Background: Intraoperative lidocaine is widely used in controlling discomfort during cataract surgery. However, recent studies have confirmed the toxic effect of lidocaine on ganglion cells. Ropivacaine is an anesthetic recently introduced in clinical practice that couples a long anesthetic effect with a mild vasoconstrictive action. Objective: The aim of this study was an in vitro evaluation of the efficacy of ropivacaine in reducing the degenerative effects usually observed during lidocaine treatment. Methods: Ropivacaine and lidocaine toxicity has been evaluated in murine fibroblasts 3T6 by measuring percentage of cell death, cell growth inhibition, and DNA degradation. The choice of this cellular line is motivated by the presence of a complete apoptotic system that can be assimilated to the endothelium precursor cells. Results: We observed that lidocaine 0.25% decreases cell viability and causes DNA degradation in murine fibroblasts 3T6, whereas ropivacaine 0.5% does not cause any cellular or molecular degenerative effect. Conclusions: Our in vitro studies confirm that ropivacaine is less toxic than lidocaine to these cells. Therefore, in vivo studies in the anterior chamber could be useful to evaluate the effects of ropivacaine versus lidocaine in intracameral anesthesia in cataract surgery. © 2011 Vergani & Rusconi, publisher and licensee Adis Data Information BV

Gianfranco Scarsella

Marcella Nebbioso

Marco Tafani

Nicola Pescosolido

PubMed

Abstract Background: Intraoperative lidocaine is widely used in controlling discomfort during cataract surgery. However, recent studies have confirmed the toxic effect of lidocaine on ganglion cells. Ropivacaine is an anesthetic recently introduced in clinical practice that couples a long anesthetic effect with a mild vasoconstrictive action. Objective: The aim of this study was an in vitro evaluation of the efficacy of ropivacaine in reducing the degenerative effects usually observed during lidocaine treatment. Methods: Ropivacaine and lidocaine toxicity has been evaluated in murine fibroblasts 3T6 by measuring percentage of cell death, cell growth inhibition, and DNA degradation. The choice of this cellular line is motivated by the presence of a complete apoptotic system that can be assimilated to the endothelium precursor cells. Results:We observed that lidocaine 0.25 % decreases cell viability and causes DNA degradation in murine fibroblasts 3T6, whereas ropivacaine 0.5 % does not cause any cellular or molecular degenerative effect. Conclusions: Our in vitro studies confirm that ropivacaine is less toxic than lidocaine to these cells. Therefore, in vivo studies in the anterior chamber could be useful to evaluate the effects of ropivacaine versus lidocaine in intracameral anesthesia in cataract surgery

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Cataract surgery complications: an in vitro model of toxic effects of Ropivacaine and lidocaine

PESCOSOLIDO, Nicola

SCARSELLA, Gianfranco

TAFANI, MARCO

NEBBIOSO, Marcella

Archivio della ricerca- Università di Roma La Sapienza

Cataract Surgery ComplicationsAn In Vitro Model of Toxic Effects of Ropivacaine and LidocaineNicola Pescosolido,1 Gianfranco Scarsella,2 Marco Tafani3 and Marcella Nebbioso41 Department of Geriatric Sciences, Sapienza University of Rome, Rome, Italy2 Department of Biology and Biotechnology Charles Darwin, Sapienza University of Rome, Rome, Italy3 Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy4 Department of Sense Organs, Centre of Ocular Electrophysiology, Sapienza University of Rome,Rome, ItalyAbstract Background: Intraoperative lidocaine is widely used in controlling discomfortduring cataract surgery. However, recent studies have confirmed the toxiceffect of lidocaine on ganglion cells. Ropivacaine is an anesthetic recentlyintroduced in clinical practice that couples a long anesthetic effect with a mildvasoconstrictive action.Objective: The aim of this study was an in vitro evaluation of the efficacyof ropivacaine in reducing the degenerative effects usually observed duringlidocaine treatment.Methods: Ropivacaine and lidocaine toxicity has been evaluated in murinefibroblasts 3T6 by measuring percentage of cell death, cell growth inhibition,and DNA degradation. The choice of this cellular line is motivated bythe presence of a complete apoptotic system that can be assimilated to theendothelium precursor cells.Results:We observed that lidocaine 0.25% decreases cell viability and causesDNA degradation in murine fibroblasts 3T6, whereas ropivacaine 0.5% doesnot cause any cellular or molecular degenerative effect.Conclusions: Our in vitro studies confirm that ropivacaine is less toxic thanlidocaine to these cells. Therefore, in vivo studies in the anterior chambercould be useful to evaluate the effects of ropivacaine versus lidocaine inintracameral anesthesia in cataract surgery.IntroductionIn the last few years, the frequency of cataractsurgery has greatly increased, possibly due to anincrease in life expectancy.[1] The current anes-thesia practice does not call for total anesthesiaexcept in pediatrics and handicapped subjects.Use of local anesthesia (retrobulbar, peribulbar,sub-Tenon’s) is limited, even though it is im-portant in eye surgery of anxious subjects andelderly patients who cannot easily control ocularmovements. To prevent severe but rare compli-cations, such us globe perforation, optic nerveinjury, muscular paresis, diplopia, eyelid ptosis,SHORT COMMUNICATION Drugs R D 2011; 11 (4): 303-3071179-6901/11/0004-0303ª 2011 Pescosolido et al., publisher and licensee Adis Data Information BV. This is an open access article published underthe terms of the Creative Commons License ‘‘Attribution-NonCommercial-NoDerivative 3.0’’(http://creativecommons.org/licenses/by-nc-nd/3.0/) which permits non-commercial use, distribution,and reproduction, provided the original work is properly cited and not altered.and retinal vascular occlusion, safer anesthesiatechniques were investigated. Fichman[2] has in-troduced topical anesthesia with xylocaine, whichhas proved to be an effective and safe techniquewith good postoperative recovery.[2-4] However,intraoperative discomfort and pain, particularlyduring phacoemulsification and intraocular lensimplantation, have necessitated the associationwith subconjunctival, retrobulbar, or peribulbaranesthesia.[5,6] Gills et al.[7] have reported en-couraging results in reducing intraoperativediscomfort during cataract surgery through theintracameral injection of 1% unpreserved lido-caine in the presence of topical anesthesia. Thesame authors have found in their studies thatintraocular lidocaine does not damage cornealendothelium (cell count and corneal thickness).These data were confirmed by Kim et al.[8]in both human endothelium and laboratoryanimals; however, a transient endothelial celledema was observed during lidocaine infusion.This was easily evaluated by specular microscopyand electronic scansion microscopy.[8] Gillset al.[9] have reported a transient loss of vision in4 cases in a total of over 15 000 patients. Thiswas essentially due do a dialysis of the posteriorcapsule with lidocaine interfering with ganglioncell function. Recent in vitro and in vivo studieshave confirmed the toxic effect of lidocaineon ganglion cells.[10,11] Furthermore, the shortduration of the local anesthetic makes adminis-tration of a second dose during cataract surgerynecessary, thus increasing the risk of toxicity.Ropivacaine, which has recently been introducedin clinical surgery, can represent a valid alterna-tive to lidocaine in intracameral anesthesia. Infact, ropivacaine presents the speculative advan-tages of a long-duration and bland vasoconstrictiveeffect.[12]In our study, we investigated an in vitroevaluation of the efficacy of the local toxicity ofropivacaine versus lidocaine by utilizing murinefibroblasts 3T6 in culture. This cell line waschosen because of its similarities to the endothe-lium precursors and because apoptosis canbe easily induced and controlled.[13,14] In fact,occurrence of apoptotic phenomena is diagnosticof generalized toxic effect at cellular level.[15]Materials and MethodsCell CulturesThe murine 3T6 fibroblasts are a stable line ofderma fibroblasts. These fibroblasts comparewell with endothelial cell precursors. Cell cul-tures were routinely maintained on Dulbecco’smodified Eagle’s medium (DMEM) and 10%heat-inactivated fetal bovine serum. Cells weremaintained at 37C in a humidified atmosphereof 5% CO2 and 95% air.Unless stated otherwise, to assess apoptoticand/or necrotic phenomena, 5 · 105 cells wereplated in 10 cm Petri dishes and left untreated for24 hours before experimentation began.Treatment ProtocolsRopivacaine (Naropine) is a well tolerated,effective, long-duration, local anesthetic. Ropi-vacaine in low concentrations, unlike lidocaine,reduces cutaneous blood flow and producesbland vasoconstriction.[12] Ropivacaine andlidocaine were dissolved in isotonic salinebuffer (phosphate buffered saline [PBS]) andmade up to a final concentration of 10mmol/Land 5mmol/L, respectively. To assess toxicity,cells were exposed to ropivacaine or lidocaine for24 hours.Measurement of Cell ViabilityCell viability as well as cell growth was meas-ured by trypan blue exclusion. Briefly, 5 · 105cells were centrifuged for 5 minutes at 100 g. Thecell pellet was resuspended in 1mL PBS. Anequal amount of cell suspension and 0.4% trypanblue were mixed. After an incubation time of 3–5minutes, a drop of cell suspension/trypan bluewas applied to a hemacytometer. Unstained(viable) and stained (unviable) cells were countedand the percentage of viability determined.DNA DegradationThe effect of the treatment was monitoredafter extraction of DNA according to Hirt[16] gelelectrophoresis and visualization with ethidiumbromide.304 Pescosolido et al.ª 2011 Pescosolido et al., publisher and licensee Adis Data Information BV. Drugs R D 2011; 11 (4)Statistical AnalysisQuantitative values were presented as mean–SD. Significant differences between sets of valuesfor control and test groups were assessed by a one-way ANOVA using a studentNewman-Keuls posthoc test. A p-value refers to a comparison of ameasured parameter in the experimental groupwith that of the appropriate control. Significancewas set at p< 0.05.Results and DiscussionThe effects of lidocaine and ropivacainetreatment on cell viability are shown in figure 1a.In particular, cells were treated for 24 hours withlidocaine 5mmol/L. After 24 hours, the mediumwas replaced and cell viability measured after48 hours. Figure 1a shows that during such treat-ment, lidocaine decreased cell viability to 65%.A partial cellular recovery was observed after72 hours and 96 hours with a cell viability of 82%and 84%, respectively (figure 1a). By contrast,during the treatment, ropivacaine 10mmol/L, cellviability did not alter and remained about 95%(figure 1a). Lidocaine and ropivacaine concen-trations were chosen from a dose-response analysisof doses ranging from 1 to 20mmol/L (unpub-lished observations, Professor Scarsella).Cytotoxic effects of lidocaine and ropivacainewere determined by measuring cell proliferation.Figure 1b shows that, compared with the pro-liferation curve of untreated cells, lidocainecaused a significant inhibition. In particular, after24 hours, there was almost a 50% decrease in thenumber of cells due to the toxic effect (figure 1b).By contrast, ropivacaine caused only an initialinhibition of cell proliferation that was reducedafter 96 hours of treatment (figure 1b); a timewhere the differences between ropivacaine andlidocaine were evident. Finally, figure 1c showsthat lidocaine caused a DNA degradation re-presented by an evident smear of heterogeneousfragments after extraction and gel electro-phoresis. The production of these fragments istypical in necrotic phenomena (figure 1c, lane d).By contrast, ropivacaine caused only a minordegradation of DNA (figure 1c, lane b).Intracameral injection of lidocaine 1% in ad-dition to topical anesthesia was an importantNumber of viable cells*****†*†5.0 × 1060 24 48 72 96Time (h)b4.5 × 1064.0 × 1063.5 × 1063.0 × 1062.5 × 1062.0 × 1061.5 × 1061.0 × 1065.0 × 1050.0 × 10−0ControlRopivacaineLidocaine100Viability (%)908070†††605048 72Time (h)96aRopivacaineLidocainea b dccFig. 1. Cytotoxic effects of lidocaine and ropivacaine. (a) Percentage cell viability after 48, 72, and 96 hours. (b) Number of viablecells after 0, 24, 48, 72, and 96 hours. (c) Gel electrophoresis (lane a shows the DNA molecular weight marker; lane b showsropivacaine-treated cells; lane c shows control untreated cells; and lane d shows lidocaine-treated cells). * p <0.05 vs control, - p <0.05 vsropivacaine.Toxic Effects of Ropivacaine and Lidocaine 305ª 2011 Pescosolido et al., publisher and licensee Adis Data Information BV. Drugs R D 2011; 11 (4)advancement in ocular anesthesia during cataractsurgery.[7,9] However, safer and newer drugs arebeing investigated in ocular anesthesia to improvesurgical techniques. In particular, ropivacainemay represent a safer alternative to lidocaine,thanks to its long-term efficacy and modest vaso-constrictive action, which is usually an advanta-geous side effect in anterior segment surgery.By contrast, lidocaine causes vasodilation and hasa short anesthetic effect (about 10 minutes).Therefore, supplemental injections of anesthetic inanterior chamber surgery to obtain the desiredanesthesia are often reported, thereby increasingthe risk of toxicity and impairing surgical proce-dure. The duration of the anesthetic effect isrelated to the extent of protein binding, whichin ropivacaine is 94%, while in lidocaine it isabout 64%. In addition, in ropivacaine, there is amarked difference in effect between sensitive andmotor blocks that enables surgeons to use very lowdoses of the drug to inhibit only sensitive func-tion. Therefore, the long duration and the vaso-constrictive action typical of ropivacaine are adefinite advantage in anterior chamber surgery.Our results indicate that, in addition to the dura-tion and vasoconstriction effects reported in thisstudy, ropivacaine has the advantage that it doesnot cause any cellular damage.In particular, some authors have observed thatperfusion of lidocaine l% in human and murineendothelial corneal cells causes a transient cel-lular edema.[8,17] In our experimental model,comparable with endothelial cells, ropivacaineversus lidocaine does not present degenerativeeffects even at a concentration as high as10mmol/L.ConclusionsIn the light of our results, neither drug causedapoptosis; however, lidocaine did give rise toevident necrosis. Therefore, lidocaine may haverelevant cytotoxic effects for the endothelium incataract surgery. Our data are in agreement withthe cytotoxic effects observed by Grosskreutzet al.[10] both in vitro and in vivo. It is there-fore reasonable to conclude that ropivacaineis safer than lidocaine because, at very low con-centrations, it can assure long duration of actioncoupled with mild but useful vasoconstriction,without causing any degenerative effects on en-dothelial cells.AcknowledgmentsFinancial support by the Italian Ministry of Universityand Scientific and Technological Research is acknowledged.The authors have declared no conflicts of interest that aredirectly relevant to the content of this study.References1. Ashwin PT, Shah S, Wolffsohn JS. Advances in cataractsurgery. Clin Exp Optom 2009; 92: 333-422. Fichman RA. Use of topical anesthesia alone in cataractsurgery. J Cataract Refract Surg 1996; 22: 612-43. Hustead RF, Hamilton RC. Pharmacology. In: Gills JP,Hustead RF, Sanders DR, editors. Ophthalmic anesthesia.Thorofare (NJ): Slack, Inck., 1993: 69-1024. Borazan M, Karalezli A, Oto S, et al. Comparison of abupivacaine 0.5% and lidocaine 2% mixture with levo-bupivacaine 0.75% and ropivacaine 1% in peribulbaranaesthesia for cataract surgery with phacoemulsification.Acta Ophthalmol Scand 2007; 85: 844-75. Patel BC, Burns TA, Crandall A, et al. A comparison oftopical and retrobulbar anesthesia for cataract surgery.Ophthalmology 1996; 103: 1196-2036. Leaming DV. Practice styles and preferences of ASCRSmembers: 2003 survey. J Cataract Refract Surg 2004; 30:892-9007. Gills JP, Cherchio M, Raanam MG. Unpreservedlidocaine to control discomfort during IOL surgeryunder topical anesthesia. J Cataract Refr Surg 1997; 23:545-508. Kim T, Holley G, Lee J, et al. The effects of intraocularlidocaine on the corneal endothelium. Ophthalmology1998; 105: 125-309. Gills JP, Jonsons DE, Cherchio M. Anestesia intraoculare.In: Davis DB, Mandel MR, editors. Oftalmologia clinicadel Nord America. Rome: Anestesia Oftalmologia, Ver-duci Editore, 1998; 10 (3): 65-7110. Grosskreutz CL, Katowitz WR, Freeman EE, et al. Lido-caine toxicity to rat retinal ganglion cells. Curr Eye Res1999; 18: 363-711. Borazan M, Karalezli A, Akova YA, et al. Comparativeclinical trial of topical anaesthetic agents for cataract sur-gery with phacoemulsification: lidocaine 2% drops, levo-bupivacaine 0.75% drops, and ropivacaine 1% drops. Eye(Lond) 2008; 22: 425-912. McClure JH. Ropivacaine. Br J Anaesth 1996 Feb; 76 (2):300-713. Bonincontro A, Iacoangeli A, Melucci-Vigo G, et al.Apoptosis dependent decrease of the intramembrane iontraffic in cultured mouse fibroblasts shown by conductivitydispersion. Biosci Rep 1997; 17: 547-56306 Pescosolido et al.ª 2011 Pescosolido et al., publisher and licensee Adis Data Information BV. Drugs R D 2011; 11 (4)14. Pescosolido N, Del BiancoG, De FeoG, et al. Induced acuteocular hypertension: mode of retinal cell degeneration.Acta Ophthalmol Scan 1998; 227 (Suppl.): 20-115. BorazanM, Karalezli A, Oto S, et al. Induction of apoptosisof rabbit corneal endothelial cells by preservative-free li-docaine hydrochloride 2%, ropivacaine 1%, or levobupi-vacaine 0.75%. J Cataract Refract Surg 2009; 35: 753-816. Hirt B. Selective extraction of polyoma DNA from infectedmouse cell cultures. J Mol Biol 1967; 14; 26: 365-917. Shah AR, Diwan RP, Vasavada AR, et al. Corneal endo-thelial safety of intracameral preservative-free 1% xylo-caine. Indian J Ophthalmol 2004; 52: 133-8Correspondence: DrMarcella Nebbioso, Department of SenseOrgans, Centre of Ocular Electrophysiology, viale delpoliclinico 155, 00161 Rome, Italy.E-mail: marcella.nebbioso@uniroma1.itToxic Effects of Ropivacaine and Lidocaine 307ª 2011 Pescosolido et al., publisher and licensee Adis Data Information BV. Drugs R D 2011; 11 (4)

Cataract surgery complications: An in vitro model of toxic effects of Ropivacaine and lidocaine

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Cataract surgery complications: An in vitro model of toxic effects of Ropivacaine and lidocaine

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