Viscoanesthesia Part I: Toxicity to corneal endothelial cells in a rabbit model Rupal H. Trivedi, MD, Liliana Werner, MD, PhD, David J. Apple, MD, Andrea M. Izak, MD, Suresh K. Pandey, MD, Tamer A. Macky, MD Purpose: To evaluate the toxicity of a solution combining sodium hyaluronate 1.5% with lidocaine (0.5%, 1.0%, or 1.65%) to the rabbit corneal endothelium. Setting: Center for Research on Ocular Therapeutics and Biodevices, Storm Eye Institute, Medical University of South Carolina, Charleston, South Carolina, USA. Methods: Each rabbit cornea was excised, and the endothelium was exposed to 1 of the following solutions for 20 minutes: viscoanesthetic solution (0.5%, 1.0%, or 1.65% lidocaine in sodium hyaluronate 1.5%; 5 corneas each), sodium hyaluronate 1.5% (n ⫽ 5), balanced salt solution (BSS姞) (n ⫽ 5), mitomycin-C 0.02% (n ⫽ 2), dextran 15% (n ⫽ 2), or distilled water (n ⫽ 2). The endothelium was then stained with trypan blue and alizarin red. Two corneas were stained immediately after excision. Cell morphology and damage to the corneal endothelium were analyzed by microscopic examination. Results: The endothelium in the corneas of the viscoanesthetic groups was comparable to that in the sodium hyaluronate 1.5% and the BSS groups and to the corneas not exposed to any solution. In some areas of the 1.0% and the 1.65% viscoanesthesia groups, the corneal endothelial cells presented irregular intercellular borders. Staining with trypan blue, which indicates cellular damage, was observed in some linear areas corresponding to corneal folds in all groups. The folds were probably caused during manipulation for corneal excision and staining. The corneal endothelium was destroyed in the mitomycin group. In the dextran and distilled-water groups, morphological alterations probably resulting from osmotic changes were observed. Conclusion: The 3 concentrations of viscoanesthetic solutions appeared to be safe to rabbit corneal endothelium. J Cataract Refract Surg 2003; 29:550 –555 © 2003 ASCRS and ESCRS
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ntraocular lidocaine has recently gained attention as an effective adjunct to topical anesthesia during cataract surgery. Topical anesthesia was used in combination with intracameral lidocaine by 37% of respondents to the 1999 survey of members of the American Society of Cataract and Refractive Surgery,1 by 40% of responAccepted for publication July 3, 2002. Reprint requests to Liliana Werner, MD, PhD, John A. Moran Eye Center, 5th Floor, 50 North Medical Drive, Salt Lake City, Utah 84132, USA. E-mail:
[email protected]. © 2003 ASCRS and ESCRS Published by Elsevier Science Inc.
dents to the 2000 survey,2 and by 80% of respondents to the 2001 survey.3 Injection of lidocaine into the anterior chamber intraoperatively is claimed to help reduce patient discomfort during phacoemulsification and iris manipulation.4 Studies of the safety and efficacy of intracameral lidocaine have been reported.5–19 However, ophthalmologists who perform cataract surgery using topical and/or intracameral anesthesia must work quickly to finish the surgery before the anesthesia wears off and the patient begins to experience discomfort.20 If a patient reports discomfort during the 0886-3350/03/$–see front matter doi:10.1016/S0886-3350(02)01601-2
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procedure, the surgeon can adopt 1 of the following strategies: (1) forge ahead and finish the procedure as quickly as possible, not providing additional anesthesia; (2) ask the anesthesiologist to administer an intravenous narcotic analgesic; or (3) interrupt the procedure and administer a parabulbar or posterior orbital block, aware that this might generate unwanted external pressure on an open globe. Most surgeons consider supplementing the topical block with additional drops if this happens, or they might consider intracameral supplementation. If they choose either of the latter options, they must be aware of the likelihood of endothelial toxicity from an anesthetic agent that enters or is injected into the eye. Ciba Vision Corp. recently developed a solution combining an ophthalmic viscosurgical device (OVD) with lidocaine. This would not only eliminate the need for extra surgical steps for intracameral injection of lidocaine but also help maintain the anesthetic effect longer. The indispensable role of OVDs in ophthalmic surgery in maintaining the depth of the anterior chamber, keeping the capsular bag expanded, and protecting intraocular structures, especially the corneal endothelium, remains unchanged.21 However, by incorporating lidocaine into the OVD, the risk for toxicity to the corneal endothelial cells may increase from the direct and prolonged contact of lidocaine with these cells. This 3-part study evaluated the safety of this new solution to intraocular structures. Part I used the principles of vital staining of the corneal endothelium in an in vitro animal model to determine the toxicity of the viscoanesthetic solution to the corneal endothelium.5,22 Part II evaluated the toxicity of viscoanesthetic solutions to uveal tissues and retina after phacoemulsification.23 Part III evaluated surgical aspects such as injection and aspiration of the viscoanesthetic solutions and compared them with those of currently available OVDs.24
lium. Immediately after the eyes were enucleated, the corneas and a 2.0 mm rim of sclera were removed with a blade and a partial-thickness scleral section was created with scissors. Care was taken to avoid touching the center of the corneal endothelium during manipulation. The iris diaphragm was peeled from the cornea, which was placed in a corneal cup, endothelial side up. The excavation of the cup corresponded to the corneal curvature. Table 1 shows the solution groups in the study. The viscoanesthetic solutions had 3 concentrations of lidocaine. Table 2 presents some characteristics of these solutions according to the manufacturer. Some corneas were also exposed to the same viscoelastic solution without lidocaine (Ophthalin Plus威). Two negative controls (without toxicity) were used: the “no-solution,” in which the corneas were stained immediately after excision, and balanced salt solution (BSS威). The positive controls were mitomycin-C 0.02% (toxic control), dextran 15% (hyperosmotic solution), and distilled water (hypoosmotic solution). Each cornea was exposed to 1 of the solutions for 20 minutes at room temperature except in the no-solution group. The 20-minute exposure was selected because it is about the time the corneal endothelium is exposed to intracameral lidocaine during phacoemulsification and intraocular lens (IOL) implantation. After the cornea was rinsed in saline 0.9%, the endothelium was immediately stained using a modification of the method of Spence and Peyman22: immersion in trypan blue 0.25% in saline for 90 seconds, 2 rinses in saline 0.9%, immersion in alizarin red 1% for 60 seconds, and a final rinse in saline 0.9%. Six to 8 peripheral radial incisions were made in each cornea, which was placed between a glass slide and a plastic coverslip for examination under a standard light microscope. The amount of endothelial damage was assessed on photographs taken at a standard magnification of ⫻400. Five photomicrographs from the center of each cornea were anaTable 1. Solutions used in the study and the number of corneas in each group.
Solution
Number of Corneas Exposed
Na hyaluronate 1.5%/lidocaine 0.5%
5
Materials and Methods
Na hyaluronate 1.5%/lidocaine 1.0%
5
Fifteen Dutch Belted, pigmented rabbits weighing 2.0 to 2.5 kg were anesthetized with an intramuscular injection of xylazine (5 to 8 mg/kg) and ketamine hydrochloride (35 to 44 mg/kg). The care and treatment of animals conformed to the ARVO Statement for Use of Animals in Ophthalmic and Vision Research. The rabbits were killed by an injection of pentobarbital sodium into an ear vein. The globes were enucleated. The model of Werner et al.5 was used to determine the toxicity of lidocaine (Xylocaine威) to the corneal endothe-
Na hyaluronate 1.5%/lidocaine 1.65%
5
Na hyaluronate 1.5% (Ophthalin Plus)
5
No solution
2
BSS
2
Mitomycin-C (0.02%)
2
Dextran 15%
2
Distilled water
2
Na ⫽ sodium; BSS ⫽ balanced salt solution
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Table 2. Characteristics of the viscoelastic and viscoanesthetic solutions. Formula
Ophthalin Plus
NaHa-Lid 0.5%
NaHa-Lid 1.0%
NaHa-Lid 1.65%
14.90
15.4
15.7
16.1
0
0.49
0.97
1.65
9.27
8.19
7.17
9.70
NaHa, mg/mL Lidocaine, % Chloride as NaCl, mg/mL Osmolality, mOsm/kg
335
309
288
380
pH
7.42
7.40
7.35
7.14
NaHa ⫽ sodium hyaluronate; Lid ⫽ lidocaine
lyzed, and the areas of damaged cells (trypan blue staining) and cell loss (alizarin red staining) were evaluated in each photomicrograph.
Results With the viscoanesthetic solutions and Ophthalin Plus, large areas of the corneal endothelium in the 5 corneas in each group were comparable to those in the negative controls (Figures 1 and 2). In some areas, the 6
Figure 1. (Trivedi) Negative, nontoxic control (no solution or BSS). In the no-solution group, the corneas were stained immediately after excision.
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corners of the endothelial cells showed slight edema, appearing as dots (Figure 2, large arrows). This was observed in the Ophthalin Plus group and in the 3 viscoanesthetic groups. In some areas of the 1% and 1.65% viscoanesthetic groups, the corneal endothelial cells presented irregular intercellular borders (Figure 2, small arrows). Trypan blue staining, indicating cellular damage, was observed in some linear areas corresponding to corneal folds in all groups, including the no-solution group (Figure 3).
Figure 2. (Trivedi) Experimental solutions. In most corneas, the aspect of the corneal endothelial cells was similar to that in the negative controls. The large arrows indicate cells presenting slight edema and the small arrows, cells presenting irregular intercellular borders.
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Figure 3. (Trivedi) Linear areas of staining with trypan blue were observed in all groups. Destruction of the endothelial cells was observed in the mitomycin-C group, and osmotic effects were observed in the dextran and distilled-water groups.
Figure 3 also shows the effect on rabbit corneal endothelium of exposure to mitomycin-C 0.02%, dextran 15%, and distilled water. Mitomycin caused almost complete destruction of the endothelial cells. The intercellular borders were irregular after exposure to dextran and thickened (edema) after exposure to distilled water.
Discussion Gills and coauthors4 report that with intracameral anesthesia, approximately one fourth of their patients experienced some pressure or discomfort during IOL implantation. Although intracameral anesthesia has been shown to be efficacious, the concern about additional intraoperative pain and discomfort control has led ophthalmic surgeons to experiment with other techniques. Whereas some surgeons have been comfortable with the level of anesthesia produced by intracameral anesthesia, many have thought the duration to be too short. Intraocular lidocaine theoretically diffuses into the iris and ciliary body to provide additional local anesthesia, which would be useful in cases that require manipulation of intraocular tissue (eg, iris and lens). However, intracameral lidocaine has the tendency to wash away easily during phacoemulsification. If the surgery is longer than normal, the effect may wear off. As mentioned, the surgeon can repeat the injection of intracameral anesthetic solution. However, this may not be efficacious. Studies report a limited role of intracameral
solutions once OVDs are injected into the anterior chamber because the solutions might not reach the target organs in sufficient concentration.25,26 Fenzl and Gills25 report that an OVD injected before the introduction of lidocaine significantly impedes the lidocaine from accessing the iris root and the ciliary body. Premixing the anesthetic solutions in the OVDs theoretically maximizes the chances that they will reach the target organs. This hypothesis is supported by the observation that the anterior capsule of white cataracts often does not stain well with trypan blue in the presence of an OVD in the anterior chamber.27 However, trypan blue premixed with sodium hyaluronate 1% in a 2 mL syringe or in the viscoelastic disposable injector in a 1:1 ratio provides better visibility. Some authors have used this technique without significant surgical and postoperative adverse experiences. However, they note that there might be a risk for corneal decompensation after intraocular use of self-mixed solutions.27 The intracameral use of any new solution raises concerns about its effect on the corneal endothelium. In this study, we evaluated the safety of viscoanesthetic solutions on rabbit corneal endothelium. A rabbit model was chosen because of the gross morphologic similarities between rabbit and human corneas and the general familiarity of the medical community with this model. Also, the effects of intracameral anesthesia have been studied in rabbit eyes.5 The toxicity of viscoanesthetic solutions to the rabbit corneal endothelium was assayed using vital staining
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with trypan blue and alizarin red. In normal eyes, the endothelial cell is impervious to trypan blue and does not stain. However, in the presence of damage to the cell membrane, trypan blue will penetrate the cells, producing blue-stained cytoplasms and nuclei. Exclusion of trypan blue suggests preserved cellular integrity. Alizarin red stains intercellular borders and bare Descemet’s membrane. Therefore, combined staining with trypan blue and alizarin red allows the analysis of endothelium cell morphology and integrity and the identification of areas with cell damage or loss.5,22 No evidence of endothelial cell damage (staining with trypan blue) or endothelial cell loss (staining with alizarin red) was found with the viscoanesthetic solutions used in this study. Trypan blue staining was observed in some linear areas only, corresponding to corneal folds in all groups (controls and viscoanesthetic groups). These were probably caused during manipulation for corneal excision and staining. The morphological aspect in Figure 2 (irregular intercellular borders) is similar to that in endothelial cells after exposure to lidocaine solutions as described by Werner et al.5 We do not believe this effect is due to direct toxicity but rather comes from differences in the ionic composition, pH, and osmolality of the solutions used. Balanced salt solution has a pH of 6.96 and an osmolality of 310 mOsmol/Kg. Its composition is close to that of the aqueous humor, with all the essential ions (ie, potassium and calcium). Thus, after 20 minutes of direct exposure with solutions that have a different composition than the aqueous humor, some morphologic alterations would be expected.28,29 Studies of irrigating solutions for intraocular surgery demonstrate their efficacy is critically dependent on their composition. Waring and coauthors30 list the factors that decrease barrier and pump functions of the corneal endothelium. Among them are the use of calcium-free solutions; substances such as diamide that cause oxidation of intracellular glutathione; solutions outside the pH range of 6.8 to 8.2; preservatives such as sodium bisulfite, thimerosal, and benzalkonium chloride; substances such as Ouabain, which causes inhibition of ATPase, and bromacetazolamide, which inhibits the carbonic anhydrase within the endothelial cells; bicarbonate-free solutions; and solutions with an osmolarity between 200 and 400 mOsm without the essential ions.
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Our experimental model demonstrated that the morphological alterations in the experimental groups were not associated with cell nonviability (lack of trypan blue staining), but it did not assess how these alterations are associated with impaired endothelial functions. However, Yagoubi and coauthors29 evaluated the effects of sodium chloride 0.9%, Hartmann’s solution, BSS, and BSS威Plus on the corneal endothelium of rabbits. Irrigation with 1 of the solutions for 90 minutes was followed by a 6-hour perfusion with tissue culture medium; during this time, endothelial function was assessed by measuring the swelling and thinning rates in the corneas, respectively, in the absence and presence of bicarbonate ions. All solutions caused some change in the endothelial cell morphology, but none had an apparent effect on the barrier properties or pump function of the endothelium. Our study showed that lidocaine premixed with an OVD appears to be safe to the rabbit corneal endothelium. Effects of the prolonged contact of lidocaine contained in viscoanesthetic solutions with intraocular structures such as the uvea and retina are evaluated in Part II of this study.23 Further in vivo clinical studies are needed to determine the efficacy and long-term effects of viscoanesthesia on intraocular tissues.
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20. Judge AJ, Najafi K, Lee DA, Miller KM. Corneal endothelial toxicity of topical anesthesia. Ophthalmology 1997; 104:1373–1379 21. Liesegang TJ. Viscoelastic substances in ophthalmology. Surv Ophthalmol 1990; 34:268 –293 22. Spence DJ, Peyman GA. A new technique for the vital staining of the corneal endothelium. Invest Ophthalmol 1976; 15:1000 –1002 23. Macky TA, Werner L, Apple DJ, et al. Viscoanesthesia. Part II: toxicity to intraocular structures after phacoemulsification in a rabbit model. J Cataract Refract Surg 2003: 29:556 –562 24. Pandey SK, Werner L, Apple DJ, et al. Viscoanesthesia. Part III: removal time of OVD/viscoanesthetic solutions from the capsular bag of postmortem human eyes. J Cataract Refract Surg 2003; 29:563–567 25. Fenzl RE, Gills JP. Intracameral lidocaine in routine phacoemulsification [letter]. Ophthalmology 2000; 107: 1803–1804 26. Eleftheriadis H, Liu C. Influence of ophthalmic viscosurgical device on the effects of intracameral anesthesia and stains [letter]. J Cataract Refract Surg 2001; 27:11–12 ¨ , Erakgu¨n T, Gu¨ler C. Trypan blue mixed 27. Kayikic¸iog˘lu O with sodium hyaluronate for capsulorhexis [letter]. J Cataract Refract Surg 2001; 27:970 28. Werner LP, Legeais J-M, Durand J, et al. Endothelial damage caused by uncoated and fluorocarbon-coated poly(methyl methacrylate) intraocular lenses. J Cataract Refract Surg 1997; 23:1013–1019 29. Yagoubi MI, Armitage WJ, Diamond J, Easty DL. Effects of irrigation solutions on corneal endothelial function. Br J Ophthalmol 1994; 78:302–306 30. Waring GO III, Bourne WM, Edelhauser HF, Kenyon KR. The corneal endothelium; normal and pathologic structure and function. Ophthalmology 1982; 89: 531–590 From the Center for Research on Ocular Therapeutics and Biodevices, Storm Eye Institute, Medical University of South Carolina, Charleston, South Carolina, USA. Presented in part at the Symposium on Cataract, IOL and Refractive Surgery, Philadelphia, Pennsylvania, USA, June 2002. Supported in part by an unrestricted grant from Research to Prevent Blindness, Inc., New York, New York, USA. None of the authors has a financial or proprietary interest in any product mentioned.
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