Corneal Endothelial Cell Damage by Free Radicals Associated With Ultrasound Oscillation FREE

Phacoemulsification has become the most commonly used technique in cataract surgery. Although both the safety and surgical outcome of phacoemulsification have dramatically improved owing to recent technical advances, phacoemulsification-associated corneal injury still remains an important complication. Several factors have been postulated to cause the injury, including excessive duration of the phacoemulsification, trauma due to the impact of the lens nuclear fragments, air bubbles, and mechanical and heat damage. In addition, it has been well established that ultrasound oscillations in water induce acoustic cavitations, which lead to the thermal dissociation of water vapors into hydroxyl radicals and hydrogen atoms.¹ In fact, some experimental studies in ophthalmology have shown that phacoemulsification results in the formation of free radical species.^2- 4 Using electron spin resonance analysis, we have also demonstrated the formation of hydroxyl radicals in the anterior chamber during phacoemulsification.⁵ In addition, there are some recent studies that have shown that the phacoemulsification-associated endothelial injury may be caused by free radicals.^6- 9 Based on these findings, it seems likely that the corneal endothelium can be damaged by free radicals during phacoemulsification because free radical species, hydroxyl radicals in particular, are highly reactive and can cause lethal injuries to cells.¹⁰ The purpose of this study was to investigate the effects of free radicals on the corneal endothelium with special emphasis on histologic and immunohistochemical analyses after performing ultrasound oscillation in the anterior chambers of rabbit eyes.

Male Japanese white rabbits, weighing 2.5 to 3.0 kg, were purchased from Saitama Experimental Animal Supply (Saitama, Japan). The animals were kept individually under standardized laboratory conditions and were given tap water and food ad libitum. The treatment and handling of the rabbits was in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and the Nippon Medical School Animal Experiment Guideline.

The rabbits were anesthetized with an intravenous injection of pentobarbital (30 mg/kg), and topical oxybuprocaine hydrochloride was used for local anesthesia. A limbal incision was made with a disposable 3.0-mm surgical blade, and the phacoemulsification probe (Prestige; AMO, Santa Ana, California) was introduced into the center of the anterior chamber through the incision without touching any ocular structures. The phacoemulsification probe was used only to create the ultrasound oscillation, but the lens was not emulsified. The ultrasound oscillations were performed for 30 seconds at 100% power with continuous irrigation (25 mL/min) with Balanced Salt Solution (BSS) Plus (Alcon Laboratories Inc, Fort Worth, Texas). In an earlier study, we showed that hydroxyl radicals were generated under these conditions.⁵

The rabbits were assigned to 1 of 4 treatment groups: (1) no treatment (control group); (2) only BSS irrigation; (3) ultrasound oscillations and BSS irrigation; and (4) ultrasound oscillations and irrigation with BSS containing 0.001 M of ascorbic acid. At 15 minutes; 3, 6, 12, 24, 36, 48, and 72 hours; and 1 week after treatment, the rabbits were euthanized with intravenous sodium pentobarbital (65 mg/kg), and the aqueous humor was collected using a tuberculin syringe with a 26-gauge needle. Special care was taken to avoid blood contamination. The aqueous humor was immediately frozen and stored at − 80°C. The corneas were then carefully removed. There were 4 to 7 rabbits for each time in each group.

The excised corneas were fixed in paraformaldehyde, 4%, overnight at 4°C, washed 5 times for 10 minutes each in phosphate-buffered saline (PBS), and treated with acetone for 5 minutes. They were then washed 3 times for 10 minutes each in PBS, incubated in proteinase K (20 μg/mL) for 30 minutes at room temperature, and then incubated in a 1:50 dilution of a monoclonal antibody against 8-hydroxy-2-deoxyguanosine (8-OHdG) (Japan Institute for the Control of Aging, Shizuoka, Japan), a marker of DNA damage by oxidative stress, overnight at 4°C. The corneas were then washed with PBS 3 times for 10 minutes and incubated with fluorescein isothiocyanate rabbit antimouse IgG (Zymed, San Francisco, California) for 1 hour at room temperature. Finally, they were washed with PBS 3 times for 10 minutes each and mounted on slides. The samples were examined with a fluorescence microscope.

Corneas were fixed overnight in paraformaldehyde, 4%, at 4°C. They were then washed 5 times for 10 minutes each in PBS, treated with acetone for 5 minutes, washed 2 times for 10 minutes in PBS, digested with proteinase K (20 μg/mL) for 30 minutes at room temperature, and washed 2 times for 10 minutes each in distilled water. The procedures followed the protocol supplied with the in situ apoptosis detection kit (Trevigen Inc, Gaithersburg, Maryland). The samples were equilibrated in terminal deoxynucleotidyl transferase–labeling buffer for 5 minutes and labeled with biotinylated nucleotide triphosphates using deoxynucleotidyl transferase for 1 hour at 37°C. The reaction was ended with deoxynucleotidyl transferase stop buffer for 5 minutes. The corneas were washed in PBS 3 times for 10 minutes and incubated with Fluorescein Avidin DCS (Vector Laboratories, Burlingame, California) for 1 hour. After washing 3 times with PBS, the samples were mounted on slides. The samples were examined with a fluorescence microscope.

Corneal tissues were fixed overnight in glutaraldehyde, 4%, postfixed in osmium tetroxide, 1%, and dehydrated through a graded alcohol series. For scanning electron microscopy, the tissues were freeze-dried using the t-butyl alcohol freeze-drying method,¹¹ sputter coated with palladium platinum, and viewed with a S3000N microscope (Hitachi, Tokyo, Japan) operated at 20 kV. For transmission electron microscopy, the samples were embedded in glycerol polyglycidyl ether, and ultrathin sections were cut on an ultramicrotome with a diamond knife and stained with uranyl acetate and lead citrate. The sections were examined at 80 kV with a transmission electron microscope.

The concentrations of lipid peroxides in the aqueous humor were measured by the hemoglobin–methylene blue method described by Yagi,¹² which selectively determines the absolute quantity of lipid peroxides and is specific for lipid peroxides. Statistical analysis of lipid peroxides was performed with a Mann-Whitney U test using SPSS software (SPSS Inc, Chicago, Illinois). P < .05 was considered significant.

None of the corneal endothelial cells from the rabbits in the control and BSS groups were 8-OHdG positive. In the ultrasound group, 8-OHdG–positive cells were also not present at 3 hours (Figure 1A) but were detected at 24 hours (Figure 1B). Cells positive for 8-OHdG were not observed at 48 hours. In the ascorbic acid group, there were no 8-OHdG–positive cells (Figure 1C and D).

The time course of detecting cells positive for terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) was similar to that for the 8-OHdG–positive cells. None of the corneal endothelial cells in either the control or the BSS groups were TUNEL positive. This was also true for the ultrasound group at 3 hours (Figure 2A). However, TUNEL-positive cells were present at 24 hours (Figure 2B); TUNEL-positive cells were not observed at 48 hours in the ultrasound group. In the ascorbic acid group, TUNEL-positive cells were not seen at any time (Figure 2C and D).

In controls, the corneal endothelial cells were predominantly hexagonal, and the cell surface had microvilli and ruffled apical membranes in the periphery (Figure 3A). In the BSS group, a few microvilli were present at 24 hours (Figure 3B), but the sizes of the cells were almost uniform and were hexagonal, similar to the controls at all times. In the ultrasound group, a few empty spaces were observed in the corneal endothelial layer, and a few necrotic cells were seen at 15 minutes to 3 hours (Figure 3C). At 6 to 24 hours, the number of necrotic cells increased, and at 24 hours (Figure 3D), many microvilli were present on the endothelial cell surface. Afterwards, the number of necrotic cells was reduced. At 48 and 72 hours (Figure 2E), the cell sizes were different, and the density of microvilli was similar to that in the control group. In the ascorbic acid group, a few microvilli were present at 24 hours (Figure 3F), but the corneal endothelial cells were almost uniformly hexagonal, and there were no necrotic cells.

In controls, the shape of the nuclei were irregular with scattered heterochromatin. The cytoplasm had a well-developed rough endoplasmic reticulum and many mitochondria (Figure 4A). In the BSS group, the corneal endothelial cells appeared similar to those in the control group, and cell damage was not observed. In the ultrasound group, cell injury was already noted at 15 minutes with severe cellular edema at 3 to 6 hours. In the early period (15 minutes to 6 hours), some of the corneal endothelial cells appeared necrotic, the plasma membrane was damaged, organelles were swollen, and heterochromatin was not detected in the nuclei (Figure 4B). At 12 to 24 hours (Figure 4C), the nuclear membranes and lumen of the rough endoplasmic reticulum in the shrunken cells were swollen. The nuclei did not have heterochromatin condensation. The microvilli were extended and quite prominent. Early signs of necrosis were observed with the plasma membrane damaged, though many organelles and nuclei appeared normal (Figure 4D). At 48 and 72 hours (Figure 4E), the cells almost appeared normal, but there still was some cellular edema. In the ascorbic acid group, slight cellular edema was observed, but most of the cells appeared almost normal (Figure 4F and G).

The results of the lipid peroxide assays are shown in Figure 5. In the ultrasound group, lipid peroxide levels at 12 to 24 hours were significantly higher than those seen at 15 minutes to 6 hours (P < .05). Because corneal endothelial cells appeared almost normal histologically in the BSS and the ascorbic acid groups, we did not measure the lipid peroxide levels in these groups.

The results of this study clearly demonstrated that 1 of the causes of corneal endothelial damage by ultrasound is free radicals. One of the most commonly used markers for DNA damage by oxidative stress is 8-OHdG.^13- 17 In our study, immunohistochemistry clearly demonstrated that 8-OHdG–positive endothelial cells were only found in the ultrasound group, which indicates that the induced DNA damage of the endothelial cells after ultrasound oscillation in the anterior chamber occurred because of free radicals. Also, because TUNEL-positive cells were only detected in the ultrasound group at times similar to those in the 8-OHdG–positive cells, we inferred an association between these 2 phenomena. Several studies have shown that oxidative stress can induce apoptosis in many kinds of cells.^18- 22 The coincidence noted thereby strongly suggests that the apoptosis was induced by the oxidative damage caused by the free radicals.

The most important targets of free radicals are unit membranes, eg, plasma, mitochondrial, rough endoplasmic reticulum, and nuclear membranes. They can also act on DNA. Free radicals initiate toxic reactions at the plasma membrane and lead to cellular edema or necrosis.^10,23- 24 In our study, scanning electron microscopy showed alterations on the surface of the corneal endothelial cells while transmission electron microscopy demonstrated alterations in the fine structure of the cells. The cellular edema and necrotic cells with plasma membrane damage, swollen organelles, and absence of heterochromatin in the nuclei that were observed at the early stage (15 minutes to 6 hours) in the ultrasound group suggest that the free radicals produced by the ultrasound oscillation induced direct damage of the plasma membranes.

Cellular atrophy and necrosis were also found ultrastructurally in the late stage (12-24 hours) in the ultrasound group. In addition, 8-OHdG–positive cells were present only at 24 hours. Although typical apoptotic cells were not detected in the ultrastructural observations, TUNEL-positive cells were present immunohistologically at times similar to those seen for 8-OHdG. Thus, the alterations of the cells at the late stage were different from those at the early stage, indicating that there was a 2-phase pattern for injury in the ultrasound group. From in vivo studies, Pan and Sato²⁵ reported that mild damage appeared in endothelial cells in the aorta during the early stage after hydrogen peroxide administration, with more extensive damage seen in the late stage. Zhang et al²⁶ and Peters et al²⁷ have reported that oxygen free radicals contributed to the ischemia/reperfusion damage in a 2-phase pattern in vivo. Their results suggest that the tissue damage induced by transient free radicals occurs in a 2-phase pattern, which supports our results.

The unsaturated bonds of cholesterol and fatty acids in the membranes can readily react with free radicals and undergo peroxidation. This process can become autocatalytic after initiation and will yield lipid peroxides. The loss of the fatty acids of cellular membranes, the formation of lipid peroxides, plus the uptake of oxygen by lipid-containing structures all suggest that peroxidation occurs. Thus, this leads to lipid peroxide–caused peroxidation once again, which enhances the cellular disorder.^10,23,28 Lipid peroxides have a sufficient lifetime, which means that they can migrate and damage other cellular components, including DNA, apart from the membranes.²⁹

The lipid peroxide levels at the later stage were higher than those at the early stage. In addition, 8-OHdG–positive cells, TUNEL-positive cells, and necrosis were detected in the later stage in the ultrasound group. These findings suggest that the latter phase of the corneal endothelial damage may be caused by the effects of lipid peroxides. Necrosis in the late stage may occur in the cells that have been damaged previously by lipid peroxides and at a time when the cellular edema from the early stage has yet to completely heal.

Some investigators have reported on the effect of the oxidative stress on the corneal endothelium by electron microscopic observations. Injection of hydrogen peroxide into the anterior chamber in vivo can cause swelling of the cytoplasm, disruption of the organelles in the corneal endothelial cells,³⁰ shrinkage of the cells, and disruption of the intercellular junctions of the Descemet membrane.³¹ Riboflavin-UV-A–mediated exposure in vivo can induce chromatin condensation, apoptotic bodies, and shrinkage of endothelial cells.³² Reactive oxygen species associated with photosensitizers can induce apoptosis and necrosis in the corneal endothelium in vitro.³³ However, to the best of our knowledge, there have not been any studies that have investigated the oxidative injury at different times, which would indicate that there is a 2-phase pattern for the damage in the corneal endothelial cells.

Ascorbic acid is a well-established antioxidant. Rubowitz et al⁶ have performed ultrasound oscillation in the anterior chambers of rabbit eyes while irrigating the chamber with a BSS of 0.001 M ascorbic acid. Their specular microscopic and histopathologic observations showed a 70% decrease in the number of corneal endothelial cell loss with irrigation with BSS containing 0.001 M of ascorbic acid compared with BSS-only irrigation. Their findings demonstrated that ultrasound oscillation in the anterior chamber can cause endothelial damages through free radical formation by using ascorbic acid as a scavenger. Because of their findings and because the natural concentration of ascorbic acid in human vitreous is approximately 0.001 M, we used the same concentration of ascorbic acid in the irrigating solution.³⁴ In the ascorbic acid group, 8-OHdG–positive cells and apoptotic cells were not detected, and most of the cells were ultrastructurally normal at all times. These results not only support the findings of Rubowitz et al⁶ but are in fact due to the oxidative insult by the free radicals. In addition, our results suggest that the anti–free radical effects of the glutathione in BSS are not sufficient but that ascorbic acid (0.001 M) can counteract the oxidative insult by the ultrasound-induced free radicals. Earlier,³⁵ we demonstrated by electron spin resonance analysis that ophthalmic visosurgical devices inhibit the effects of free radical formation. Therefore, clinicians should pay more attention to the retention of ophthalmic visosurgical devices in the anterior chamber during ultrasound oscillation.

In conclusion, we examined the corneal endothelial cell damage associated with ultrasound oscillation in the anterior chamber. The appearance of 8-OHdG–positive cells and the 2-phase pattern of damage indicate that the corneal endothelial cell can be damaged by transient production of free radicals. While other clinical insults—such as trauma by the impact of lens nuclear fragments, heat injury, air bubbles, or turbulence of the solution—can occur, clinicians should be aware of the effects of free radicals, as the oxidative stress can cause lethal damage to corneal endothelial cells.

Correspondence: Hiroshi Takahashi, MD, PhD, Department of Ophthalmology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan (tash@nms.ac.jp).

Submitted for Publication: August 27, 2007; final revision received November 8, 2007; accepted November 28, 2007.

Author Contributions: All of the authors had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Financial Disclosure: None reported.