LABORATORY SCIENCES. Free Radicals in Phacoemulsification and Aspiration Procedures. the most popular cataract surgery owing to the establishment

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1 LABORATORY SCIENCES Free Radicals in Phacoemulsification and Aspiration Procedures Hiroshi Takahashi, MD; Atsuhiro Sakamoto, MD; Ryoki Takahashi, PhD; Takeo Ohmura, PhD; Shigeto Shimmura, MD; Kunitoshi Ohara, MD Objectives: To detect free radicals in phacoemulsification and aspiration procedures using electron-spin resonance and to observe the effect of ophthalmic viscosurgical devices (viscoelastic agents) on free radical intensity. Methods: (1) A test tube containing BSS Plus (Alcon Laboratories, Inc, Fort Worth, Tex) with 1% of the spin-trapping agent, 5,5 -dimethyl-1-pyrroline N-oxide, without irrigation and aspiration, was exposed to ultrasound (0% for seconds). A preparation of hyaluronate sodium (Healon [a cohesive agent that contains 1% hyaluronate sodium] {Pharmacia, Uppsala, Sweden} or Viscoat [a dispersive agent that contains 3% hyaluronate sodium and 4% chondroitin sulfate] {Alcon Laboratories, Inc}) was added to the solution to observe inhibitory effects. (2) To simulate a clinical procedure, an eye model with irrigation and aspiration of a combination of 1% 5,5 -dimethyl-1-pyrroline N-oxide and BSS Plus, 25 ml/min, as the irrigating solution was exposed to ultrasound (for,, or seconds). Healon or Viscoat was injected into the anterior chamber. Free radicals were measured by an electron-spin resonance spectrometer. Results: (1) A characteristic signal corresponding to hydroxyl radicals was detected. Similar inhibition by Healon and Viscoat was observed. (2) Two ophthalmic viscosurgical devices similarly suppressed the signal at seconds. The inhibition by Healon ceased at seconds, whereas Viscoat suppressed the signal throughout the time course. Conclusions: Phacoemulsification produces hydroxyl radicals in the anterior chamber even with irrigation and aspiration. The effect of ophthalmic viscosurgical devices on free radicals depends on the retention of the materials within the anterior chamber. Clinical Relevance: There are complications associated with phacoemulsification. Arch Ophthalmol. 02;1: From the Departments of Ophthalmology (Drs H. Takahashi and Ohara) and Anesthesiology (Dr Sakamoto), Nippon Medical School, Tokyo; Sagami Laboratory, Wakamoto Pharmaceutical Co, Ltd, Kanagawa (Drs R. Takahashi and Ohmura); and the Department of Ophthalmology, Tokyo Dental College, Chiba (Dr Shimmura), Japan. None of the authors has any commercial or proprietary interest in the products or companies described in this article. PHACOEMULSIFICATION and aspiration (PEA) has become the most popular cataract surgery owing to the establishment of safe surgical techniques and the development of associated instruments. Corneal endothelial damage, however, can still be a serious complication because excessive damage can lead to irreversible bullous keratopathy. Surgeons need to be aware of this particular problem to prevent endothelial damage, especially when functional reservoirs are poor. Several causes that lead to damage have been documented, and include items such as mechanical or heat injuries. Recently, free radical formation due to ultrasound (US) has been postulated to be another cause of the damage. In experimental situations, free radical formation caused by US in conjunction with commercially available PEA devices has been reported. 1-3 Considering the oxidative insult to the endothelial cells caused by free radicals, their presence in the anterior chamber may be one of the most harmful factors during these procedures. 4,5 With regard to protecting the endothelium from mechanical injuries, the effectiveness of ophthalmic viscosurgical devices (OVDs), the new term recommended by the International Organization for Standardization for viscoelastic agents, 6 has been well documented. The major ingredient of OVDs is hyaluronate sodium, which is known to be a free radical scavenger. Several studies 7,8 have revealed that hyaluronate sodium plays an important protective role against the oxidative damage in patients with arthritis. Hyaluronate sodium injection therapy into the joint cavity was introduced with the expectation that it would provide an anti free radical effect. 9 Other ophthalmic studies,11 have also reported on the protective properties of hyaluronate sodium on oxidative stress in the corneal endothelium. Thus, OVDs can be expected to provide 1348

2 some anti free radical effect during PEA procedures. One study 2 has shown that Healon (a cohesive agent that contains 1% hyaluronate sodium) (Pharmacia, Uppsala, Sweden), when added to the irrigating solution, reduced the free radical concentration caused by US. To our knowledge, however, there has been no study to investigate free radical intensity during standard PEA procedures in which the OVD is injected into the anterior chamber followed by US with irrigation and aspiration (I/A). It is reasonable to assume that the free radical concentration will be affected by the continuous irrigation. In addition, because commercially available OVDs have different properties regarding their retention in the anterior chamber during PEA, the anti free radical effect of OVDs is likely to depend on their behavior during I/A. In this study, we followed standard PEA procedures in an eye model and measured free radical signals with the electron-spin resonance (ESR) procedure. The kinetics of the free radical intensity and the effects of several OVDs during clinical PEA were also demonstrated by our study. Control 1% Dimethyl Sulfoxide % Dimethyl Sulfoxide Healon, 0.1 mg/ml Healon, 0.3 mg/ml Viscoat, 0.1 mg/ml METHODS Viscoat, 0.3 mg/ml IN VITRO STUDY The method for free radical detection by ESR has been described previously. 12 All measurements were repeated 5 times. For a spintrapping agent, 5,5 -dimethyl-1-pyrroline N-oxide (DMPO) (Sigma-Aldrich Japan, Tokyo) was used. Before each experiment, nitrogen gas was bubbled into the solution for 15 minutes to purge oxygen and prevent nonspecific oxidation of the DMPO. To confirm the results of previous studies, 1-3 we first performed an in vitro experiment. In 2-mL plastic test tubes, 1.5 ml of a combination of 1% DMPO and BSS Plus (Alcon Laboratories, Inc, Fort Worth, Tex) solution was prepared (control). The US probe of a commercially available PEA device (Series Ten Thousand Phacoemulsifier; Alcon Laboratories, Inc) was placed in the center of the tube, and US was performed at a power level of 0% for seconds without I/A. Immediately after US, 0 µl of the solution was transferred to a flat quartz ESR cuvette. The cuvette was then placed in an ESR spectrometer (model JES-RE3X; JEOL, Tokyo), and the signal of the spin adducts, the hydroxyl radical ( OH) trapped by DMPO, was measured by double integration using a computer software program. To observe the effect of an OH-specific scavenger, 1% or % dimethyl sulfoxide (Sigma-Aldrich Japan) was added to the solution before US. For observation of the effects of the OVDs, Healon or Viscoat (a dispersive agent that contains 3% hyaluronate sodium and 4% chondroitin sulfate) (Alcon Laboratories, Inc) was added to the solutions to achieve either a 0.1- or a 0.3-mg/mL final concentration before US. EYE MODEL STUDY To detect free radicals in conditions simulating a standard clinical PEA procedure, we used a commercially available eye model (Marty System; Iatrotech, Menlo Park, Calif) that was developed for use as a training procedure for various ophthalmic surgical procedures, including PEA. BSS Plus containing 1% DMPO was used as an irrigating solution. The PEA probe was inserted through a 3.2-mm incision, and the tip was fixed at the center and on the iris plane of the model eye. PEA was performed for,, or seconds with 0% US power level. The following experimental protocols were examined: (1) control, no I/A and no OVD; (2) BSS Plus group, I/A (25 ml/min) and no OVD; (3) Figure 1. Representative signals in the in vitro experiment. Spectrometer settings were as follows: modulation frequency, 0 khz; microwave frequency, 9.4 GHz; microwave power, mw; scan time, 1 seconds; time constant, 0.3 seconds; and receiver gain, Healon is a cohesive agent that contains 1% hyaluronate sodium, and Viscoat is a dispersive agent that contains 3% hyaluronate sodium and 4% chondroitin sulfate. DMPO indicates 5,5 -dimethyl-1-pyrroline N-oxide; OH, hydroxyl radical; and and, the third and fourth signals, respectively, of the manganese in the electron-spin resonance spectra. Healon group, I/A (25 ml/min) and injection of 0.3 ml of Healon into the anterior chamber before US; and (4) Viscoat group, I/A (25 ml/min) and injection of 0.3 ml of Viscoat into the anterior chamber before US. After PEA, 0-µL samples of the solutions were collected from the anterior chambers, and free radical intensity was determined as previously described. DATA ANALYSIS The intensities of the signals were calculated through image analysis after standardization using the amplitudes of the Manganese signal. Statistical analysis of the digitized data was performed by the Dunnett test and the t test, and P.05 was considered significant. RESULTS IN VITRO STUDY The ESR spin adducts of the sample revealed the characteristic quartet signal pattern (Figure 1), which is specific for OH. The hyperfine coupling constants for the spin adduct ( N =1.49 and H =1.49 milli-tesla) are consistent with those for OH according to a previous report. 13 Superoxide-related signals were not detected. There was a dose-dependent inhibition observed with the addition of dimethyl sulfoxide that supports the fact that 1349

3 Signal Intensity, Arbitrary Unit Control 1 Dimethyl Sulfoxide, % the signals corresponded to OH signals. Ophthalmic viscosurgical devices also suppressed the signals in a dosedependent manner. The suppression was similar for the 2 agents tested (Figure 2). EYE MODEL STUDY Although the change was smaller compared with the in vitro experiments, the presence of the quartet signal was also revealed by ESR in the eye model experiments (Figure 3). In the control, signals increased and reached a plateau at seconds. In the BSS Plus group, signals were enhanced in a time-dependent fashion, but the intensity at seconds was not significantly different from that at seconds. In the Healon group, although the signals at seconds were significantly smaller than for the BSS Plus group, signals at to seconds were similar to those of the BSS Plus group. On the contrary, Viscoat significantly suppressed the signals throughout the entire time course (Figure 4). COMMENT The mechanism of free radical formation by US is thought to occur as follows. Ultrasound in aqueous solutions induces acoustic cavitation that causes gas bubbles to collapse, leading to the thermal dissociation of water vapor into OH and hydrogen atoms. 14 Free radical formation associated with clinical PEA, therefore, seems inevitable. In fact, several studies have demonstrated ophthalmic PEA device-related free radical formation. Shimmura et al 1 first described free radical formation in vitro, and Holst et al 2 demonstrated this phenomenon in vivo. Both studies, however, used the chemiluminescence technique that, while suitable for detecting superoxides, does not detect OH signals, the most potent of the free radical species. Because OH is highly reactive and short-lived, measurements are achieved by using radical trap agents and the detection of these radical adducts by ESR. Recently, Cameron et al 3 reported on the ability to detect OH formation through ESR. They applied US in a test chamber Healon, mg/ml Viscoat, mg/ml Figure 2. Signal intensities shown with an arbitrary unit in the in vitro experiment. Dimethyl sulfoxide, Healon (a cohesive agent that contains 1% hyaluronate sodium), and Viscoat (a dispersive agent that contains 3% hyaluronate sodium and 4% chondroitin sulfate) significantly suppressed the signal vs control (P.05, each agent vs control). The difference between Healon and Viscoat was not significant (P=.78). with a closed circulation loop in which the same solution was recirculated. During clinical PEA, however, there is I/A of the solution at various rates. Thus, the aqueous humor is continuously replaced by the irrigating solution. Consequently, the actual free radical concentration in the anterior chamber is determined by the ratio of its production and subsequent clearance. During clinical PEA, an OVD is injected into the anterior chamber before US. An OVD reduces free radical concentration caused by US when added to the test solution in vitro and in vivo. 2 Furthermore, because each of the OVDs has different properties regarding its retention in the anterior chamber during PEA, the individual behavior of each agent during PEA may alter the net result that occurs. Considering these factors, we sought to simulate the clinical PEA procedures in an eye model and detect free radicals via ESR. In the in vitro experiment, in which the reacted solution was collected intact, notable signals for OH were detected, confirming the results of a previous report. 3 Ophthalmic viscosurgical devices suppressed the free radical intensity in a dose-dependent fashion, suggesting that the OVD, a radical scavenger itself, functions as an alternate reactant for the radicals and consequently reduces free radical concentration in the aqueous solution. Interestingly, there was no significant difference between the 2 OVDs when compared at the same concentration. Healon contains only 1% hyaluronate sodium, while Viscoat comprises 3% hyaluronate sodium and 4% chondroitin sulfate, which is also known as a free radical scavenger. 15,16 These results indicate that the anti free radical effects of hyaluronate sodium and chondroitin sulfate are not synergetic, at least in the present experimental setup. In the eye model study, the experimental conditions for the control (no I/A and no OVD) were almost the same as for the in vitro study. Signals increased until seconds but then seemed to reach a plateau, suggesting that the DMPO trapping mechanism was saturated. In the BSS Plus group, in which I/A continuously replaced the reacted solution, smaller but characteristic signals of OH were detected. To our knowledge, this is the first ESR evidence that during PEA there is existence of OH in the anterior chamber even with I/A. Although signals increased in a time-dependent manner, the intensity at seconds was not significantly different from that at seconds. Cameron et al 3 reported that, in their closed circulation system, the OH concentration was proportional to US duration. Present results indicate that with I/A, free radicals may reach a stable concentration because of a constant production and clearance ratio. In the Healon and the Viscoat groups, similar inhibition of the signals was observed at seconds. At and seconds, however, the suppression compared with the BSS Plus group was significant only in the Viscoat group. This indicates that Healon was flushed out by I/A by the -second point, while Viscoat was retained in the anterior chamber long enough to exhibit the effect for at least seconds. These data confirm the results from previous studies concerning the retention of OVDs during PEA. Assia et al 17 experimentally compared the removal time for several OVDs from the anterior chamber due to I/A. They 1350

4 A B BSS Plus Group Time, s Control Group Time, s Viscoat Group Time, s Healon Group Time, s Figure 3. Representative signals in the eye model experiment. A, Control and BSS Plus groups. B, Healon (a cohesive agent that contains 1% hyaluronate sodium) and Viscoat (a dispersive agent that contains 3% hyaluronate sodium and 4% chondroitin sulfate) groups. Even with irrigation and aspiration, ultrasound produced the characteristic quartet signals in the BSS Plus group. DMPO indicates 5,5 -dimethyl-1-pyrroline N-oxide; OH, hydroxyl radical; and and, the third and fourth signals, respectively, of the manganese in the electron-spin resonance spectra. Signal Intensity, Arbitrary Unit Control Group BSS Plus Group Healon Group Viscoat Group Time, s Figure 4. Signal intensities shown with an arbitrary unit in the eye model experiment. Inhibition by Healon (a cohesive agent that contains 1% hyaluronate sodium) was significant only at seconds (Healon vs BSS Plus group, P=.002), while suppression by Viscoat (a dispersive agent that contains 3% hyaluronate sodium and 4% chondroitin sulfate) was significant even at seconds (Viscoat vs BSS Plus group, P=.004). The differences at and seconds were not significant for the control (P=.58), BSS Plus (P=.13), and Healon (P=.36) groups. Data are given as mean±sd. The asterisk indicates P.05. found that the removal time for the cohesive agent, Healon, was to 25 seconds, while that for the dispersive agent, Viscoat, was 3.5 minutes. Poyer et al 18 quantitatively measured the vacuum levels when bolus removal of the materials occurred, and showed that such a phenomenon was commonly observed with cohesive agents, including Healon, but not with Viscoat. Viscoat coats the endothelial cells to a thicker degree than any other agents after a PEA procedure. 19 In this study, the longest US duration was seconds in the eye model experiment, while it is usually longer than seconds during clinical PEA. Presumably, the longer US time would cause the more enhanced signals. Yet, it is likely that Viscoat would remain in the anterior chamber to some extent even after a longer duration and consequently inhibit the signals, considering the results of the previously cited reports The evidence so far is that the more dispersive the agent is, the more retention that is seen in the anterior chamber during and after PEA. The present results suggest that the anti free radical effect of the OVD depends on its retention in the anterior chamber during PEA. Thus, Viscoat, among all of the commercially available OVDs, seems to be the most effective agent for the protection of the endothelium from free radicals. Recently, a new OVD was introduced that has similar properties to Viscoat with regard to retention in the anterior chamber. Thus, it may also have a comparable effect to Viscoat with regard to free radical concentration. In conclusion, we demonstrated that OH production can be documented by ESR in conditions that simulate clinical PEA procedures and that the OVD anti free radical effects seen are correlated to the retention times of the OVD within the anterior chamber during the procedure. However, many radical scavengers exist within the anterior chamber in vivo and may play a part in the free radical concentration. 21,22 Furthermore, during clinical PEA procedures, there are many other factors that can cause endothelial damage, including shock wave injury, fluid-flow turbulence injury, and thermal injury. We do not have any direct evidence as to how harmful the radicals may actually be to the endothelial cells. Thus, further studies are needed to elucidate the actual damage caused by the free radicals associated with clinical PEA. 1351

5 Submitted for publication December 18, 01; final revision received March 27, 02; accepted April, 02. Corresponding author and reprints: Hiroshi Takahashi, MD, Department of Ophthalmology, Nippon Medical School, Sendagi, Bunkyo-ku, Tokyo , Japan ( REFERENCES 1. Shimmura S, Tsubota K, Oguchi Y, Fukumura D, Suematsu M, Tsuchiya M. Oxiradical-dependent photoemission induced by a phacoemulsification probe. Invest Ophthalmol Vis Sci. 1992;33: Holst A, Rolfsen W, Svensson B, Ollinger K, Lundgren B. Formation of free radicals during phacoemulsification. Curr Eye Res. 1993;12: Cameron MD, Poyer JF, Aust SD. Identification of free radicals produced during phacoemulsification. J Cataract Refract Surg. 01;27: Hull DS, Green K, Thomas L, Alderman N. Hydrogen peroxide mediated corneal endothelial damage: induction by oxygen free radical. Invest Ophthalmol Vis Sci. 1984;25: Hull DS, Green K. Oxygen free radicals and corneal endothelium. Lens Eye Toxic Res. 1989;6: Arshinoff S. New terminology: ophthalmic viscosurgical devices. J Cataract Refract Surg. 00;26: Greenwald RA, Moy WW. Effect of oxygen-derived free radicals on hyaluronic acid. Arthritis Rheum. 1980;23: Greenwald RA. Oxygen radicals, inflammation, and arthritis: pathophysiological considerations and implications for treatment. Semin Arthritis Rheum. 1991;: Dougados M. Sodium hyaluronate therapy in osteoarthritis: arguments for a potential beneficial structural effect. Semin Arthritis Rheum. 00;: Artola A, Alio JL, Bellot JL, Ruiz JM. Protective properties of viscoelastic substances (sodium hyaluronate and 2% hydroxymethylcellulose) against experimental free radical damage to the corneal endothelium. Cornea. 1993;12: Artola A, Alio JL, Bellot JL, Ruiz JM. Lipid peroxidation in the iris and its protection by means of viscoelastic substances (sodium hyaluronate and hydroxypropylmethylcellulose). Ophthalmic Res. 1993;25: Shimmura S, Masumizu T, Nakai Y, et al. Excimer laser induced hydroxyl radical formation and keratocyte death in vitro. Invest Ophthalmol Vis Sci. 1999;40: Buettner GR. Spin trapping: ESR parameters of spin adducts. Free Radic Biol Med. 1987;3: Riesz P, Kondo T. Free radical formation induced by ultrasound and its biological implications. Free Radic Biol Med. 1992;13: Albertini R, Rindi S, Passi A, et al. The effect of cornea proteoglycans on liposome peroxidation. Arch Biochem Biophys. 1996;327: Albertini R, De Luca G, Passi A, Moratti R, Abuja PM. Chondroitin-4-sulfate protects high-density lipoprotein against copper-dependent oxidation. Arch Biochem Biophys. 1999;365: Assia EI, Apple DJ, Lim ES, Morgan RC, Tsai JC. Removal of viscoelastic materials after experimental cataract surgery in vitro. J Cataract Refract Surg. 1992; 18: Poyer JF, Chan KY, Arshinoff SA. New method to measure the retention of viscoelastic agents on a rabbit corneal endothelial cell line after irrigation and aspiration. J Cataract Refract Surg. 1998;24: McDermott ML, Hazlett LD, Barrett RP, Lambert RJ. Viscoelastic adherence to corneal endothelium following phacoemulsification. J Cataract Refract Surg. 1998; 24: Holzer MP, Tetz MR, Auffarth GU, Welt R, Volcker HE. Effect of Healon5 and 4 other viscoelastic substances on intraocular pressure and endothelium after cataract surgery. J Cataract Refract Surg. 01;27: Rose RC, Richer SP, Bode AM. Ocular oxidants and antioxidant protection. Proc Soc Exp Biol Med. 1998;217: Richer SP, Rose RC. Water soluble antioxidants in mammalian aqueous humor: interaction with UV B and hydrogen peroxide. Vision Res. 1998;38: Notice to the Authors of Reports From Clinical Trials T he Journal of the American Medical Association ( JAMA) and the Archives of Ophthalmology function as an editorial consortium. With one submission and one set of reviews, your clinical trial manuscript will be considered for publication in both JAMA and the Archives of Ophthalmology. Submit your paper to the journal of your choice according to the appropriate Instructions for Authors and the following guidelines will apply: 1. If your manuscript is accepted by JAMA, it will be considered for an editorial or commentary in JAMA. Your abstract will also be published in the Archives of Ophthalmology with a commentary or editorial. 2. If your manuscript is accepted by the Archives of Ophthalmology, it will be considered for an editorial or commentary in the Archives of Ophthalmology. Your abstract will also be considered for publication in JAMA. 1352