Purpose. To provide evidence that free radical damage is a component of postischemic retinal injury; to determine whether antioxidant enzymes, superoxide ...
Protection of Rabbit Retina from Ischemic Injury by Superoxide Dismutase and Catalase •
Mala S. Nayak, Mihori Kita, and Michael F. Marmot
Purpose. To provide evidence that free radical damage is a component of postischemic retinal injury; to determine whether antioxidant enzymes, superoxide dismutase (SOD) and catalase, can protect the retina from ischemic injury. Methods. Total retinal ischemia for 60 or 75 min was produced in Dutch rabbits by raising intraocular pressure. Retinal recovery was monitored with the electroretinogram. Enzymes were administered as an intravenous bolus dose 2-3 min before restoration of circulation. Results. In eyes subjected to 60 min ischemia, the amplitude of the a-wave 4 hours after reperfusion averaged 114.9% of baseline value in control rabbits and 126.5% in SOD-treated animals. The b-wave amplitude at this time was 79.3% and 106.8% in control rabbits and SOD-treated rabbits, respectively. After an ischemic insult of 75 min, at 4 hours the a-wave amplitude was 89.2% of baseline in control eyes, 108.8% in SOD-treated eyes, 159.6% in eyes that received a combination of SOD and catalase, and 149.8% in catalase-treated eyes. The amplitude of the b-wave was reduced to 47.8% in control eyes and 44.8% in SOD-treated eyes, but recovered to 92.3% in rabbits that received the combination therapy and 98.8% in animals that received catalase alone. Conclusions. These findings suggest that free radical generation is involved in ischemic tissue damage. The fact that antioxidant enzymes can be protective has implications for the treatment of acute ischemic diseases of the retina. Invest Ophthalmol Vis Sci 1993; 34:2018-2022.
v-Jxygen-derived free radicals, such as superoxide and the more reactive hydroxyl radical, are believed to be responsible for a large part of the damage that occurs after an ischemic insult.12 Once generated they cause extensive tissue damage directly and indirectly by causing the release of excitatory amino acids (EAA).3 Tissues are normally protected from oxidative damage by the presence of enzymes such as superoxide dismutase (SOD), that brings about the dismuta-
Frmn the Department of Ophthalmology, Stanford University School of Medicine, Stanford, California. Supported in part by NIH-NEI Research Grant EY-0167S (MFM) and the Delta Gamma FidUnuship Fund (MSN). Submitted for publication March 13, 1992; accepted August 20, 1992. Proprietary interest Category: N Reprint requests: Michael F. Mannor, Department of Ophthalmology, Stanford University School of Medicine, Stanford, CA 94305-530S.
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tion of superoxide radicals, and catalase, that removes the hydrogen peroxide formed as a consequence.2 However, after ischemia the load of free radicals can exceed the defensive capacity of cells and lead to cell death. SOD, alone or in combination with catalase, has been found in experiments to be effective in renal4 and myocardial ischemia,5>6>7 in preventing an increase in vascular permeability in the lung after air embolism,8 or in the intestine after vascular hypotension,9 and in improving the survival of acute island skin flaps.10 The aim of our experiments was to determine the efficacy of SOD and catalase in preventing ischemic damage to the retina in rabbits, using the scotopic electroretinogram (ERG) as an in vivo measure of the physiologic viability of the photoreceptors and inner retinal neurons. Investigative Ophthalmology & Visual Science, May 1993, Vol. 34, No. 6 Copyright © Association for Research in Vision and Ophthalmology
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Protection from Retinal Ischemia b wave recovery 60 minutes ischemia
MATERIALS AND METHODS All experiments adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Experiments were performed on 46 pigmented Dutch rabbits weighing 1.2-1.8 kg. The rabbits were pretreated with acepromazine maleate (2 mg/kg) and xylazine hydrochloride (2 mg/kg) administered intramuscularly, and anesthetized with an intraperitoneal injection of urethane (1 g/kg). The pupils were dilated with 1% atropine sulfate and 10% phenylephrine hydrochloride, and the rabbits allowed to adapt to the dark for 40 min before a baseline ERG was recorded. We produced retinal ischemia by elevating the intraocular pressure." 12 Under a red light, the anterior chamber in both eyes was cannulated. The cannula consisted of a 2.5-inch heparinized polythene tube (internal diameter 0.030 inch, outer diameter 0.048 inch), attached to a 21-gauge needle, that was connected to a saline bag, the height of which could be adjusted depending on the intraocular pressure desired. A 24-gauge catheter was inserted in the marginal ear vein and Hushed with heparinized saline to maintain patency. The rabbit was again allowed to adapt to the dark for 40 min with the saline bag adjusted so that normal intraocular pressure was maintained, after which an ERG was recorded. The intraocular pressure in one of the eyes was rapidly increased by raising the saline bag to obtain a pressure of 150 mm of Hg. A flat ERG and blanching of the fundus vessels on ophthalmoscopy confirmed retinal ischemia. Throughout the experiment normal intraocular pressure was maintained in the contralata wave recovery 60 minutes ischemia 180170160" 150140130120110100 •; 908070605040-; 3020 0
180170 J 160 150 140 130 120 110 100 90 80 70 60
SOD (n-8) Controls (n>9)
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Reperfusion time (mins)
FIGURE 2. Effect of superoxide dismutase on the amplitude of the b-wave after 60 min ischemia. The data are represented as in Figure I.
eral eye, which served as a control to monitor the general condition of the rabbit. Superoxide dismutase 6.5 mg/kg (3000 U/mg, Sigma Chemical Co, St. Louis, MO) or catalase 5 mg/ kg (sold as 2000-5000 U/mg, Sigma), were prepared in 3 ml normal saline, and injected as an intravenous bolus 2-3 min before releasing ischemia. At the end of the ischemic period the saline bagwas lowered to normalize the intraocular pressure. During reperfusion ERGs were recorded in both eyes every 15 min during the first hour and hourly thereafter for 4 hours. To record ERGs the rabbit was positioned in a box, with a photostimulator lamp (Model PS 22, Grass Medical Instruments, Quincy, MA) placed 15 cm in front of each eye and used at the maximum intensity setting. Monopolar ERG jet electrodes (Universo SA, Switzerland) were placed on each cornea and a silver wire in the conjunctival cul de sac served as a reference. The signals were differentially amplified and displayed on a storage oscilloscope. RESULTS
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Reperfusion time (mins)
FIGURE l. Effect of superoxicle dismutase on the amplitude of the a-vvave after 60 min ischemia. The amplitude of the a-wave is expressed as a percentage of the preischemic postcannulation value to give % recovery. Data points are mean ± SEM. Recovery during the first hour followed the same pattern as subsequent recovery and for clarity these points were not included in the figure.
The average postcannulation preischemic amplitudes of the a- and b-waves were 113 ± 32 and 312 ± 78 /xV (±SD) respectively. Because of the variation between animals we have expressed the recovery data as a percentage of this baseline preischemic control value in each animal. The recovery patterns after 60 min ischemia are shown in Figures 1 and 2. Intravenous SOD enhanced the recovery of both a- and b-waves, though its effect on the b-wave was more marked. The amplitude of the a-wave 4 hr after reperfusion was 114.9 ± 12.9% in control rabbits and 126.5 ± 8.1% in SOD-treated rabbits (not statistically significant). The improvement in
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Investigative Ophthalmology 8c Visual Science, May 1993, Vol. 34, No. 6
the b-vvave after treatment with SOD was statistically significant (P < 0.02) at all points 1-4 hours after reperfusion. The b-wave amplitude 4 hours after the commencement of reperfusion was 106.8 ± 6.0% with SOD treatment in contrast to 79.3 ± 6.7% in untreated rabbits. Because ERG recovery after 60 min of ischemia was relatively good in the control eyes, we also tested the effects of antioxidant therapy after a 75 min ischemic insult. These results are seen in Figures 3 and 4. The recovery in control eyes was much less after 75 min than after 60 min of ischemia. After the longer ischemic insult, SOD enhanced a-vvave recovery only slightly (not statistically significant) and had no effect on the b-wave. However, the combination of SOD and catalase had a dramatic protective effect as did catalase alone. The a wave 4 hours after reperfusion was 159.6 ± 1 2.7% in rabbits that were treated with a combination of SOD and catalase, and 149.8 ± 11.5% in catalase-treated rabbits in contrast to 89.2 ± 6.8% in control eyes. Both effects were statistically significant at 1-4 hours (P £ 0.02). The b-wave recovered to 92.3 ± 7.7% of baseline in rabbits that received the combination therapy and 98.8 ± 11.8% in rabbits that received catalase alone relative to 47.8 ± 2.2% in control eyes. These effects were also statistically significant (P 6)
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Reperfusion time (mins)
4. Effect of superoxide dismutase, superoxide dismutase + catalase and catalase alone on the amplitude of the b-wave after 75 min ischemia. The data are represented as in Figure 1. FIGURE
are generated in excess after an acute ischemic attack.12 They are formed in the postischemic period when the circulation is re-established and molecular oxygen is brought to the tissues. Superoxide radicals are typically generated by the reaction: Xanthine + H9O + 2O.;
xanthine oxidase Uric acid + 2O9 ~ + 2H +
SOD, a protective enzyme, ubiquitously distributed in animal cells, reduces the toxicity of these superoxide free radicals by bringing about their dismutation: 2O 2 " + 2H +
SOD
• O2 + H 2 O 2
However, the hydrogen peroxide formed during the removal of superoxide radicals is capable of tissue damage either directly, as it is an oxidant with good diffusional ability, or by its reduction to form the hydroxyl radical. Accumulation of hydrogen peroxide in tissues is prevented by intrinsic enzyme systems such as catalase and various peroxidases, which reduce peroxide to water:
100 i Controls (n-12)
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2H 2 O 2
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2H2O + O2
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:
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FIGURE 3. Effect, of superoxide disinutase, superoxide disinutase + catalase and caialase alone on the amplitude of the a-wave after 75 min ischemia. The data are represented as in Figure I.
The suppression of the ERG seen after ischemia may be a combination of direct effects of ischemia (breakdown of ATP, loss of sodium pump activity, and resultant disturbance of ionic balance), and indirect effects involving the generation of oxygen-free radicals during the reperfusion stage. Free radicals are, by virtue of their powerful oxidizing properties, capable of causing extensive tissue injury. They cause peroxidation of lipids in the cell membrane, modification of
Protection from Retinal Ischemia
proteins and nucleic acids, and changes in cellular calcium homeostasis.2 It was reported recently that, in addition to causing tissue damage directly, free radicals are also responsible for stimulating the release of EAA.3 These amino acids combine with specific receptors on the cell membrane to increase its permeability resulting in an increase in the influx of ions such as sodium and calcium into the cell.13 The increase in the intracellular calcium concentration that occurs after free radical release therefore may either be caused by the free radicals themselves or may occur via EAA. Calcium stimulates a host of intracellular lytic enzymes and xanthine oxidase. A vicious circle is thereby set up because the latter stimulates the release of more free radicals. The combined effects of free radical release, osmotic swelling of the cell (caused by the influx of sodium ions into the cell), and activation of lytic enzymes lead to severe damage to the cell and ultimately to cell death. Damage to tissues by oxygen-derived free radicals is normally minimized by tissue enzymes such as SOD and catalase. However, the endogenous stores of these enzymes may be unable to cope with the flood of free radicals generated after a period of ischemia. In this study we have examined whether exogenously administered SOD and catalase could enhance resistance to retinal ischemia. We found that SOD enhanced both a- and b-wave recovery after 60 min of ischemia. After an ischemic insult of 75 min, it improved the a-wave only minimally and did not have any effect on the b-wave. It is possible that as the duration of ischemia increases, the production of superoxide radicals and the damage to tissues caused by them exceeds the ability of SOD administered in our dose levels to counter the effects. Because catalase was useful at 75 min it appears that hydrogen peroxide, either by itself or by reduction to the hydroxyl radical, is a source of considerable damage to ischemic tissue. The a-wave reflects photoreceptor activity, whereas the b-wave reflects neuronal activity in the inner retina. We observed that in many experiments (both control eyes and treated eyes) the amplitude of the a-wave during the recovery period exceeded the preischemic baseline values. This phenomenon was observed only to a minor degree with the b-wave (after antioxidant therapy). Slower and incomplete recovery of the b-wave relative to a hyper-response in the awave has been observed previously12 and may reflect a greater sensitivity of inner retinal neurons to oxygenfree radical damage, or the greater proximity of the photoreceptors to the choroidal blood flow. The different degrees to which antioxidants affected the aand b-wave responses suggests that both photoreceptors and inner retinal neurons may be susceptible to oxidative reperfusion injury and possible therapeutic rescue.
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An intriguing question raised by these experiments involves the exact site of action of these enzymes in the postischemic period. Oxygen-derived free radicals are highly reactive, and once generated they react almost immediately with cellular lipids and proteins.14 Conversely, neither SOD nor catalase can penetrate across cell membranes.15 How are these enzymes effective in ischemia if they are unable to gain access to the cell? One possible explanation is that the reactive oxygen species may be generated primarily within the vasculature where the protective enzymes are available. It has been demonstrated that endothelial cells are susceptible to damage by free radicals16 and ERG changes could result in part from fluid (and ionic) shifts that follow endothelial blood-retina barrier damage. The retinal vasculature is unlikely to be of relevance in the rabbit, because there is so little of it, but choriocapillary damage and injury to the RPE barrier would lead to changes in the subretinal space. SOD and catalase have some theoretical advantages over dextromethorphan, which has been shown to be effective in protecting the retina against ischemic injury.17 Dextromethorphan is an N-methyl-D-aspartate receptor antagonist that blocks the EAA receptor sites on the cell membrane. However, because oxygenderived free radicals cause the release of EAA, SOD and catalase, which deactivate free radicals, may prevent their release. Another advantage of SOD and catalase is their ease of administration (a single intravenous injection was adequate in our experiments), in contrast to dextromethorphan which was given as an intravenous infusion. These enzymes also appear to be relatively safe. SOD possesses anti-inflammatory activity,1819 and has been used in veterinary practice in the management of arthritis,2021 in clinical trials in humans in degenerative joint diseases,2223 and radiation cystitis in patients with carcinoma of the cervix.2425 There are less data on the safety of catalase, but no local or systemic toxicity was observed during our experiments. The ERG in the opposite nonischemic eye remained normal during the course of the experiment. The ischemic eye appeared normal except for corneal edema that was probably caused by the prolonged elevation in intraocular pressure. The use of these enzymes in clinical practice will depend on further studies. During these experiments we monitored ERG recovery for only 4 hours after ischemia and it remains to be shown whether the difference between treated and untreated eyes will be maintained weeks or months after the ischemic insult. A possible limitation to the clinical use of these enzymes relates to their pharmacokinetic properties. Both SOD and catalase are rapidly cleared by the kidneys leading to short half lives after intravenous injection, which vary from 6-10 min in rats15'26 to 25 min in humans.23 The half-life is longer after intramuscular and subcutaneous injection. A short half-life might
Protection from Retinal Ischemia limit their use in clinical practice if free radicals continue to be released or remain active for hours after an ischemic episode. However, because the enzymes were effective in our experiments, it is possible that oxygenfree radicals are generated very rapidly after the onset of reperfusion so that the enzymes need only be present in the tissues during this phase of massive free radical production. Of course this scenario would reduce the value of therapy severely unless drugs could be given immediately during or after a vascular occlusion, which will rarely be possible clinically. Key Words retina, ischemia, oxygen-derived free radicals, superoxide dismutase, catalase References 1. McCord JM. Oxygen-derived free radicals in postischemic tissue injury. N EnglJ Med. 1 985;312:159-163. 2. Ernster L: Biochemistry of reoxygenation injury. Crit Care Med. 1988; 16:947-953. 3. Pellegrini-Giampietro DE, Cherici G, Alesiani M, Caria V, Moroni F. Excitatory amino acid release and free radical formation may cooperate in the genesis of ischemia-induced neuronal damage. J Neurosci. 1990; 10:1035-1041. 4. Baker CL, Cony RJ, Autor AP. Oxygen free radical induced damage in kidneys subjected to warm ischemia and reperfusion. Ann Surg. 1985;202:628-64]. 5. Shlafer M, Kane PF, Kirsh MM. Superoxide dismutase plus catalase enhances the efficacy of hypothermic cardioplegia to protect the globally ischemic, reperfused heart. J Thome Cardiovasc Surg. 1982; 83:830839. 6. Jolly SR, Kane WJ, Bailie MB, Abrams CD, Lucchesi BR: Canine myocardial reperfusion injury. Its reduction by the combined administration of superoxide dismutase and catalase. Circ Res. 1984;54:277-285. 7. Gross GJ, Farber NE, Hardman HF, Warltier DC. Beneficial actions of superoxide dismutase and catalase in stunned myocardium of dogs. Am J Physiol. 1986; 250:H372-H377. 8. Flick MR, Hoeffel J, Staub NC. Superoxide dismutase prevents increased lung vascular permeability after air emboli in unanesthetized sheep. Fed Proc. 1981; 40:405. 9. Parks DA, Bulkley GB, Granger DN, Hamilton SR, McCord JM. Ischemic injury in the cat small intestine: role of superoxide radicals. Gastroenterology. 1982; 82:9-15. 10. Im MJ, Manson PN, Bulkley GB, Hoopes JE. Effects of superoxide dismutase and allopurinol on the survival of acute island skin flaps. Ann Surg. 1985; 201:357-359. 1 1. Popp C. Die retinafunktion nach intraocularer is-
2022 chamie. Albrecht von Graefes Archivfur Ophlhalmologie.
1955; 156:395-403. 12. Foulds WS, Johnson NF. Rabbit ERG during recovery from induced ischemia. Trans Ophthal Soc UK. 1974;94:383-393. 13. Rothman SM, Olney JW. Glutamate and the pathophysiology of hypoxic- ischemic brain damage. Ann Neurol. 1986; 19:105-1 11. 14. Hutchinson F. The distance that a radical formed by ionizing radiation can diffuse in a yeast cell. Radial Res. 1957; 7:473-483. 15. BeckmanJS, Minor RL, White CW, Repine JE, Rosen GM, Freeman BA. Superoxide dismutase and catalase conjugated to polythene glycol increases endothelial enzyme activity and oxidant resistance. / Biol Chem. 1988;263:6884-6892. .16. Bishop CT, Mirza Z, Crapo JD, Freeman BA. Free radical damage to cultured porcine aortic endothelial cells and lung fibroblasts: modulation by culture conditions. In Vitro Cell Dev Biol. 1985; 21:229-236. 17. Yoon YH, Marmor MF: Dextromethorphan protects retina against ischemic injury in vivo. Arch Ophthalmol. 1989; 107:409-411. 18. McCord JM, Wong K, Stokes SH, Petrone WF, English DK. A mechanism for the anti-inflammatory activity of superoxide dismutase. In: Autor AP, ed. Pathology of Oxygen. New York: Academic Press; 1982:75-83. 19. Petrone WF, English DK, Wong K, McCord JM. Free radicals and inflammation: superoxide-dependant activation of a neutrophil chemotactic factor in plasma. Proc Nail, Acad Sci USA. 1980; 77:1159-1163. 20. Decker WE, Edmundson AH, Hill HE, Holmes RA, Padmore CL, Warren HH, Wood WC. Local administration of orgotein in horses. Modern Veterinary Practice. 1974; 55:773-774. 21. Ahlengard S, Tufvesson G, Petterson H, Anderson T: Treatment of traumatic arthritis in the horse with intra-articular orgotein Palosein). British Equine Veterinary Journal. 1978; 10:122-124. 22. Lund-Olsen K, Menander KB. Orgotein: A new antiinflammatory metallo- protein drug: preliminary evaluation of clinical efficacy and safety in degenerative joint disease. Curr Ther Res. 1974; 16:706-71 7. 23. Huber W, Menander-Huber KB. Orgotein. Clinics in Rheumatic Diseases. 1980; 6:465-498. 24. Marberger H, Bartsch G, Huber W, Menander KB, Schulte TL: Orgotein: a new drug for the treatment of radiation cystitis. Curr Ther Res. 1975; 18:466-475. 25. Edsmyr F, Huber W, Menander KB. Orgotein efficacy in ameliorating side effects due to radiation therapy. I. double-blind, placebo controlled trial in patients with bladder tumors. Cwr Ther Res. 1976; 19:198211. 26. Pyatak PS, Abuchowski A, Davis FF. Preparation of a polythene glycol: superoxide dismutase adduct, and an examination of its blood circulating life and anti-inflammatory activity. Res Commun Chem Pathol Pharma-
col. 1980;29:l 13-127.
