A C T A O P H T H A L M O L O G I C A V O L . 5 4 1 9 7 6

The Department of Ophthalmology (Head: M . N . Luxenberg)

and the Department of Physiology (Head: R . C . Little)

Medical College of Georgia, Augusta, Georgia, USA

ELECTROPHYSIOLOGICAL AND ANATOMICAL EFFECTS

OF CETY LPY RI D I N I UM CHLORIDE ON THE RABBIT CORNEA

BY

KEITH GREEN

The effects of cetylpyridinium chloride on the trans-corneal potential difference and the surface anatomy of the cornea have been examined. Concentrations of cetylpyridinium chloride from 0.21 m M to 2 mM were used for either 1 or 2 minute exposure times on the in vitro and in vivo cornea for the electrophysiology studies. The potential difference of the in vitro cornea showed a concentration and exposure-time dependent de- crease, the in vivo cornea shows a qualitatively similar behaviour although quantitatively less. The fall in potential difference is preceeded by a hyper- polarization. The scanning electromicroscopy reveals a loss of microvilli and microplicae as well as surface pitting, with some exposure of cells underlying the superficial epithelium. These changes occur in a dose-de- pendent manner. The effect of cetylpyridinium chloride on the cornea is to enhance the permeability of the superficial cells by destroying the cell membranes and causing lysis of the cells.

Key words: cetylpyridinium chloride - cationic surfactant - rabbit - cornea - scanning electron microscopy - electropotential.

Received December 4, 1975.

145 Acta ophthal. 54, 2 10

Keith Green

In 1971, Green & TOnjum described the effects of various agents, including two cationic surfactants (benzalkonium chloride (BA) and cetylpyridinium chloride (CPC)), on corneal permeability to fluorescein. It was found that both agents caused a marked increase in permeability in conjunction with a lysis of the outermost cell membranes; indeed, the effects of CPC, at 2 mM, were most pro- nounced but only one concentration of CPC was employed.

Recently, some studies on drug penetration into the eye have examined the relationship between drug-protein interaction in the tear film and the effect of CPC on releasing adsorbed drug from teal film proteins following drug instillation onto the eye (Milrkelson, Chrai & Robinson 1973). These authors suggested that the 10-fold increase in biological activity of pilocarpine in the presence of CPC was the result of competitive inhibition of drug-protein inter- action causing release of adsorbed drug. The concentration of CPC used was 0.56 mM (0.020/0) compared to the 2 mM used by Green & Tsnjum (1971), and it was suggested (Mikkelson, Chrai & Robinson 1973) that the concentration difference, together with the difference in technique, was responsible for the alternative interpretation of CPC effects. Other studies (Smolen, Park & Williams 1975) have indicated that drug penetration depends on the fixed charges both within and upon the cornea, and that substances such as benzalko- nium chloride affect drug binding sites on the corneal surface.

In order to fully investigate the effect of CPC on the cornea, both electro- physiological and anatomical studies were performed on both in vivo and in vitro rabbit corneas.

Materials and Methods

Adult, healthy albino rabbits, 2-4 kg, of either sex were used.

Electrophysiology. The procedures for both in vitro and in vivo determinations of corneal potential followed those given previously (Green & Tsnjum 1975). Corneas were mounted in chambers which allowed a 15 mmHg hydrostatic pressure to be maintained on the posterior surface. The chamber was immersed in a beaker containing sufficient Krebs-bicarbonate Ringer, with glucose at 5 mg/ml (pH 7.4) to cover the exposed corneal surface. The potential difference (PD) was measured with a Heath EU-SOB recorder to an accuracy of k 0.1 mV.

Experiments were made with normal Ringer initially on both sides of the cornea. After 10 to 15 min, all tissues exhibited a stable PD. An identical Ringer solution containing either 0.21 (0.075 mg/ml), 0.56 (0.2 mg/ml) or 2 mM (0.7 16 mg/ml) cetylpyridinium chloride (CPC) was placed in another beaker

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Corneal Effects of Cetylpyridiniiim Chloride

and the chamber placed in this solution for either 1 or 2 min. The exposed surface of the cornea, and the chamber, was washed with a t least 200 ml of normal Ringer before reimmersion in a beaker containing Ringer alone. One series of experiments followed the ensuing steps: in Ringer until the PD was stable, into a solution of 2 0 / 0 pilocarpine hydrochloride and 100 mgo/o bovine serum albumin, again until a stable PD was reached (less than 3 min), then exposure for 1 min to a pilocarpine-albumin solution containing 0.56 mM CPC followed by a washing phase and return to normal Ringer. The trans-corneal PD was measured during all phases, except the washing phase. At least 4 corneas were used a t each concentration and exposure time.

In vivo experiments were also performed in a manner identical to that described previously (Green & Tsnjum 1975) except that CPC at 0.56 mM or 2 mM was utilized in the corneal cup. The exposure time to either concentration was either 1 or 2 min.

Anntomy. All three concentrations of CPC were used in the in vivo situation in the following regimens: 3 drops, with each drop every 30 seconds and 10 drops, each applied every 30 seconds. Control corneas received 10 drops of 0.90/0 saline solution, 1 drop every 30 seconds. Fifty microliter drops were applied to each cornea. The corneas were quickly removed from the eye and immersed in 30/0 glutaraldehyde. The fixed corneas were then prepared for scanning electron microscopy and examined at Mid-America Microanalysis Lab. Inc., where the microscope operator was not aware which specimen was under examination.

Results

Electroplzysiology. Cetylpyridinium chloride (CPC) at all concentrations and exposure times examined, both in vivo and in vitro, caused an initial hyper- polarization of the potential difference (PD) within 12 to 20 seconds after ex- posure. The PD then fell rapidly, in a concentration-dependent manner, to a minimum (Table I). Generally, longer exposure times caused greater falls in PD. The recovery rate of PD to normal pre-treatment levels was also exposure- time and concentration-dependent (Table I).

In vitro experiments. Normal values of PD for the different experimental series ranged from 2.03 k 0.40 to 4.75 f 0.54 (SE) mV. The lowest concentration of CPC (0.21 mM) caused a brief hyperpolarization of 12 to 20 seconds duration, followed by an exposure-time dependent reduction in PD (Fig. 1) . As the con- centration was increased, the maximal reduction in PD, as a percentage of the

147 10"

Keith Green

Concentration and time

Table I . Values of hyperpolarization, percentage reduction and recovery of corneal potential

difference after exposure of the epithelial surface of the in vitro rabbit cornea to cetylpyridinium chloride.

Hyper- polarization

010 of original PD

Maximal reduction

"0 of original PD

I

O i o recovery

010 of original PD

Time of after 2 h: minimum

PD (min)

14 0.21 mM 1 min

2 min

16 0 . 5 6 m ~ 1 min 2 min

2 rnM 1 min 15

0 . 5 6 m ~ 1 min 340

t 2 O i o pilocarpine + 100 mg o!u albumin

58 G8 30 75 48 60

90 25 75 93 13 45

100 0 120

59 45 30

- 0 10 20 30 40 50 60 70 80 90 100 110 120

TIME (MINUTES)

F i g . 1 . Effect of 0.21 mM cetylpyridinium chloride on the potential difference across the isolated rabbit cornea. PD, potential difference (mV). 0-0, 1 min exposure to CPC; 0-0,

2 min exposure to CPC, Values are the mean ? SEM.

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Corneal Ef lec ts o j Cetylpyridinizrm Chloride

1

0 10 20 30 40 50 60 70 80 90 Kx) 110 120 TIME (MINUTES)

F i g . 2. Effect of 0.56 mM cetylpyridinium chloride on the potential difference across the isolated

rabbit cornea. For explanation of symbols see legend to Fig. 1 .

- 2.0 >

€ 8 - b

1.0 - '

0 10 20 30 40 50 60 70 80 90 100 I10 120

TIME (MINUTES)

Fig. 3. Effect of 2 mM cetylpyridinium chloride on the potential difference across the isolated

rabbit cornea. The exposure time was 1 min. Values are the mean f SEM.

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Keith Green

Y

4.0

3.0

2.0

.

-

original PD, also increased (Fig. 2) until at 2 mM the PD was abolished at 120 min (Fig. 3). The results with 2 0 / 0 pilocarpine, 100 mgO/o albumin and 0.56 mM CPC in the epithelial bathing solution reveal some differences from the data obtained with Ringer alone in the bathing solution. Transfer of the cornea from normal Ringer to Ringer, pilocarpine and albumin resulted in a fall of PD of approximately 40°/0 (2.65 & 0.40 to 1.45 f 0.21 m v ; mean k SE

of 4 corneas), indicating that this solution alone produced a change in epithelial permeability. The hyperpolarization phase in the presence of CPC is both greater and of longer duration than with CPC alone (Fig. 4) but the maximum depres- sion in PD is similar to that seen with 0.56 mM CPC alone (Table I ) .

Zn v i v o experiments. A similar pattern to the in uitro experiments is also seen in the in vivo experiments. The reduction in PD is concentration-dependent since 2 mM causes a greater fall than 0.56 mM (Figs. 5 and 6). The exposure time appeared to have no effect on the magnitude of the PD fall, except at earlier times. With 2 mM CPC for both 1 and 2 min exposure there was still a hyperpolarization at 2 min, which was followed by a fall in PD by approximately

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Corneal Effects o j Cetylpyridiniztm Chloride

2 -

I -

7 r

3t

01 " " " " " " ' ' & I 2 3 4 5 6 7 8 91011 121314 15

TIME (MINUTES)

Fig. 5. Effect of 0.56 mM cetylpyridinium chloride on the potential difference across the in vivo

rabbit cornea. For explanation of symbols see legend to Fig. 1

7r

0 I 2 3 4 5 6 7 8 9 0 I I 1213 14 15

TIME (MINUTES)

F i g . 6'. Effect of 2 mM cetylpyridinium chloride on the potential difference across the in vivo

rabbit cornea. For explanation of symbols see legend to Fig. 1 .

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Keith Green

the same degree. With 0.56 mM CPC the initial hyperpolarization only lasted for 1 min, even during 2 min exposure, and the PD fell less than with 2 mM CPC (Figs. 5 and 6).

Anatomy. The normal anatomical structure of the epithelium is seen in Fig. 7a and b, where microvillae and microplicae are abundant on the surface of the epi- thelial cells. With the regimen of one drop x 3, at 0.21 mM there were few changes, except possibly more ‘dark’ cells, with these cells having fewer or flatter microplicae. At 0.56 mM, however, there is loss of microplicae from almost all cells (Fig. 8a), whereas at 2 mM there is also exposure of underlying cells (Fig. 8b). With the regimen of 1 dropx 10, some surface pitting is seen at 0.21 mM (Fig. ga), and at 0.56 mM all cells seem to be altered, with apparent holes in the cell membrane (Fig. 9b), at 2 mM, there is little further change in appearance except that most cells are smooth, having lost their microplicae (Fig. 10).

Discussion

In many respects the effects of cetylpyridinium chloride (CPC) on the electro- physiology and anatomy of the rabbit cornea parallel those of benzalkonium chloride (BA) which have been reported earlier (Green & Tsnjum 1971, 1975; Tsnjum 1975a,b). The electrophysiological responses, with an initial hyper- polarization followed by a fall in potential difference (PD) are very similar to those found with BA. The recovery of PD also appears to be concentration and exposure-time dependent (Table I ) . The initial hyperpolarization appears to be faster than that seen with BA, and this may be a reflection of the much smaller molecular size of CPC since the smaller compound would be able to penetrate the corneal epithelium more readily than the much larger BA molecule. Com- paring BA (Green & Tsnjum 1971, 1975; Tsnjum 1975a), to CPC indicates that CPC is far more active, at least at the concentrations tested.

As with BA, the in vivo effects of the surfactant are less than those seen with the in vitro preparation, but the changes are qualitatively similar. The reduction in PD is much less in vivo and recovery is complete within 15 to 30 min com- pared to the more profound in vitro effects. Presumably the more rapid recovery

Fig. 7 rr and b. Scanning electron micrograph of normal corneal epithelium. Note the presence of ‘dark’ cells. All cells have an abundance of microvilli and microplicae. a) x 5000, b) x 10 000.

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Corneal Effects of Cetylbyridinium Chloride

a

b

153

Keith Green

of the PD and the less marked reduction in PD in vivo compared to the in vitro cornea reflects a difference in the metabolic status of the in vivo and in vitro cornea, and the ability of the in vivo cornea to reestablish the barrier function of the epithelium.

The electrical responses of the tissue to CPC in pilocarpine-albumin solution is of interest since the hyperpolarization phase is not only substantially greater but is also of longer duration. One explanation for this effect is related to the placement of the hypertonic solution on the anterior surface of the corneal epithelium. Such a procedure in other tissues, such as frog skin (Ussing & Wind- hager 1964) and toad bladder (Urakabe, Handler & Orloff 1970; Wade, Revel & DiScala 1973; DiBona & Civan 1973) has been shown to increase the per- meability of these membranes to a variety of solutes. The decrease in PD when the cornea is moved from Ringer alone to the pilocarpine-albumin solution indicates that the permeability of the epithelium is also increased by the hyper- tonic solution on the outer surface.

Schoffeniels, Gilles & Dandrifosse (1962) and Webb (1965) observed an initial hyperpolarization of the isolated frog skin with surfactants. The hyperpolariza- tion was thought to be the result of a disruption of cellular integrity caused by interactions of the surfactant with membrane-located calcium. The membrane permeability is thereby increased giving rise to a transient potassium efflux which causes the hyperpolarization. The hyperpolarization seen with CPC is presumably related to this increased potassium efflux. The excessive hyper- polarization when pilocarpine and albumin are on the epithelial surface is prob- ably related to the fact that the intercellular channels of the epithelium are dilated with hypertonic solutions. Thus, CPC is able to penetrate into the deeper layers of the epithelium causing a greater hyperpolarization as well as a hyper- polarization of more cells. Saladino, Hawkins & Trump (1971) examined the effects of CPC on toad bladder but did not observe such a hyperpolarization. They did, however, note an increased oxygen uptake at a time following sur- factant treatment equivalent to the electrical changes seen here. The toad bladder recovered from low doses (10.5) of CPC, whereas high doses (10-3 M) caused

F i g . 8 a and b. Scanning electron micrograph of corneal epithelium after CPC treatment. a) 1 drop of 0.56 mM CPC 3 times at 30 second intervals. Note loss of microplicae from cells. An apparently normal cell remains. x 5000. b) 1 drop of 2 mM CPC, 3 times at 30 second intervals. There is loss of superficial cells over much of the epithelium causing exposure of underlying cells. x 1000.

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Corneal Effects o f Cetylkyridinium Chloride

a

b

155

Keith Green

apparently irreversible damage (Saladino, Hawkins & Trump 197 1 ) . Similarly, with the cornea, the PD recovered from 0.21 mM (2.1 x 10-4 M) but not from a 1 min exposure to 2 mM ( 2 x 1 0 - 3 M).

The data of Mikkelson, Chrai & Robinson (1973) can, therefore, be explained on the basis of the enhancement of permeability caused by CPC since all con- centrations tested here (which span the concentration used by Mikkelson, Chrai 8c Robinson by at least three-fold in each direction) caused an increase in passive epithelial permeability even in uivo. The anatomical studies, particularly, were performed with drops of CPC applied to the in vivo corneal surface in the normal manner, whereas the electrophysiological studies, by necessity, required techniques which departed from the normal physiological condition. Both the electrophysiological and anatomical data indicates that the PD is reduced by a brealcdown of the epithelial barrier located in the superficial epithelial cells (Green 1969), which creates a shunt pathway for the movement of solutes and solvent across the membrane. The recovery of the PD is related to the recovery of the barrier function, presumably at a deeper location, in the corneal epi- thelium. Previously, we (Green 8c Tsnjum 1971) described a single dose-effect of CPC ( 2 mM) on fluorescein permeability, and found a 28-fold increase in permeability to the dye. The present data substantiate this finding and expand the dose range.

While it is obviously true that surfactants, such as CPC and BAC (Green 8c Tenjum 1975), affect the electrical characteristics of the cornea, and may affect the fixed surface and tissue charges on the tissue, it is equally obvious that the major effect of such agents is to enhance the penetration of drugs, particularly ionic forms, across the relatively impermeable epithelium. This effect is even more pronounced when one considers the clinical situation under which a drug is applied topically to the eye, namely one drop which is subjected to immediate dilution and loss via the lacrymal system.

The effects of CPC have been examined on the electrophysiology and anatomy of the rabbit cornea. The primary effect of CPC, at the concentrations tested here, is to eliminate the physiological and anatomical barrier to fluid movement

Fig. 9 a and b. Scanning electron micrograph of corneal epithelium after CPC treatment. a) 1 drop of 0.21 mM CPC, 10 times at 30 second intervals. Surface pitting is evident on several cells. x 1000. b) 1 drop of 0.56 m CPC, 10 times at 30 second intervals. Apparent holes in cell surface are seen, membrane appears to be 'lattice-like'. x 10 000.

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Corneal Effects of Cetylpyridinium Chloride

a

b

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Keith Green

Fig. 10. Scanning electron micrograph of corneal epithelium after 2 mM CPC treatment. Cells are smooth with loss of most microvilli and exposure of underlying cells. There is

expansion of intercellular spaces. x 2000.

in the corneal epithelium by destroying the cell membranes and causing lysis of the cells. The sanction of the use of CPC in ophthalmic preparations as a protein-drug unbinding agent would, on the basis of the present data , appear to be premature. Only long-term testing to determine the toxicity of CPC can assure the safety of this surfactant in ophthalmic preparations.

Acknowledgments

This study was supported in part by U. S. Public Health Service Research Grant, EY 01413, from the National Eye Institute. The electron microscopy was performed by Mid-America Microanalysis Laboratory, Inc., Milwaukee, Wisconsin.

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Corneal Effects of Cetylpyridinium Chloride

References

DiBona D. R. & Civan M. M. (1973) Pathways for movement of ions and water across toad urinary bladder. I. Anatomic site of transepithelial shunt pathways. J. Membr. Biol. 12, 101-128.

Green K. (1969) Anatomic study of water movement through rabbit corneal epithelium. Amer. J. Ophthal. 67, 110-1 16.

Green K. & Tenjum A. M. (1971) Influence of various agents on corneal permeability. Amer. J. Ophthal. 72, 897-905.

Green K. & Tenjum A. M. (1975) The effect of benzalkonium chloride on the electro- potential of the rabbit cornea. Acta Ophthal. (Kbh.) 53, 348-357.

Mikkelson T. J., Chrai S. S. & Robinson J. R. (1973) Competitive inhibition of drug- protein interaction in eye fluids and tissues. /. Pharm. Sci. 62, 1942-1945.

Saladino A. J., Hawkins H. K. & Trump B. F. (1971) Ion movement in cell injury. Amer. J. Path. 64, 271-294.

Schoffeniels E., Gilles R. & Dandrifosse G. (1962) Action des detergents et du calcium sur le potential electrique de la peau de grenouille isolte. Arch. int. Physiol. Pharm. 20, 335-344.

Smolen V. F., Park C. S. & Williams E. J. (1975) Drug-biomolecule interactions: Bio- electrometric study of the mechanism of carbachol interactions with cornea and its relation to miotic activity. /. Pharm. Sci. 64, 520-528.

Tenjum A. M. (1974) Permeability of horseradish peroxidase in the rabbit corneal epi- thelium. Acta ophthal. (Kbh.) 52, 650-658.

Tenjum A. M. (1975a) Permeability of rabbit corneal epithelium to horseradish peroxi- dase after the influence of benzalkonium chloride. Acta ophthal. (Kbh.) 53, 335-347.

Tenjum A. M. (1975b) Effects of benzalkonium chloride upon the corneal epithelium studied with scanning electron microscopy. Acta ophthal. (Kbh.) 53, 358-366.

Urakabe S., Handler J. S. & Orloff J. (1970) Effect of hypertonicity on permeability properties of toad bladder. Amer. J. Physiol. 210, 1179-1187.

Ussing H. H. & Windhager E. E. (1964) Nature of shunt path and active sodium trans- port path through frog skin epithelium. Acta physiol. scand. 61, 484-504.

Wade J. B., Revel J. P. & DiScala V. A. (1973) Effect of osmotic gradients on inter- cellular junctions of the toad bladder. Amer. J. Physiol. 224, 407-415.

Webb G. D. (1965) The effects of surfactants on the potential, shortcircuit current, and ion fluxes across the isolated frog skin. Acta physiol. scand. 63, 337-384.

Author’s address: K. Green, Ph.D., Department of Ophthalmology, 3 D 1 1 , R & E Building, Medical College of Georgia, Augusta, GA 30902.

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