Toxicity of topical anesthetic agents to human keratocytes in vivo1

Toxicity of topical anesthetic agents to human keratocytes in vivo Luciane B. Moreira, MD, Ngamjit Kasetsuwan, MD, Daniel Sanchez, MD, Sujil S. Shah, MD, Laurie LaBree, MS, Peter J. McDonnell, MD ABSTRACT Purpose: To test the potential toxicity on human keratocytes of topical anesthetic agents used after photorefractive keratectomy (PRK) to reduce or eliminate pain. Setting: Department of Ophthalmology, Doheny Eye Institute, University of Southern California, Los Angeles, California, USA. Methods: Cultured human keratocytes were incubated with commercially available tetracaine and proparacaine at reduced concentrations of 0.001%, 0.01%, 0.1%, and 0.25%. Evaluations were performed by phase-contrast microscopy and tetrazolium salt colorimetric assay every 2 hours for 12 hours after adding 1 of the anesthetic agents to the media. Results: After time of incubation and concentration were adjusted, both drugs reduced overall cell viability; however, tetracaine produced a larger decrease in cell viability than proparacaine (P ⫽ .008). For both drugs, significant differences were found among concentrations for and across time (P ⬍ .001 and P ⫽ .004, respectively). Conclusion: Both tetracaine and proparacaine had toxic effects on stromal keratocytes related not only to drug concentrations but also to time exposure. These findings underscore the widespread concern that anesthetic drugs may affect corneal stromal wound healing after PRK. J Cataract Refract Surg 1999; 25:975–980 © 1999 ASCRS and ESCRS

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xcimer laser photorefractive keratectomy (PRK) has been used to correct myopia, hyperopia, and astigmatism. Moderate to severe postoperative pain is a significant complication of PRK, especially during the first 24 hours after surgery.1–3 The most common means of pain management has been a therapeutic bandage contact lens in combination with a topical nonsteroidal anti-inflammatory drug and an oral analgesic. Many patients are uncomfortable with the lens and have fitting problems. Because pain after PRK remains a problem, new and more effective methods must be developed.

Correspondence to Peter J. McDonnell, MD, University of California, Irvine, Department of Ophthalmology, Gottschalk Medical Plaza, 2000 Medical Plaza Drive #2004, Irvine, California 92697-4380, USA. © 1999 ASCRS and ESCRS Published by Elsevier Science Inc.

Topical anesthetic agents have a role in ophthalmic examination and diagnosis. The 2 most common are proparacaine and tetracaine. Tetracaine is a para-aminobenzoic acid ester anesthetic agent. The unpreserved form is commercially available and may be less toxic than the preserved form. Proparacaine hydrochloride is a benzoic acid ester-derived topical anesthetic agent.4 These agents are for short-term use only because chronic use is associated with toxic effects on ocular tissues.5 When the use of a topical anesthetic agent after surgery is considered, the possible toxicity of that agent must be considered. Some data suggest these agents produce a modest delay in epithelial healing.2 The effects of these anesthetic agents on the stromal keratocytes (the key cells involved in the stromal reparative response), 0886-3350/99/$–see front matter PII S0886-3350(99)00075-9

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however, remain unexplored. We report a comparative in vitro analysis of keratocyte cytotoxicity using currently available topical anesthetic solutions for the eye.

Materials and Methods Keratocyte Culture The central 8.0 mm of a fresh human cornea was obtained from Lions Doheny Eye Bank, Los Angeles, California. The epithelial and endothelial layers were removed by scraping with a #15 scalpel. The stroma was minced, cultured in Eagle’s modified minimal essential medium (MEM, Irvine Scientific) supplemented with fetal bovine serum 20%; 100 IU/mL penicillin G, and 100 IU/mL streptomycin (all from Irvine Scientific) in 35.0 mm petri dishes (Becton Dickinson). The samples were incubated at 37°C in carbon dioxide 5%. When cells reached confluence, they were trypsinized with trypsin 0.05% (Irvine Scientific) for 3 minutes, centrifuged, and resuspended. The cells were then added to MEM 20% and transferred to 75 mL culture flasks (Corning Glass Works). After 3 passages, keratocytes were transferred to 96-well plates (Becton Dickinson). Ten cells in 100 ␮L of MEM 20% were plated in 96well plates and incubated for 48 hours before the drugs were added. Drug Preparation and Addition Commercially available eyedrops of preservativefree tetracaine hydrochloride 0.5% (AK-T-CAINE™ PF) and proparacaine hydrochloride 0.5% (Ophthetic威) were tested. Each anesthetic agent was dissolved separately in MEM 20% at each of 4 different concentrations (0.001%, 0.01%, 0.1%, and 0.25%). The control group received only MEM 20%. The pH of the drugs was evaluated with pHydrion Paper dual range Jumbo (Micro Essential Laboratory). After the MEM 20% was removed, 1 of the anesthetic solutions or MEM 20% alone (control) was added to each well. Each drug concentration, as well as the control, was tested 8 times. Drug Sensitivity Test Cytotoxicity was assessed 15, 30, and 60 minutes after the addition of the drugs and then every 2 hours for 12 hours. 976

A tetrazolium salt, XTT [sodium (2,3-bis[2-methoxy-4-nitro-5-sulphophenyl]-2H-tetrazolium-5carboxanilide, inner salt] (Boehringer Mannheim), was used. This colorimetric assay is based on the bioreduction of a tetrazolium salt to an intensely colored formazan and is directly related to the activity of cellular mitochondrial enzymes.6 For each test, the solution in the wells was removed and 100 ␮L of MEM 20% with 50 ␮L of freshly prepared XTT were added. After 2 hours of incubation with XTT at 37°C in carbon dioxide 5%, the spectrophotometric absorbance of the samples was measured with an enzyme-linked immunoabsorbant assay reader. Morphological Evaluation Keratocyte cultures were monitored every 2 hours with a phase-contrast microscope equipped with a photographic control system. The morphologic evaluation was performed by a pair of experienced observers (L.M.B., D.S.) at each time point. The observers were masked as to treatment group. The observers reached a consensus grading of the cellular morphology at each time point. Cytotoxic drug effects on cell morphology were assessed semiquantitatively according to the following criteria: 0 ⫽ none (spindle-shaped cells; multiple long, delicate processes; no cytoplasmic granularity; multiple intercellular contacts); ⫹ ⫽ mild (spindle-shaped cells; short, thickened processes in fewer than one third of the cells; cytoplasmic granules in fewer than one third of the cells; reduced intercellular contacts); ⫹⫹ ⫽ moderate (triangular or polygonal cells; short, thickened processes in more than one third of the cells; cytoplasmic granules in more than one third of the cells; few intercellular contacts; moderate cell detachment); ⫹⫹⫹ ⫽ severe (round cells; no processes; no cell contact; severe cell detachment). Statistical Analysis An analysis of covariance (ANCOVA), adjusted for concentration level and time, was used to compare proparacaine and tetracaine. An analysis of variance (ANOVA) was used to test for differences between concentrations and for trends across time. Multiple comparison t tests were used for pairwise comparisons, adjusting the alpha level for the number of comparisons. The vari-

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able of interest was the number of cells expressed as a percentage of the control means.

Results Proparacaine The XTT assay demonstrated significant differences among concentrations of proparacaine (P ⬍ .001, ANOVA). The greatest toxicity occurred at concentrations of 0.1% and 0.25%, with less toxicity at lower concentrations of 0.001% and 0.01% (P ⬍ .001, multiple comparison t test). The ANOVA for differences across time was significant (P ⫽ ⬍ .001). The cytotoxicity was significantly greater at 1 and 10 hours than at 15 minutes and 4 hours (P ⬍ .01, multiple comparison t test). Tetracaine Significant differences were found among the various concentrations (P ⬍ .001, ANOVA). Concentrations of 0.1% and 0.25% were not significantly different (P ⬎ .05) but had greater toxicity than 0.001% and 0.01% (P ⬍ .001). In addition, the toxicity at 0.01% was significantly greater than that at 0.001% (P ⬍ .001). There were significant differences across time (P ⬍ .001, ANOVA).

XTT and Group Differences After adjusting for time and concentration, tetracaine had a greater overall toxic effect than proparacaine (P ⫽ .001, ANCOVA) (Figure 1). Phase-Contrast Microscopy Fifteen minutes after addition of either anesthetic agent at the described concentrations of 0.001% and 0.01%, keratocytes were mildly altered in morphology (Figure 2). At 0.1% and 0.25%, these cells showed moderate to severe signs of cytotoxicity. Two hours after the addition of proparacaine or tetracaine 0.001%, phase-contrast microscopy revealed mild morphologic changes. Cells exposed to tetracaine at a concentration of 0.01% showed moderate changes in the cytoskeleton. At higher concentrations of 0.1% and 0.25% of proparacaine and tetracaine, severe signs of cytotoxicity were found. These results did not change over time.

Discussion It is essential to minimize pain after any surgical procedure. Drugs used for this purpose should be effective but should also have minimal side effects.7 In patients who have had PRK, the epithelial barrier

Figure 1. (Moreira) Graph comparing XTT sensibility and time.

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Figure 2. (Moreira) Phase-contrast microscopy showing mild morphologic change 15 minutes after addition of tetracaine 0.01% to the keratocyte culture (left) and no morphologic changes in the control group (right).

properties are lost, leaving the stroma exposed.7 Although sensory nerve endings may be completely ablated, confocal microscopy has shown that the nerve plexus in the anterior stroma may remain intact, accounting for the severe pain of some patients.7 Techniques used for pain control after PRK vary greatly, and no study has reported complete pain control in all patients to date. Some studies describe the successful use of an anesthetic agent for the first 24 hours after PRK without significant effect on the cornea.7 Because of the known toxic effect of anesthetic agents that can occur with chronic use, concern continues about their routine use in this setting. However, many studies support the idea that the toxic effect occurs only when these agents are used in an uncontrolled and unsupervised manner for more than 3 days.2,5 A single-dose application of tetracaine or proparacaine in the rabbit eye has no toxic effect.8 When abuse does occur, there are no pathognomonic signs; in the early stages, any effects are always mild and reversible within 1 to 3 hours.9,10 If abuse continues, stromal keratitis, granularity, infiltration, stromal edema, and Descemet’s membrane folds can be observed after 1 week of chronic use.8,10,11 The removal of epithelium during PRK is of concern since it might increase the stromal concentration and the toxicity of topically applied drugs. In this study, we found a rapid onset of keratocyte cytotoxicity within 15 minutes at anesthetic concentrations of 0.1% and 0.25%. Previous studies have documented toxicity of corneal epithelial cells after 30 to 60 minutes of treat978

ment with local anesthetic agents. Minimal cell damage occurred after 15 minutes.12 We observed moderate to severe alteration of keratocyte morphology with phase-contrast microscopy 15 minutes after the administration of the anesthetic agents at 0.25%, suggesting that keratocytes are more sensitive to these agents. The exact mechanism of this toxicity is unclear; it may be a direct toxic effect of the anesthetic agent on the cornea. Tetracaine, for example, directly damages the cell membrane, eventually causing cell death.9 The cell shape changes that occur indicate tetracaine affects the cytoskeleton before plasma membrane integrity, causing loss of cell viability.11 Both tetracaine and proparacaine work by blocking sodium conduction across nerve cell membranes.5 Anesthetic agents elevate calcium ions before cytotoxicity occurs, and disruptions in calcium homeostasis may contribute to their toxicity.12 Their toxicity may result from a direct effect of the anesthetic agent on the corneal cells and impairment of the trophic action of nerve fibers.13 Chronic use of topical anesthetic drugs has been shown to cause permanent corneal scarring and decreased vision.13 Our experiments confirm that topical anesthetic agents affect the cytoskeleton of keratocytes within 15 minutes. Similar to our findings with keratocytes in vitro, other findings suggest that tetracaine is more damaging than proparacaine to the corneal epithelium.12 Tetracaine also causes a rise in calcium ions at much lower concentrations than proparacaine; this dual effect on mitochondrial integrity and calcium homeostasis may

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Table 1. Calculated concentrations of anesthetic agents used in vitro; based on Grant and Acosta.12 Variable

because we evaluated these agents only in relation to their effects on keratocytes.

Percentage

Concentration applied to the eye

0.5

Initial dilution in the eye (1/3)

0.2

First T1/2 (7 to 13 minutes)

0.08

Second T1/2 (14 to 26 minutes)

0.04

In vitro concentrations used in this study

0.25; 0.1; 0.01; 0.001

explain the increased toxicity of tetracaine over proparacaine.12 Bartfield and coauthors4 showed that instillation of proparacaine eyedrops is less painful than instillation of tetracaine and the resulting anesthesia lasts longer. These properties may make proparacaine preferable to tetracaine. Topically applied ophthalmic medication and the accompanying preservatives may damage ocular tissues.8 Gasset and coauthors,14 studying preservative toxicity, demonstrated that benzalkonium chloride had the most cytotoxic effect, but this preservative was relatively safe and effective (at a concentration of 0.04%).8,14 The use of preservative-free medication has the added advantage of avoiding possible allergy.2 We used proparacaine with 0.01% of benzalkonium chloride and preservative-free tetracaine. The concentrations of local anesthetic agents used in this study resemble the actual in vivo concentrations of these medications that may result after the dilution with tear secretion and loss in the lacrimal drainage system (approximately one third the concentration of commercially prepared drugs11,12) (Table 1). There is a continuous turnover of the ocular tear film at a rate of 16% per minute, and the conjunctival cul-de-sac can hold only a fraction of the volume of a regularly dispensed eyedrop.15 Because of the rapid turnover of tear film, we began our evaluation of the toxic effect after 15 minutes. When the drug sensitivity was analyzed with XTT, tetracaine demonstrated significantly greater toxicity than proparacaine, but the effects of both these commercially available anesthetic agents on stromal keratocytes were related to drug concentration and to time exposure. Whether 1 of these anesthetics agents is superior to the other cannot be determined on the basis of this study

References 1. Seiler T, Wollensak J. Myopic photorefractive keratectomy with the excimer laser; one-year follow-up. Ophthalmology 1991; 98:1156 –1163 2. Verma S, Marshall J. Control of pain after photorefractive keratectomy. J Refract Surg 1996; 12:358 –364 3. Arshinoff SA, Mills MD, Haber S. The pharmacotherapy of photorefractive keratectomy. J Cataract Refract Surg 1996; 22:1037–1044 4. Bartfield JM, Holmes TJ, Raccio-Robak N. A comparison of proparacaine and tetracaine eye anesthetics. Acad Emerg Med 1994; 1:364 –367 5. Zagelbaum BM, Tostanoski JR, Hochman MA, Hersh PS. Topical lidocaine and proparacaine abuse. Am J Emerg Med 1994; 12:96 –97 6. Scudiero DA, Shoemaker RH, Paull KD, et al. Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Res 1988; 48:4827– 4833 7. Verma S, Corbett MC, Marshall J. A prospective, randomized, double-masked trial to evaluate the role of topical anesthetics in controlling pain after photorefractive keratectomy. Ophthalmology 1995; 102: 1918 –1924 8. Burstein NL. Corneal cytotoxicity of topically applied drugs, vehicles and preservatives. Surv Ophthalmol 1980; 25:15–30 9. Boljka M, Kolar G, Vidensek J. Toxic side effects of local anaesthetics on the human cornea. Br J Ophthalmol 1994; 78:386 –389 10. Rocha G, Brunette I, Le Franc¸ois M. Severe toxic keratopathy secondary to topical anesthetic abuse. Can J Ophthalmol 1995; 30:198 –202 11. Grant RL, Acosta D. Comparative toxicity of tetracaine, proparacaine and cocaine evaluated with primary cultures of rabbit corneal epithelial cells. Exp Eye Res 1994; 58:469 – 478 12. Grant RL, Acosta D Jr. A digitized fluorescence imaging study on the effects of local anesthetics on cytosolic calcium and mitochondrial membrane potential in cultured rabbit corneal epithelial cells. Toxicol Appl Pharmacol 1994; 129:23–35 13. Rapuano CJ. Topical anesthetic abuse: a case report of bilateral corneal ring infiltrates. J Ophthalmic Nurs Technol 1990; 9:94 –95 14. Gasset AR, Ishii Y, Kaufman HE, Miller T. Cytotoxicity of ophthalmic preservatives. Am J Ophthalmol 1974; 78: 98 –105 15. Tang-Liu DDS, Schwob DL, Usansky JI, Gordon

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YJ. Comparative tear concentrations over time of ofloxacin and tobramycin in human eyes. Clin Pharmacol Ther 1994; 55:284 –292

ment of Preventive Medicine (LaBree), University of Southern California, Los Angeles and the Department of Ophthalmology, University of California, Irvine (McDonnell), California, USA.

Accepted for publication March 8, 1999.

Supported in part by an unrestricted grant from Research to Prevent Blindness, Inc., New York, New York, and by a core grant, EY03040, from the National Institutes of Health, Bethesda, Maryland, USA.

From the Doheny Eye Institute and Department of Ophthalmology (Moreira, Kasetsuwan, Sanchez, Shah, McDonnell) and the Depart-

None of the authors has a financial or proprietary interest in any product mentioned.

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