Cytotoxicity of proparacaine to human corneal endothelial cells in vitro.

Proparacaine is a widely used topical anesthetic in ophthalmic optometry and surgery, and has been reported to have cytotoxic effects on rabbit corneal endothelial cells after prolonged and repeated usage. Since rabbit is an exceptive mammal whose corneal endothelial cells still maintaining proliferation abilities even in adulthood, whether proparacaine has cytotoxic effects on human corneal endothelial (HCE) cells need to be further verified. Our objectives in the present study were to investigate the cytotoxicity to HCE cells of proparacaine and its underlying mechanisms in vitro and verify the cytotoxicity using cat corneal endothelial (CCE) cells in an in vivo model of cat corneas. Cytotoxic evaluation results indicated that a dose- and time-dependent toxic response of HCE cells to proparacaine over 0.03125% was rated based on morphology and viability, and a toxic response of CCE cells to 0.5% (clinical applied dosage) proparacaine was also rated based on cell density and histology. Importantly, treatment with proparacaine resulted in significant elevation of plasma membrane permeability, cell cycle arrest at S phase, fragmentation of genomic DNA, formation of apoptotic bodies, and externalization of phosphatidylserine (PS) of HCE cells. Moreover, proparacaine demonstrated disrupting effects on mitochondrial transmembrane potential (MTP) of HCE cells and activating effects on caspase-3, -8 and -9. This study demonstrates that proparacaine has notable cytotoxicity to both HCE cells in vitro and CCE cells in vivo, and its dose- and time-dependent cytotoxicity to HCE cells is achieved by inducing apoptosis via a mitochondrion-mediated caspase-dependent pathway. These findings provide new insights into the cytotoxicity and apoptosis-inducing effect of local anesthetics which should be used with great caution in the eye clinic.

Early amniotic membrane transplantation for toxic keratopathy secondary to topical proparacaine abuse: a report of seven cases.

PURPOSE: The aim of this study is to evaluate the effect of early amniotic membrane transplantation (AMT) in patients with topical proparacaine-related toxic keratopathy. MATERIALS AND METHODS: Between 2008 and 2011, eight eyes of seven patients with toxic keratopathy related to 0.5% proparacaine abuse underwent early AMT (within 1 to 5 days following the diagnosis). Clinical findings and treatment outcomes of these cases were evaluated retrospectively. RESULTS: The median time of topical anesthetic abuse until admission to our clinic was 28 (10-112) days. One case was referred due to achanthamoeba keratitis; two cases due to intractable corneal ulcer and melting. At initial examination, visual acuities varied between hand motions and 0.4 (in decimal notation). Biomicroscopic evaluation at presentation revealed epithelial defects, corneal ulcers, stromal ring infiltrate, stromal edema and corneal melting with varying degrees. At third month after AMT, the visual acuities varied between hand motions and 0.9, and all the patients had corneal stromal opacities with varying densities. One patient, who did not respond to medical and surgical treatment and developed secondary infections that invaded intraocular structures, underwent evisceration. CONCLUSION: Topical anesthetic abuse can lead to serious ocular complications. After proper diagnosis, the first step of treatment is the cessation of drug abuse. In addition to medical treatment, early AMT has an advantage of early pain relief and consequential elimination of the need for topical anesthetic instillation.

The systemic toxicity of equipotent proxymetacaine, oxybuprocaine, and bupivacaine during continuous intravenous infusion in rats.

BACKGROUND: Although proxymetacaine and oxybuprocaine produce topical ocular and spinal anesthesia, they have never been tested as cutaneous anesthetics. We compared cutaneous analgesia of proxymetacaine and oxybuprocaine with bupivacaine and tested their central nervous system and cardiovascular toxicity. METHODS: After blockade of cutaneous trunci muscle reflex with subcutaneous injections, we evaluated the local anesthetic effect of proxymetacaine and oxybuprocaine on cutaneous analgesia in rats. After i.v. infusions of equipotent doses of oxybuprocaine, proxymetacaine, and bupivacaine, we observed the onset time of seizure, apnea, and impending death and monitored mean arterial blood pressure and heart rate. RESULTS: Proxymetacaine and oxybuprocaine acted like bupivacaine and produced dose-related cutaneous analgesia. On a 50% effective dose basis, the ranks of potencies were proxymetacaine > oxybuprocaine > bupivacaine (P < 0.01). Under equipotent doses, the infusion times of proxymetacaine or oxybuprocaine required to cause seizure, apnea, and impending death were longer than that of bupivacaine (P < 0.05). The decrease in mean arterial blood pressure and heart rate was slower with oxybuprocaine and proxymetacaine compared with bupivacaine (P < 0.05 for the differences) at equipotent doses. CONCLUSIONS: Oxybuprocaine and proxymetacaine were more potent at producing cutaneous anesthesia but were less potent than bupivacaine at producing central nervous system and cardiovascular toxicity.

Epithelial activity of hexokinase and glucose-6-phosphate dehydrogenase in cultured bovine lenses recovering from pharmaceutical-induced optical damage.

PURPOSE: In a previous toxicological study, cultured bovine lenses exposed to three topical anesthetics displayed distinct patterns of optical damage and recovery. This work investigated the epithelial activity of the metabolic enzymes hexokinase (HK) and glucose-6-phosphate dehydrogenase (G6PD) in lenses recovering from anesthetic-induced damage. METHODS: Cultured bovine lenses were exposed to the anesthetics Alcaine, Fluress and Fluoracaine for 2 h. An automated laser scanner was used to determine the focal length variability (FLV) of the lenses at time-points up to 24 h following their return to fresh culture medium. The epithelial enzyme activities for HK and G6PD were then assayed at the 24 h time-point. RESULTS: Lenses exposed to Alcaine displayed an abrupt increase in FLV, while Fluoracaine treated lenses exhibited optical damage at a slower rate. The FLV in these two groups recovered to near-control levels after 24 h. Fluress treated lenses did not differ in FLV from controls at any time. The activities of both HK and G6PD were significantly reduced in epithelial samples from each of the three anesthetic treatment groups, relative to controls. CONCLUSIONS: These results show that lens optical quality can recover despite a severe reduction in epithelial HK and G6PD activity, indicating that the optical function of the lens may not be directly related to epithelial metabolic activity. The ScanTox In Vitro Assay System provides an objective measure of lens optical quality, enabling a direct comparison of optical damage and recovery to lens biochemical changes.

Toxicity of topical anesthetic agents to human keratocytes in vivo.

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.

Corneal endothelial toxicity of topical anesthesia.

PURPOSE: To determine the relative corneal endothelial toxicities of the following topical anesthetic agents: bupivacaine HCl 0.75%, unpreserved lidocaine HCl 4%, proparacaine HCl 0.5%, and tetracaine HCl 0.5%. METHODS: The experiment was conducted using pigmented rabbits. Approximately nine animals each were randomly assigned to eight groups. Right eyes received injections of 0.2 ml of one of the four anesthetic agents at one of two concentrations and left eyes received injections of 0.2 ml of balanced salt solution. Corneal thickness and clarity were measured before surgery and on postoperative days 1, 3, and 7. RESULTS: A statistically significant increase (P < 0.05) in corneal thickness and opacification over preoperative measurements was noted with injections of bupivacaine, lidocaine, and proparacaine, controlling for changes occurring in control eyes from surgery alone. Proparacaine was statistically more toxic than were the others. The toxicity of tetracaine was statistically indistinguishable from balanced salt solution, although mild toxicity was evident clinically. Injection of 1:10 dilutions of the same anesthetic agents failed to produce a statistically significant increase in corneal thickness or opacification on any postoperative examination. CONCLUSIONS: Anterior chamber injection of bupivacaine HCl 0.75%, unpreserved lidocaine HCl 4%, and proparacaine HCl 0.5% produces corneal thickening and opacification that is clinically and statistically significant. Tetracaine HCl 0.5% injection produces corneal thickening and opacification that is clinically apparent in some eyes but statistically insignificant. Ophthalmic surgeons should be aware of the potential for endothelial cell injury if anesthetic agents enter or are injected into the eye during cataract surgery in the concentrations supplied commercially.

A digitized fluorescence imaging study on the effects of local anesthetics on cytosolic calcium and mitochondrial membrane potential in cultured rabbit corneal epithelial cells.

It has been documented by several investigators that local anesthetics displace calcium from calcium binding sites and alter the functioning of different calcium regulating systems. Local anesthetics have also been shown to have adverse effects on mitochondrial function and interact with cytoskeletal elements. Few studies have addressed the role that a potential disturbance of calcium homeostasis and mitochondrial function may have on the toxicity caused by local anesthetics in corneal epithelial cells. This investigation was undertaken to evaluate the effects of tetracaine (TTC), proparacaine (PPC), and cocaine (CC) on cytosolic calcium and mitochondrial membrane potential in primary cultures of rabbit corneal epithelial cells. Previous studies by our laboratory documented that the local anesthetics produce toxicity after 30 to 60 min of treatment. In this study, the cells were treated for 15 min, a time when minimal cell damage occurred. The following concentrations of local anesthetics were used to treat the cells: TTC, 0.5-2.5 mM; PPC, 1-5 mM; and CC, 4-10 mM. We utilized the technology of digitized fluorescence imaging to measure changes in intracellular calcium ([Ca2+]i) with fura-2 and mitochondrial membrane potential (delta psi) with rhodamine 123. A dose-dependent increase in [Ca2+]i was evident after treatment with each local anesthetic. Concentrations equal or greater than 2.5 mM TTC dissipated delta psi. A rise in [Ca2+]i preceded any loss of delta psi caused by TTC. PPC at high concentrations (4-5 mM) occasionally dissipated delta psi but this was not a consistent finding. The effects of CC on delta psi could not be evaluated accurately because of the extensive morphological alterations that occurred after treatment. We conclude that TTC, PPC, and CC elevate [Ca2+]i before cytotoxicity occurs and disruptions in calcium homeostasis may contribute to their toxicity.

Comparative toxicity of tetracaine, proparacaine and cocaine evaluated with primary cultures of rabbit corneal epithelial cells.

Cocaine was first used as a topical anesthetic for the eye by Dr Carl Koller in 1884. It became evident that this agent produced erosion of the corneal epithelium in high doses or with repeated use. Synthetic local anesthetics such as tetracaine and proparacaine were developed which were more potent and less toxic than cocaine, but still produced corneal epithelium defects if used chronically. This investigation was undertaken to compare and rank the cytotoxicity of the most commonly used ocular local anesthetics, tetracaine, proparacaine and cocaine, with primary cultures of rabbit corneal epithelial cells. Cultures were exposed to either low concentrations of local anesthetics for 4-24 hr or to higher concentrations of local anesthetics for 15-120 min. Plasma membrane integrity was evaluated by measuring leakage of the cytosolic enzyme, lactate dehydrogenase, into the medium. Cell shape changes were evaluated by observing morphological changes. Mitochondrial dehydrogenase activity and cell viability were assessed by measuring 3-(4,5-dimethythiazol-2yl)-2,5-diphenyl tetrazolium bromide reduction. The cytotoxicity of the local anesthetics as evaluated by the lactate dehydrogenase leakage cytotoxicity test was ranked as follows: tetracaine (EC50 = 0.96 mM) >> proparacaine (EC50 = 4.4 mM) > cocaine (EC50 = 9.7 mM). The mitochondrial reduction assay seemed to be more sensitive than the lactate dehydrogenase leakage test in predicting toxicity: tetracaine (EC50 = 0.81 mM) >> proparacaine (EC50 = 3.4 mM) > cocaine (EC50 = 7.1 mM). When corneal epithelial cells were treated with local anesthetics, marked morphological changes occurred at concentrations that did not cause a decrease in viability. This was especially true for cocaine-treated cells. Tetracaine and proparacaine have the same anesthetic potency in vivo, although tetracaine is considered to be more irritating than proparacaine. This in vitro study showed that tetracaine was approximately four times more toxic than proparacaine. Cocaine was less toxic in vitro than proparacaine and tetracaine when compared on an equimolar basis, but in vivo it may be more toxic because of the higher concentrations that must be used to obtain the same degree of anesthesia as well as its marked effects on cell morphology.

Topical bupivacaine and proparacaine: a comparison of toxicity, onset of action, and duration of action.

Bupivacaine is a local ocular anesthetic with a long duration of action when administered by retrobulbar injection. To determine the potential for the use of bupivacaine as a topical ocular anesthetic, the onset and duration of action and toxicity of various concentrations of bupivacaine were studied after instillation in rabbit eyes. The onset and duration of action were not significantly different from that of topical 0.5% proparacaine. Increasing the pH of the bupivacaine solution from 5.7 to 6.5 nearly doubled the duration of action, but the increase was not sufficient to be clinically important. Slit lamp biomicroscopic examination and scanning electron microscopy showed that bupivacaine was less toxic to the corneal epithelium than 0.5% proparacaine. Healing after keratectomy was significantly more rapid in eyes treated with 0.75% bupivacaine, compared with eyes treated with 0.5% proparacaine. These results suggest that bupivacaine may be less toxic to the corneal epithelium than proparacaine and could be clinically useful for topical ocular anesthesia, particularly if pharmacologic modifications can increase the duration of anesthesia provided by this drug.

Effects of proparacaine on actin cytoskeleton of corneal epithelium.

Chronic use of proparacaine, a topical ocular anesthetic, is associated with punctate keratopathy and delayed epithelial wound healing. Spreading corneal epithelial cells normally elaborate cytoplasmic arrays of actin-rich stress fibers which insert onto the inner surface of the cell membrane at discrete adhesion complexes. As actin is implicated in cell-to-substratum adhesion and cell motility, the effects of proparacaine on the actin cytoskeleton of corneal epithelial cells were studied in vitro. Spreading rat corneal epithelial cells in tissue culture were treated with proparacaine hydrochloride. At the lowest drug concentration used (0.01 mM), no effects were seen on the actin cytoskeleton. At 1.0 mM, some disruption of stress fibers was evident and actin was redistributed in a diffuse fashion. Many of the intact stress fibers had abnormal morphology, distribution, and orientation. Scanning electron microscopy showed a loss of cell extensions and cell-to-substratum adhesiveness at the leading epithelial edge. Above 1.0 mM, cell spreading was completely abolished and most cells detached from the substratum. After a washout period with drug-free media, these effects were reversible at concentrations of 1.0 mM or less. We postulate that one mechanism by which proparacaine inhibits corneal epithelial migration and adhesion is through alteration of the actin cytoskeleton.

A permeability test for acute corneal toxicity.

The penetration of sulforhodamine B (SRB) into the two corneas of a freshly killed mouse was measured after one eye was briefly exposed to a solution of a possibly toxic substance. The ratio of the penetrations provides a numerical index of acute toxicity to the corneal epithelium. With a group of 6 animals, a 50% rise in the index can be distinguished at the 95% confidence level; the maximum rise achievable in practice seems to be about 30. NaOH and benzalkonium chloride (BAK) gave graded increases in the value as their concentrations rose; the rise in index corresponded roughly to the injury caused to the living eye.

Morphological changes of the corneal epithelium after application of topical anaesthetic ointments.

The effect of topical anaesthetic ointments (4% cocaine, 4% xylocaine, 0.5% proparacaine, 0.2% oxybuprocaine) on the corneal epithelium of rabbits was examined using a scanning electron microscope. Even after a single application, the more toxic effect of cocaine compared to the other topical anaesthetics was evident. Cocaine caused disruption of both the plasma membrane and the cytoplasm. After a single application, the other preparations caused a marked decrease in the microvilli and microplicae, disruption of the intercellular spaces and the prominence of the cell nucleus which under normal condition is not visible. Repeated applications caused regular cell desquamation and damage to the plasma membrane and cytoplasm. The damage also affected several cell layers. The cell reactions described are clearly a consequence of the topical anaesthetics, as the ointment base itself produced no essential cell damage. The scanning electron microscope findings were supported by results from the transmission electron microscope.