Toxicology in Vitro 18 (2004) 853–857

www.elsevier.com/locate/toxinvit

Corneal permeability in a redesigned corneal holder for thebovine cornea opacity and permeability assay

J.L. Ubels a,b,*, J.A. Ditlev a, D.P. Clousing a, P.L. Casterton c

a Department of Biology, Calvin College, 3201 Burton St., SE, Grand Rapids, MI 49546, USAb Department of Ophthalmology, Wayne State University School of Medicine, Detroit, MI 48201, USA

c 3520 Vinewood SE, Grand Rapids, MI 49546, USA

Received 1 March 2004; accepted 9 April 2004

Abstract

The bovine cornea opacity and permeability assay (BCOP) is a proposed alternative to the Draize rabbit test for potential eye

irritants. In the standard BCOP, bovine corneas are mounted in a holder on a flat surface between two identical chambers. The flat

configuration of the standard holder does not conform to the normal curved shape of the bovine cornea and it comes into direct

contact with the cornea tissue. Mounting corneas in this holder causes extensive damage to both epithelial and endothelial corneal

cell layers. Our laboratory has designed a new holder that allows the cornea to maintain its natural curvature and does not damage

the cornea. Previous tests, using both the new and standard holders, and comparing corneal opacity, hydration and endothelial

morphology, have shown that the new holder is a significant improvement over the standard holder. The present study extends the

comparisons of the new and standard holders to measurement of corneal fluorescein permeability. The permeability (ng/cm2/min) of

intact corneas, corneas with no epithelium, and corneas treated with 1% NaOH, isopropanol, acetone, 30% trichloroacetic acid or

30% sodium dodecysulfate for either 1 or 10 min was determined by measuring fluorescence of samples taken from the endothelial

chamber after 90 min epithelial exposure to 0.04% sodium fluorescein. In all trials, the redesigned holders yielded not only lower

permeability measurements but also decreased measurement variability. The data provide further evidence that the new holder is an

improvement over the standard holder and should be incorporated into a new protocol for the BCOP.

� 2004 Elsevier Ltd. All rights reserved.

Keywords: Cornea; Ocular toxicology; Bovine cornea opacity and permeability assay

1. Introduction

The bovine cornea opacity and permeability assay

(BCOP), proposed by Gautheron et al. (1992), is an in

vitro method for testing the effects of potential irritants

on the cornea of a bovine eye. Earlier studies showed

that results obtained from the BCOP assay correlatewell with data from the Draize test (Gautheron et al.,

1994; Sina, 1994), but, as with all in vitro eye irritation

tests, the BCOP assay has not yet achieved regulatory

acceptance as an alternative to animal testing. Valida-

tion of this assay as predictive of human response to

irritants requires that the test be performed under

*Corresponding author. Tel.: +1-616-526-6219; fax: +1-616-526-

6501.

E-mail address: jubels@calvin.edu (J.L. Ubels).

0887-2333/$ - see front matter � 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.tiv.2004.04.005

optimal conditions, including the use of tissue that is in

excellent condition.

Since the development of the BCOP assay, our lab-

oratories have proposed several assay modifications.

Casterton et al. (1996) proposed that a spectropho-

tometer can be used for opacity measurement. By

determining corneal light absorbance at 570 nm, moreprecise measurements of opacity can be obtained. We

have also proposed that measurement of corneal

hydration and evaluation of the corneal endothelium be

added to the protocol. Ubels et al. (2000, 2002) showed

that the standard holders in which the bovine corneas

are mounted cause excessive damage to the delicate

corneal endothelial layer because the holder flattens and

wrinkles the cornea. The standard holders also causecorneal edge damage because the holder clamps directly

onto the cornea. To address these problems we devel-

oped a new, redesigned holder that does not contact the

854 J.L. Ubels et al. / Toxicology in Vitro 18 (2004) 853–857

cornea and allows it to maintain its normal shape. We

have previously tested this holder against the standard

holder, measuring both opacity and hydration as well as

endothelial morphology (Ubels et al., 2002). The resultsof these studies indicated that the redesigned holder did

not damage the cornea during the mounting process and

that corneal opacity and hydration were similar in the

two types of holder after any given treatment.

The present study is a continuation of our evaluation

of the new holder. We have now measured the perme-

ability of the cornea to Na-fluorescein under control

conditions and following exposure to irritants in boththe standard and redesigned holders. Because the cornea

is not damaged in the new holder, it was expected that

for any condition, permeability would be lower when

using the new holder compared to the standard holder.

2. Materials and methods

2.1. Corneas

Corneas were excised from slaughterhouse-derived

bovine eyes within 2 h of enucleation. Those that were

obviously damaged or that stained with fluorescein werediscarded. Corneas with absorbance at 570 nm of

greater than 0.1 after mounting in the holders and 15

min of pre-incubation were not used in experiments.

2.2. Corneal holders––volume and permeability measure-

ments

Both the standard and redesigned corneal holders

used in this study have been previously described (Ubels

et al., 2002, 2003). The new holder is illustrated in Fig. 1.

In the standard holder, the cornea is clamped between

two chambers of approximately equal volume and hasan exposed surface area of 2.26 cm2. In the new holder

the volume of the posterior (or endothelial) chamber is

Fig. 1. The new corneal holder for the BCOP assay. The cornea with a

5 mm scleral rim is mounted with the endothelial side of the sclera

contacting the O-ring of the half-chamber on the left. The corneal

epithelium faces the half-chamber on the right. The chamber does not

make contact with the cornea.

larger than that of the anterior (or epithelial) chamber

and the exposed area of the cornea is 10.04 cm2.

In the standard BCOP protocol, corneal permeability

is determined by measuring optical density at 490 nm ofthe solution in the endothelial chamber after a 90 min

incubation with fluorescein in the epithelial chamber

(Gautheron et al., 1992; Casterton et al., 1996). This OD

reading is used directly in calculating the in vitro score.

Because of the differences in holder dimensions, this

method could not be used in comparing the standard

and redesigned chambers. Alternatively, we calculated

an actual permeability rate in ng fluorescein/cm2 ofcornea/min during the 90 min incubation period. To do

this it was necessary to know the volumes of the

chambers. This was calculated by mounting a cornea in

the chamber, introducing a known amount of methylene

blue into each side and calculating the chamber volumes

according to the dye dilution principle. To measure the

total amount of fluorescein crossing the cornea, fluo-

rescence of samples taken from the endothelial chamberwas measured in a Packard FluoroCount Microplate

Fluorometer (Packard Instruments, Meridian, CT) and

the fluorescein concentration was determined from a

standard curve.

2.3. Experimental protocol

Intact corneas or corneas with epithelium removed by

scraping with a Gill knife were mounted in the standard

and redesigned holders. Endothelial chambers were fil-

led with minimum essential medium without phenol red

(MEM, Sigma). Na-fluorescein (Sigma, St. Louis, MO)

dissolved in MEM at 0.04% was placed in the epithelialchamber and the corneas were incubated for 90 min at

35 �C. The contents of the endothelial chamber were

mixed well, fluorescence of a sample from the endothe-

lial chamber was measured and fluorescein permeability

was calculated.

To compare effects of irritants on cornea permeability

between the two chambers, corneas were mounted and

pre-incubated for 15 min in MEM. The corneal epithe-lium was then exposed to a test substance for 1 or 10

min. Following exposure, the test material was removed,

the epithelial surface was rinsed with balanced salt

solution to remove the test substance and the corneas

were incubated with the fluorescein solution for 90 min,

followed by measurement of fluorescein levels in the

endothelial chamber. Effects of acetone, isopropyl

alcohol (IPA), 1% NaOH, 30% trichloroacetic acid(TCA) and 30% sodium dodecylsulfate (SDS) on cor-

neal permeability were compared between the two cor-

neal holders. These compounds were chosen to represent

categories of organic solvents, bases, acids and surfac-

tants. Each experiment was conducted using five corneas

and the t-test ðp6 0:05Þ was used to make relevant

comparisons. Data in the figures were grouped not

45

Std. Holder*

J.L. Ubels et al. / Toxicology in Vitro 18 (2004) 853–857 855

according to related categories but by range of values

for clarity of illustration.

0

5

10

15

20

25

30

35

40

1 min.1%NaOH

10 min.1% NaOH

1 min.Acetone

10 min.Acetone

Intact

Fluo

resc

ein

Upt

ake

(ng/

cm2 /m

in)

New Holder

*

*

*

Fig. 3. Permeability of corneas exposed to 1% NaOH or acetone for

1 or 10 min. * Significantly different than permeability in new holder

(t-test, p6 0:05, n ¼ 5). Permeability of corneas treated for 10 min is

significantly different than permeability of corneas treated for 1 min in

each type of holder.

12

14

16

/min

)

Std. HolderNew Holder

*

3. Results

Under all conditions tested in this study, corneal

fluorescein permeability measurements were higher in

the standard cornea holder than in the redesignedholder. There was also a measurable flux across the in-

tact cornea when fluorescein was detected using a fluo-

rimeter. In the new holder permeability of the intact

cornea was 0.13 ng/cm2/min, but this increased about

threefold, to 0.37 ng/cm2/min in the standard holder.

Removal of the cornea epithelial barrier by scraping

resulted in a permeability of 31.7 ng/cm2/min in the new

holder, but this increased to 74.2 ng/cm2/min in thestandard holder (Fig. 2).

Acetone, isopropanol (IPA) and 1% NaOH all caused

increased corneal permeability to fluorescein with

markedly greater permeability measurements in either

holder following the 10-min exposures (Figs. 3 and 4).

Permeability after these treatments never reached the

level of permeability of a cornea without epithelium.

After 1 or 10 min treatments with NaOH or IPA, cor-neal permeability was significantly higher in the stan-

dard holder than in the new holder. Permeability was

higher in the standard holder after a 1-min treatment

with acetone. After a 10-min exposure to acetone, cor-

neal permeability was also higher in the standard holder

but the difference was not statistically significant. It is

0

20

40

60

80

100

Intact no Epi 1 min.30%SDS

10 min.30% SDS

Fluo

resc

ein

upta

ke (n

g/cm

2 /min

)

Std. Holder

New Holder

*

*

*

Fig. 2. Permeability of intact corneas, corneas with the epithelium

removed by scraping and corneas exposed to 30% SDS for 1 or 10 min.

* Permeability in the standard holder is significantly different than in

the new holder (t-test, p6 0:05, n ¼ 5).

0

2

4

6

8

10

1 min.IPA

10 min.IPA

1 min.30% TCA

10 min.30%TCA

Intact

Fluo

resc

ein

upta

ke (n

g/cm

2

*

**

Fig. 4. Permeability of corneas exposed to isopropanol (IPA) or 30%

trichloroacetic acid (TCA) for 1 or 10 min. *Significantly different than

permeability in new holder (t-test, p6 0:05, n ¼ 5). Permeability of

corneas treated with IPA for 10 min is significantly different than

permeability of corneas treated for 1 min in each type of holder.

noted, however, that the variability of the response in

the standard holder (coefficient of variation¼ 0.42) was

much greater than in the new holder (coefficient of

variation¼ 0.066).

856 J.L. Ubels et al. / Toxicology in Vitro 18 (2004) 853–857

Exposure of the cornea to 30% TCA for either 1 or 10

min had no effect on permeability in the new holder. In

the standard holder treatment with TCA for 1 min

caused an approximately threefold increase in perme-ability compared to an intact cornea (0.92 vs 0.37 ng/

cm2/min), but a 10 min exposure had no effect on per-

meability compared to the intact cornea in the standard

holder. Corneal permeability to fluorescein after TCA

treatment was always very low, but was higher in the

standard holder than in the new holder after a 1 min

exposure (Fig. 4).

The permeability of corneas exposed to 30% SDS for1 min in the standard holder increased markedly to 96.5

ng/cm2/min, a level equal to permeability of corneas

without epithelium in the standard holder (Fig. 2). In

contrast, a 1 min exposure to SDS in the new holder

increased permeability to only 2 ng/cm2/min. A 10 min

exposure to 30% SDS increased the corneal permeability

to the range of 20–30 ng/cm2/min with no significant

difference between the two holders. The coefficient ofvariation was however higher in the standard holder

(0.28) than in the new holder (0.19).

4. Discussion

This study demonstrates that corneal permeability is

lower in the re-designed corneal holder for the BCOP

assay than in the standard holder under control condi-

tions or following treatment that breaks down the epi-

thelium. It is also shown that in both holders, when

corneas are exposed to compounds that compromise the

epithelial barrier a reduced treatment time of 1 mincauses less damage to the cornea than a 10 min treat-

ment. The exception to this observation is the effect of

SDS, as discussed below.

We previously suggested that the standard corneal

holder, which clamps onto clear cornea, causes edge

damage to the epithelium and endothelium (Ubels et al.,

1998, 2000, 2002), and we have confirmed this by his-

tology (data not shown). This edge damage would beexpected to increase corneal fluorescein permeability

and the data from the present study confirms this. The

large differences in permeability between the standard

and new holders when the epithelium is removed,

damaged by scraping or exposed to chemicals suggests

however that the advantage of the new holder is not due

to elimination of edge damage alone. In our previous

studies we showed that the standard holder causesextensive damage to the corneal endothelium due to

wrinkling of the cornea (Ubels et al., 2000, 2002). It is

well known that damage to the endothelium increases

corneal permeability (Araie, 1986a,b; Watsky et al.,

1989). We therefore conclude that the lower perme-

ability of corneas with a damaged epithelium in the new

holders is largely due to elimination of the mechanical

damage to endothelium that is caused by the standard

holder. It should be noted that corneal edge damage

may also allow greater penetration of test substances

into the cornea, thereby accounting for some of thedifference between the standard and new holders. The

exception to these observations is the effect of TCA. As

previously reported (Casterton et al., 1996) and con-

firmed in the present study, the permeability of corneas

treated with TCA is very low because TCA chemically

fixes the corneal epithelium rather than breaking down

the epithelial barrier (Ubels et al., 1998).

SDS, a surfactant, has a detergent effect on cellmembranes and extracellular matrix. A 1 min exposure

to SDS in the standard holder completely removed the

corneal epithelium, which explains the high permeability

of these corneas. In the new holder it was observed that

a 1 min exposure to SDS did not completely remove the

corneal epithelium, accounting for the lower perme-

ability in the new holder. Loss of the epithelium in the

standard holder may be accelerated by edge damagewhich allows more rapid penetration of SDS to the basal

cell layer of the epithelium. The relatively low perme-

ability of the corneas in the standard holder after a 10

min exposure to SDS was unexpected; however, it was

observed that after 10 min SDS not only removed the

epithelium, but also damaged the underlying stroma.

This may have had the effect of reducing permeability

compared to corneas with epithelial damage alone.The lower permeability of corneas treated with ace-

tone, 1% NaOH or IPA for 1 min compared to 10 min in

either holder is in agreement with our previously re-

ported data on opacity and hydration after reduced

treatment times (Ubels et al., 2000). We have suggested

that the 10 min treatment with test substances in the

BCOP protocol is an exaggerated exposure compared to

that which would occur accidentally in humans. On theother hand the bovine corneal epithelium is much

thicker than the human epithelium (10–12 cell layers

compared to 5–7 cell layers) with the result that a re-

duced treatment time might lead to underestimates of

damage when bovine eyes are used to screen potential

irritants. This problem requires further investigation.

In our report introducing the redesigned holder we

were unable to demonstrate a difference in cornealopacity or hydration between the standard and new

holders (Ubels et al., 2002). It was clear, however, that

the new holder eliminates edge damage and damage to

the endothelium. Therefore we argued for use of the new

holder based on morphology, since it is essential to be-

gin an in vitro toxicology assay with tissue that is in

optimal condition. This is a principle that has been

followed for many years in corneal physiology andpharmacology (Dikstein and Maurice, 1972; Edelhauser

and Maren, 1988; Klyce and Crosson, 1985; Riley,

1985). We have now demonstrated that using the rede-

signed corneal holder does in fact provide lower

J.L. Ubels et al. / Toxicology in Vitro 18 (2004) 853–857 857

permeability measurements. The permeability values

reflect effects of treatment alone that are not affected by

mechanical damage to the cornea. We believe that these

permeability data are more accurate and less variablethan those obtained when using the standard holder.

The use of the new holder will reduce the risk of false

positives due to erroneously high corneal permeability

when using the BCOP assay to screen substances for

potential ocular irritancy.

Acknowledgements

This study was supported by a grant from the Access

Business Group, Alticor Corp., Ada MI, USA.

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