the epithelium with epithelial solution 8 (saline) in vitro. Bar indicates 1.8 mm (corrected for tissue shrinkage). The inset shows the area outlined by the box at x 125 at-stage magnification.

Figure 9. Scanning electron microscope images of part of the mid-peripheral region of the cornea1 epithelial surface after incubation in epithelial solution 8 (saline): (a) low magnification: x 200; (b) medium magnification: x 1000; (c) high magnification: x 5000. Arrowheads in (b) and (c) indicate a cell with total destruction of the surface membrane. Bar indicates: (a) 150pm; (b) 84pm; and (c) 15pm (corrected for tissue shrinkage).

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cells was evident under some of these conditions (e.g. following the use of NaCl solutions on the epithelium). In agreement with the studies of Wilson et a1.28, incubation of the epithelium with surfactant-containing solutions (e.g. Geropon AC-78 0.05% in PBS) can produce swelling at a rate in excess of 100 pm h-’ (unpublished data). The five different types of epithelial incubation solutions used in this comparative study have all been used in other studies with little data being provided on either cornea1 thickness or cornea1 hydration. Experimental protocol have either used an intact enucleated eye or an isolated cornea mounted in some form of apparatus such that the endothelium could be exposed to an aqueous solution or silicone oil. The epithelial solutions have included saline (NaCl) solutions2’,25.26.45,46,52, PBS solutions43.44,NaCliKCl solutions26, mixed salts solutions buffered with phosphate, citrate or acetate4’.42 and various formulations of bicarbonatecontaining mixed salts solutions23-26.30-40. Some of these solutions have also contained glucose (up to at least 33.9 mM>, sucrose, phosphate or Tris buffers, gluconate salts and buffers, adenosine, glutathione or even y-aminobutyric acid. The present studies provide a standardized comparison of five types of solutions on the rabbit cornea1 epithelium in vitro. In all studies, the endothelium was perfused with a non-toxic, glucose and bicarbonate-containing, mixed-salts solution. This approach should minimize any contributions of endothelial functions to the maintenance of cornea1 thickness in vitro’9.20. Cornea1 thickness and the appearance of the epithelial cells can be maintained close to those found in vivo if a high bicarbonate, isotonic mixed-salt solution is used to incubate the epithelium. A similar result can be obtained with a low bicarbonate, mixed salts solution if it is made slightly hypertonic while all other solutions produced obvious damage of the superficial cells. Reasonably stable cornea1 thickness values have been noted with the use of high bicarbonate, mixed salt solutions on the cornea1 epithelium for up to 6 h25, up to 3 h23, l-3 h22-24.26.28 or just 1 h53. Other studies have reported significant initial cornea1 swelling33,40 (at 29-50 pm h-‘) or variable initial swelling39; this initial swelling has been temporary in some preparations. Cornea1 hydration levels of nearly 83 % were reported after incubation of corneas in a similar high bicarbonate solution (equilibrated with 5% CO,-95% 02)38. In the present studies, cornea1 hydration values for freshly isolated tissue have been found to be close to 72% with the highest level of 82.2% being found for fixed button that were almost 200 pm thicker than the freshly isolated corneas. The prefixation used in the present study appears to produce wet weight values that are 5-7% smaller than those found for unfixed tissue. As a result, the tissue hydration (%H) values are slightly smaller than might be expected although it seems most unlikely that an unfixed cornea will respond in the exact same way as a glutaraldehyde-fixed cornea to trephination. Regardless, a value of 82% hydration at a cornea1 thickness of 550 pm is thus

probably an underestimate indicating that a value of 83% reported for cornea1 tissues38 means that these corneas were at least 550pm thick. Little attention has previously been given to the scanning electron microscopy appearance of the cornea1 epithelial surface after in vitro incubation in any aqueous solutions. After in vitro exposure to high bicarbonate, mixed salt solutions, one published micrograph (at x720) shows a mosaic of polygonal cells with the cells having a range of light, medium and dark electron reflexes23 similar to those found in the present study. Many of the cells in this micrograph were conspicuously decorated with crater-like features but the cells also appeared slightly oedematous (as the apical surfaces bulged outwards slightly). For another high bicarbonate, mixed salts solution, another micrograph (high magnification: c. x3000) of the cornea1 epithelial surface lacked obvious light, medium and dark contrast between the cells and the cells also showed signs of oedema38. Transmission electron micrographs prepared from corneas incubated in the same solutions showed marked irregularity to the epithelial cell surfaces38and it is unclear why these features were not evident in the scanning electron micrographs (or vice versa). The results of these studies suggest that maintenance of the thickness of a cornea1 preparation in vitro can be achieved with the use of a high bicarbonate, mixed salts solution equilibrated with 5% CO,-air for the epithelium. However, it is recognized that such solutions may not always be compatible with test chemicals and their use requires that means are available to prepare and maintain the appropriate CO, equilibria, pH and ionized calcium levels20.49,50.The use of lower bicarbonate/lower CO, levels may provide greater compatibility and flexibility in that the solutions could be supplemented on an empirical basis with another buffer to help stabilize the pH. In the present studies, a low (5 mu) bicarbonate-containing mixed salts solution (equilibrated with 0.5% CO,) was found to support a relatively stable cornea1 thickness and cornea1 hydration but only if these solutions were made slightly hypertonic (with the addition of some NaCl and KCl). This finding is consistent with a number of recent reports on the osmolality of normal rabbit tear film54,55. Similarly, the electrical impedance of bovine cornea1 epithelium was reported to be stable only if the bicarbonate-containing, epithelial-side solution was made very hypertonic (0.234 M, i.e. about 450 mOsm kg-‘) to the solution used on the endothelium3’. Only slight morphological changes were reported4’ after exposure of rabbit cornea1 epithelial surfaces to buffered balanced salt solutions (without bicarbonate) at both 330 and 407mOsm kg-‘. Most recently, the use of bicarbonate-containing artificial tear solutions were reported to promote faster recovery of the epithelial cells from a toxic challenge, compared with the use of solutions containing other potential buffering agents12.The present studies also indicate that bicarbonate

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can be beneficial. At this time, it is unknown if these bicarbonate effects reflect a specific action on the cornea1 epithelial cells or an action on the cornea1 stroma. Notwithstanding, the present studies confirm those of others22m26.28.53 that cornea1 thickness can be maintained in vitro in a specular when the cornea1 epithelial surface is incubated in a high bicarbonate-containing solution, or a low bicarbonate solution that is slightly hypertonic. Either way, these so1ution.s (but not buffered saline solutions, PBS, Na/K or NaCl) can be used as vehicles for delivery of drugs or fluorescein to the cornea for in vitro assessments of the effects of various agents or exposure times on cornea1 thickness, epithelial permeability or the scanning electron microscope studies of the cornea1 epithelial surface. More detailed evaluation of the morphological changes that can occur and how these relate to epithelial permeability are currently in progress. The present set of comparative studies confirm that saline solutions can be very cytotoxic to the cornea1 epithelial surface cells. Bergamson and Wilson22 reported a lo-fold increase in exfoliating or damaged cells (assessed by transmission electron microscopy) after 2.5 h incubation of rabbit cornea1 epithelium in 0.97% NaCl. Increases of 5% in epithelial thickness (i.e. epithelial oedema) have been reported after incubation of rabbit cornea1 epithelium in 0.9% saline for just a few minutes” while O’Leary and Wilson2’ note that exfoliation of sheets of cells could develop within 45 min of exposure of rabbit cornea1 epithelium to 1% (172 mM) NaCl. The cell damage is presumably the cause of enhanced light scattering evident, by specular reflection, in the superficial layers of the cornea1 epithelium after exposure to 0.97% NaClz6. The present scanning electron microscopy confirms this damage although the degree of damage seen might be more than that encountered in some of these previous studies since it has been implied’2.26 that cornea1 swelling was usually less than 6% over 2-3 h. It should be noted, however, that a 1% NaCl solution (171 mM) can be expected to be hypertonic to the endothelial perfusion solution so an osmotically induced maintenance of cornea1 thickness could have contributed to the lesser swelling that was encountered. However, this does no! explain why 0.88% saline (150 mM NaCl) was not found to cause a progressive increase in cornea1 thickness in an earlier study25. O’Leary and Wilson” note that the first signs of exfoliation of epithelial cells were evident by specular reflection within 45-70 min upon incubation of rabbit cornea1 epithelium with 1% NaCl. Psychophysical measurements’ or cornea1 thickness measurements’ indicate that changes in the epithelium can be induced in 20 min or less with 0.9% NaCl solutions. The epithelium of a few corneas was exposed to a 35 mM bicarbonate solution that was equilibrated with 95% O2 (5% CO,) rather than 21% 0, (5% COJ because some investigators have also used epithelial solutions that were bubbled with a gas mixture containing 95% 023’.32.3sor

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elected to use 95% 0, for solutions on both sides of isolated corneas38.39.56.None of these studies actually provide data on the oxygen tension (PO, values) of the solution in use. In the present studies, even with a glass plate being used to quickly cover/seal the epithelial-side chamber, the solution still had to be changed every 15 min to maintain the oxygen level within 5 % of the stated value. In a few sampling periods, this value was only 90% of the original value. While scanning electron micrographs did not reveal a gross cytotoxic effect of the use of such high oxygen solutions, there was evidence of increased cell exfoliation which suggests that 95% 0, is toxic to the cornea1 epithelial cells. Further studies are in progress to evaluate these effects in more detail, especially in a timedependent fashion. The present studies reveal that some relationship can be expected between the swelling behaviour of a cornea1 preparation in vitro and the appearance of the epithelial surface (as assessed by scanning electron microscopy). With the use of most (but not all) solutions that produced substantial increases in cornea1 thickness (i.e. 100-200 pm over 4 h), substantial damage was evident at the epithelial surface. Solutions that produced only marginal swelling (<5Opm) over 4 h clearly had an epithelial surface appearance that was readily distinguishable from most solutions that caused substantial swelling of the cornea. Solutions that produced a stable cornea1 thickness in vitro also resulted in a cornea1 epithelial surface that was distinguishable from those where marginal swelling occurred. However, when comparing solutions that produced only small changes on cornea1 thickness (i.e. epithelial solutions 1 and 5), a particularly noteworthy characteristic was the epithelial craters. These craters are present on about 75% of the superficial cells on freshly fixed samples16,i.e. some 25% cells, at least at the time of evaluation, may not have obvious craters with the conspicuous collars. After the use of epithelial solution 5, almost all the cells lacked conspicuous craters and even after the use of epithelial solution 1, almost half the cells examined did not have conspicuous craters on them. A possible explanation is that exfoliation has occurred at some point prior to the evaluation since recently uncovered cells have been reported to have fewer craters on them5’. More detailed kinetic studies are obviously required to clarify this issue but the apparent loss of the craters is suggestive of cell exfoliation and means that in vitro incubated corneas may readily lose some or all of their most superficial layer of cells. Acknowledgements This research was supported by an operating grant from the Natural Sciences and Engineering Research Council (NSERC) of Canada. Additional funding was by a grant from Bausch and Lomb International, Rochester, NY, USA. I wish to express my gratitude to Drs Michael Riley

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(Oakland University) and Keith Green (University of Georgia), for useful discussions, for reviewing a draft of an earlier version of the manuscript and providing many useful comments. References

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Doughty, M. J. General principles of pharmacology. In: Clinical Optometric Pharmacology and Therapeutics (ed. B. E. Onofrey), J.B. Lippincott, Philadelphia, USA, pp. l-38 (1991) Draize, J. H., Woodward, G. and Calvery, H. 0. Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes. J. Pharmcol. Exp. Ther. 70, 377-390 (1944) Freeburg, F. E., Nixon, G. A. Reer, P. J., Weaver, J. E., Bruce, R. D., Griffith, J. F. and Sanders, L. W. Human and rabbit eye responses to chemical insult. Fund. Appl. Toxicol. 7, 626-634 (1986) Conquet, P. H., Durand, G., Laillier, J. and Plazonnet, B. Evaluation of ocular irritation in the rabbit: objective versus subjective assessment. Toxicol. Appl. Pharmacol. 39, 129-139 (1977) Heywood, R. and James, R. W. Towards objectivity in the assessment of eye irritation. J. Sot. Cosmet. Chem. 29, 25-29 (1978) Green, K. and Chapman, J. M. Benzalkonium chloride kinetics in young and adult albino and pigmented rabbit eyes. J. Toxicol. Cut. Ocular Toxicol. 5, 133-142 (1986) Simon, J. M., Verges, C., Camins, J. L. and Pita-Salorio, D. Comparative analysis of treatment of the dry-eye syndrome [Spanish] Arch. Sot. Esp. Ojtalmol. 56, 185-192 (1989) Chan, R. S and Mandell, R. B. Cornea1 thickness changes from bathing solutions. Am. J. Optom. Physiol. Opt. 52, 465-469 (1975) Remole, A. Effect of saline solution immersion on cornea1 light scattering characteristics. Am. J. Optom. Physiol. Opt. 58, 435-444 (1981) Ramselaar, J. A. M.. Boot, J. P., Van Haeringen, N. J., Van Best, J. A. and Oosterhuis, J. A. Cornea1 epithelial permeability after instillation of ophthalmic solutions containing local anesthetics and preservatives. Curr. Eye Res. 7, 947-950 (1988) Lopez Bernal, D. and Ubels, J. L. Quantitative evaluation of the cornea1 epithelial barrier: effect of artificial tears and preservatives. Curr. Eye Res 10, 645-656 (1981) Lopez Bernal, D. and Ubels, J. L. Artificial tear composition and promotion of recovery of the damaged cornea1 epithelium. Cornea 12, 115-120 (1993) Rolando, M., Brezzo, V., Giordano, G., Campagna, P., Burlando, S. and Calabria, G. The effect of different benzalkonium chloride concentrations on human normal ocular surface. A controlled prospective impression cytology study. In: The Lacrimal System (eds 0. P. van Bijsterveld, M. A. Lemp and D. Spinelli), Kugler and Ghedine, Amsterdam, Netherlands, pp. 87-91 (1991) Doughty, M. J. Scanning electron microscopy evaluation of twice-daily use of a chlorobutanol-preserved artificial tear on rabbit cornea1 epithelium. Ophthal. Physiol. Opt. 12, 457-466 (1992) Doughty, M. J. Effect of the use of artificial tears on the size of the squamous cells of the rabbit cornea1 epithelium evaluated by scanning electron microscopy. Optom. Vision Sci. 69, 45 l-457 (1992)

16 Doughty, M. J. Acute effects of chlorobutanol- or benzalkonium chloride-containing artificial tears on the surface features of rabbit cornea1 epithelial cells. Optom. Vision Sci. 71, 562-572 (1994) 17 Dikstein, S. and Maurice, D. M. The metabolic basis of the fluid pump in the cornea. J. Physiol. (Lond.) 221, 29-41 (1972) 18 Edelhauser, H. F. and MacRae, S. M. Irrigating and viscous solutions. In: Surgical Pharmacology of the Eye (eds M. Sears and A. Tarkkanen). Raven Press, New York, USA, pp. 363-388 (1985) 19 Green, K. Cornea1 endolethial structure and function under normal and toxic conditions. Cell. Biol. Rev. 25, 169-207

(1990) 20 Doughty, M. J. Quantitative evaluation of the effects of a bicarbonate and glucose-free balanced salt solution on rabbit cornea1 endothelium in vitro. Optom. Vision Sci. 69, 846-857 (1992) 21 Wilson, G. and Fatt, I. Thickness changes in the epithelium of the excised rabbit cornea. Am. J. Optom. Physiol. Opt. 51, 75-83 (1974) 22 Bergmanson, J. P. G. and Wilson, G. S. Ultrastructural effects of sodium chloride on the cornea1 epithelium. Invest. Ophthalmol. Visual Sci. 30, 116-121 (1989) 23 Green, K., Livingstone, V., Bowman, K. and Hull, D. S. Chlorhexidine effects on cornea1 epithelium and endothelium. Arch. Ophthalmol. 98, 1273-1278 (1980) 24 Klyce, S. D. Stromal lactate accumulation can account for corneal oedema osmotically following epithelial hypoxia in the rabbit. J. Physiol. (Land.) 321, 49-64 (1981) 25 Riley, M. V. The role of the epithelium in control of cornea1 hydration. Exp. Eye Res. 12, 128-137 (1971) 26 Bachman, W. and Wilson, G. Essential ions for maintenance of the cornea1 epithelial surface. Invest. Ophthalmol. Visual Sci. 26, 1484-1488 (1985) 27 O’Leary, D. J. and Wilson, G. Tear-side regulation of desquamation in the rabbit cornea1 epithelium. A specular microscope study. Clin. Exp. Optom. 69, 22-26 (1986) 28 Wilson, G., Bachman, W. G. and Call, P. L. A nutritional role for tears. In: The Preocular Tear Film in Health, Disease and Contact Lens Wear (ed. F. J. Holly), The Dry Eye Institute, Lubbock, TX, USA, pp. 978-987 (1986) 29 Wilson, G. S and Chalmers, R. L. Effect of H,O, concentration and exposure time on stromal swelling; an epithelial perfusion model. Optom. Vision Sci. 67, 252-255 (1990) 30 Babbott, E. The effect of certain drugs on cornea1 impedance. Arch. Ophthalmol. 57, 425-429 (1957) 31 Hori, M. Cell membrane activity of rabbit cornea [Japanese] Folia Ophthalmol. Jpn. 20, l-6 (1969) 32 Akaike, N. and Hori, M. Effect of anions and cations on membrane potential of rabbit cornea1 epithelium. Am. J. Physiol. 219, 1811-1818 (1970) 33 Green, K. Dependence of cornea1 thickness on epithelial ion transport and stromal sodium. Am. J. Physiol. 217, 1169-1177 (1969) 34 Wiederholt, M. and Koch, M. Intracellular potentials of isolated rabbit and human cornea1 epithelium. Exp. Eye Res. 26, 629-640 (1978) 35 Midelfart, A. The effect of amiloride on Na, K and water in bovine cornea1 epithelium. Exp. Eye Res. 45, 751-762 (1987) 36 Erhart, M., Zilliox, P., De Burlet, G. L. and Andermann, G. Differences in ocular toxicity and antimicrobial activity of benzalkonium chlorides. Concepts Toxicol. 4, 145-151 (1987)

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48 Doughty, M. J. New observations on bicarbonate-pH effects on thickness changes of rabbit corneas under silicone oil in vitro. Am. J. Optom. Physiol. Opt. 62, 879-888 (1985) 49 Doughty, M. J. pH, ionized calcium and osmotic artifacts can affect determination of rabbit cornea1 deturgescence in vitro. Ophthal. Res. 23, 246-258 (1991) 50 Doughty, M. J. Postmortem evaluation of rabbit aqueous humor partial pressure of carbon dioxide, total carbon dioxide, bicarbonate, partial pressure of oxygen and ionized calcium levels. Ophthal. Res. 25, 83-90 (1993) 51 Doughty, M. J. Morphometric analysis of the surface cells of rabbit cornea1 epithelium by scanning electron microscopy. Am. J. Anat. 189, 316-328 (1990) 52 Katzin, H. M. A study of the electrical resistance properties of cornea1 epithelium. Am. J. Ophthalmol. 34, 1159-1168 (1951) 53 Beekhuis, W. H., and McCarey, B. E. Cornea1 epithelial Cl-dependent pump quantified. Exp. Eye Res. 43, 707-711 (1986) 54 Rismondo, V., Osgood, T. B., Leering, P., Hattenhauer, M. G., Ubels, J. L. and Edelhauser, H. F. Electrolyte composition of lacrimal gland fluid and tears of normal and vitamin A-deficient rabbits. CLAO J. 15, 222-229 (1989) 55 Gilbard, J. P. and Rossi, S. R. An electrolyte-based solution that increases cornea1 glycogen and conjunctival goblet-cell density in a rabbit model for keratoconjunctivitis sicca. Ophthalmology 99, 600-604 (1992) 56 Dorm, A., Maurice, D. M. and Mills, N. L. Studies on the living cornea in vitro. I. Method and physiological measurements. Arch. Ophthalmol. 62, 741-747 (1959) 57 Begley, C. G., Waggoner, P. G., Jani, N. B. and Meetz, R. E. The effects of soft contact lens disinfection solutions on rabbit cornea1 epithelium. CLAO J. 20, 52-58 (1994)