- Email: [email protected]
Protective effect of ischemic preconditioning on retinal ischemia-reperfusion injury in rats
BASIC SCIENCE STUDY
Protective effect of ischemic preconditioning on retinal ischemia-reperfusion injury in rats Davut Ozbay, * MD; Serap Ozden, * MD; Sevda Mtifttioglu, t MD; Figen Kaymaz,t MD; Volkan Yaylah,* MD; Cern Yildmm,* MD; Sinan Tathpmar,* MD ABSTRACT • RESUME Background: A short period of ischemia can induce remarkable tissue resistance to the deleterious effects of subsequent ischemia and reperfusion. We performed a study to investigate the effect of ischemic preconditioning on retinal ischemiareperfusion injury in rats. Methods: Ten Wistar albino rats were divided into two groups of five animals (I 0 eyes): one group underwent 5 minutes of ischemic preconditioning (achieved by clamping the common carotid arteries at the time of vertebral artery cauterization), and the other did not (control group). In both groups, the vertebral arteries were occluded bilaterally with an electric needle coagulator under an operating microscope. Forty-eight hours later the rats were reanesthesized, and both common carotid arteries were clamped to interrupt blood flow. The duration of ischemia was 30 minutes. The clamp was then removed to enable reperfusion for 4 hours. The animals were killed by decapitation, and retinal sections were evaluated under light and electron microscopy. The signs of ischemia-reperfusion injury (cellular degeneration, vacuolization between retinal layers, increase in retinal thickness due to edema, mononuclear cell infiltration and apoptotic cell count) were recorded. Results: Light microscopy of retinal sections from rats in the ischemic preconditioning group showed a well-preserved retinal structure. The mean thickness values (and standard deviation [SD]) for the inner nuclear layer (I 04.0 IJm [2.54 IJm] vs. 49.0 IJm [I 0.83 IJm]) and inner plexiform layer ( 134.8 IJm [I 0.13 IJm] vs. 88.5 IJm [17.46 IJm]) were significantly higher in the control group than in the preconditioning group (p =0.009), indicating increased retinal thickness in the former group due to tissue edema resulting from ischemia-reperfusion injury. The mean mononuclear cell count (6.67 [SD 1.97] vs. 2.5 [SD 1.0]) and apoptotic cell count (18.2 [SD 5.7] vs. 5.3 [SD 1.0]) were significantly higher in the control group than in the preconditioning group (p = 0.002), indicating an inhibitory effect of ischemic preconditioning on leukocyte infiltration and apoptotic cell death.
Interpretation: Ischemic preconditioning attenuated ischemia-reperfusion injury in the rat retina. From *the Department of Ophthalmology, Pamukkale University Medical Faculty, Denizli, Turkey, and tthe Department of Histology and Embryology, Hacettepe University Medical Faculty, Ankara, Turkey
Presented at the 4th International Glaucoma Symposium, Barcelona, Mar. 19-22, 2003 Correspondence to: Prof. Dr. Serap Ozden, P.K. 185, 20003 Denizli, Turkey; fax +90 258 2410040, [email protected]
Originally received Oct. 23, 2003
This article has been peer-reviewed.
Accepted for publication July 6, 2004
Can j Ophthalmol 2004;39:727-32
Ischemic preconditioning-Ozbay et al
727
Ischemic preconditioning-O zbay et al
Contexte : Une breve periode d'ischemie peut induire une remarquable resistance tissulaire aux effets nocifs d'une ischemie et d'une reperfusion subsequentes. Nous avons etudie les effets du preconditionnement ischemique sur Ia blessure de l'ischemie-reperfusion retinienne chez les rats. Methodes : Dix rats albinos Wistar ont ete repartis en deux groupes de cinq (dix yeux) : un groupe a ete Soumis a 5 minutes de preconditionnement ischemique (par clampage des arteres carotides communes lors d'une cauterisation de l'artere vertebrale); l'autre, laisse intact, a servi de temoin. Chez les deux groupes, les arteres vertebrales ont ete obliterees bilateralement avec un coagulateur electrique (aiguille), sous microscope operatoire. Quarante-huit heures apres, les rats ont ete anesthesies de nouveau et les deux arteres carotides communes ont ete clampees pour interrompre le flot sanguin. L'ischemie a dure 30 minutes. On a ensuite retire Ia clampe pour permettre Ia reperfusion pendant 4 heures. Les animaux ont ete decapites et les sections de Ia retine, evaluees sous microscopie classique et electronique. Les signes de blessure de l'ischemie-reperfusion (degenerescence cellulaire, vacuolisation entre les couches retiniennes, accroissement de l'epaisseur de Ia retine dG a l'redeme, infiltration des cellules mononucleaires et decompte des cellules apoptotiques) ont ete notes. Resultats : La microscopie classique des sections retiniennes chez les rats du groupe de preconditionnement ischemique a montre une structure retinienne bien conservee. L'epaisseur moyenne (et ecart type [ET]) de Ia couche nucleaire interne (104,0 IJm [2,54 IJm] c. 49,0 IJm [10,83 IJm]) et celle de Ia couche plexiforme interne (134,8 IJm [10,13 IJm] c. 88,5 IJm [17,46 1Jm]) etaient beaucoup plus grandes chez le groupe temoin que chez le groupe de preconditionnement (p = 0,009), ce qui indique une plus forte augmentation de l'epaisseur de Ia retine chez le premier groupe due a l'redeme tissulaire consecutif a Ia blessure causee par l'ischemie-reperfusion. Lecompte moyen des cellules mononucleaires (6,67 [ET I,97] c. 2,5 [ET I ,0]) et celui des cellules apoptotiques ( 18,2 [ET 5,7] c. 5,3 [ET I ,0]) etaient significativement superieurs dans le groupe temoin (p = 0,002), ce qui indique l'effet inhibiteur du preconditionnement ischemique sur !'infiltration des leukocytes et Ia mort des cellules apoptotiques. Interpretation : Le preconditionneme nt ischemique a attenue Ia blessure de l'ischemie-reperfu sion dans Ia retine des rats.
I
schemia is an important cause of tissue injury. Inadequate blood supply leads to tissue hypoxia and accumulation of metabolic waste products, which may cause cellular damage. The phenomenon of worsening tissue injury after perfusion is called reperfusion injury. 1 Leukocytes are thought to play a major role in ischemia-reperfusion injury. 1•2 Production of oxygen free radicals has also been reported to be an important factor in the pathophysiology of this condition. 1•3 A short period of ischemia can induce remarkable tissue resistance to the deleterious effects of subsequent prolonged ischemia and reperfusion. This phenomenon, known as ischemic preconditioning,4 was first demonstrated in canine myocardium. 5 Although the exact mechanisms underlying ischemic preconditioning are unknown, release of adenosine, de novo
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protein synthesis and opening of adenosine triphosphate-sensitive potassium channels have been proposed as possible factors. 4·6-8 The purpose of this study was to investigate the effect of ischemic preconditioning on retinal ischemiaperfusion injury in rats. METHODS
The study was carried out on 10 Wistar albino rats (20 eyes) weighing 250-350 g each, obtained from the Hifzisihha Institute Animal Laboratory, Ankara, Turkey. The rats were housed in wire-bottomed cages at room temperature with a 12-hour light/dark cycle. All animals were fed standard rat chow and were given only water for 12 hours before surgery. The care
Ischemic preconditioning-Ozbay et al
and handling .of the animals were in accordance with the guidelines of the US National Institutes of Health. The rats were divided into two groups of five animals (10 eyes): one group underwent 5 minutes of ischemic preconditioning (achieved by clamping the common carotid arteries at the time of vertebral artery cauterization), and the other did not (control group). The surgical procedure was identical in the two groups. Surgical procedure We used the four-vessel occlusion method9•10 to induce retinal ischemia. The rats were anesthesized with an intramuscular injection of ketamine hydrochloride, 50 mg/kg (Ketalar, Pfizer Canada Inc., Kirkland, Que.), and xylazine, 10 mglk:g (Rompun, Bayer, Leverkusen, Germany). Following skin shaving and preparation with 10% povidone-iodine solution, a 2-cm incision was made behind the occipital bone directly overlying the first two cervical vertebrae. The vertebral arteries were occluded bilaterally by coagulation of the first cervical vertebra with an electric needle coagulator through the alar foramina under an operating microscope. The incision was closed with interrupted 4-0 silk sutures. Forty-eight hours after the initial procedure, the rats were anesthesized as previously, and a 3-cm midline cervical incision was made. Both common carotid arteries were exposed, and a nontraumatic microvascular clamp was placed around either artery to interrupt the blood flow. The absence of blood flow in the retina was monitored by direct ophthalmoscopy. The carotid artery clipping time (i.e., duration of ischemia) was 30 minutes. The clamp was then removed and the eye allowed to reperfuse for 4 hours. Retinal reperfusion was confirmed by direct ophthalmoscopy. The cervical incision was closed with interrupted 4-0 silk sutures. At the end of the reperfusion period the animals were killed by decapitation, and the eyeballs were enucleated and fixed in 2.5% glutaraldehyde solution for histologic study. Histopathological evaluation Tissue samples were fixed by immersion in 2.5% glutaraldehyde in phosphate buffer and postfixed in 1% osmium tetroxide in the same buffer. After dehydration in ethanol gradients at room temperature, the tissue samples were embedded in araldite. Semithin
(1 J.iffi) and thin (70 nm) sections were cut, examined and photographed under a light microscope (Olympus BH2, Olympus America Inc., Melville, NY) and electron microscope (Zeiss EM9-S2, Carl Zeiss, Inc., Thornwood, NY) respectively. Semithin sections were stained with methylene blue-azure II, and ultrathin sections were stained with uranyl acetate and lead citrate. We recorded the signs of ischemia-reperfusion injury at the microscopic level (i.e., cellular degeneration, vacuolization between retinal layers, increase in retinal thickness due to edema, mononuclear cell infiltration and cell apoptosis) in the two groups. The thickness of the inner plexiform layer, inner nuclear layer and ganglion cell layer was measured by means of a compass on the photographs obtained with the light microscope. We calculated mononuclear and apoptotic cell counts as the mean value for three different regions under 40x magnification. Statistical analysis We performed statistical analysis using the MannWhitney U test. A p value less than 0.05 was accepted as statistically significant. RESULTS
In retinal tissue samples obtained from rats in the control group, light microscopy showed vacuolization due to edema in the outer nuclear layer and between the inner and outer segments of photoreceptors (Fig. 1). Prominent erythrocyte infiltration was also noted between the nuclei of the outer nuclear layer. Increased thickness of the outer plexiform layer due to edema and vacuolization in some ganglion cells within the ganglion cell layer were also observed. Mononuclear cell infiltration was present and was prominent in the ganglion cell layer. In the ischemic preconditioning group, light microscopy of semithin retinal sections showed a well-preserved retinal structure (Fig. 1). The retinal layer most affected was the ganglion cell layer. Vacuolar degeneration within the cytoplasm of ganglion cells was noted in some areas. Within the inner nuclear layer, an increase in the distance between nuclei was observed in some parts of the retina, indicating edema and mononuclear cell infiltration. Some vacuolization was noted in the inner plexiform layer; however, an increase in the thickness of this layer and mononuclear
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'
Fig. 1-Light photomicrographs of retinal sections from rats that received retinal ischemia-reperfusion injury with (preconditioning group) or without (control group) ischemic preconditioning. Left: Control group. Edema in retinal layers and infiltrative cells in ganglion cell layer, inner plexiform layer (IPL) and inner nuclear layer (INL) were prominent. Vacuoles (V) were also seen in ganglion cells and INL. Right: Preconditioning group. Note less edema compared to control group. Thickness of outer plexiform layer (OPL) was almost normal (methylene blue-azure II; magnification x20).
Table 1-Mean thickness of retinal layers and mean cell counts in rats that received ischemia-reperfusion injury with (preconditioning group) or without (control group) ischemic preconditioning Mean (and standard deviation)
Variable Inner nuclear layer thickness, !Jill Inner plexiform layer thickness, 1-1m Ganglion cell layer thickness, !Jill Mononuclear cell count Apoptotic cell count
Control group (n = 10)
Preconditioning group (n = 10)
p value
I04.0 (2.54)
49.0 (I 0.83)
0.009
134.8 (10.13)
88.5 ( 17.46)
0.009
151.4 (20.7) 6.7 ( 1.97) 18.2 (5.7)
122.0 (36.15) 2.5 (1.0) 5.3 ( 1.0)
0.295 0.002 0.002
infiltration were not prominent. The cells in the outer nuclear layer were relatively well preserved, and there were considerably fewer erythrocytes than in the control group. The inner nuclear and inner plexiform layers (but not the ganglion cell layer) were significantly thicker in the control group than in the preconditioning group (p =0.009) (Table 1). These results indicate increased retinal thickness in the control group due to tissue edema resulting from ischemia-reperfusion injury. The mean mononuclear cell count and apoptotic cell count were significantly higher in the control
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group than in the ischemic preconditioning group (p 0.002) (Table 1). In the control group, electron microscopy showed edema between the axons of the inner plexiform layer. Vacuolization within cells, disruption between the intercellular adhesions and mononuclear cells were observed in the inner nuclear layer (Fig. 2). There was also vacuolization around the nuclei of the photoreceptors that form the outer nuclear layer as well as apoptotic cells. In the preconditioning group, electron microscopic evaluation revealed vacuolar swelling in the axons of the inner plexiform layer. Some of the cells within the
=
Ischemic preconditioning-Ozbay et al
Fig. 2-Eiectron photomicrographs. Left: Control group. Infiltrative lymphocytes (L) were seen near apoptotic cells (a) in inner nuclear layer. Vacuolar degeneration was prominent in amacrine cells. Right: Ischemic preconditioning group. Most cells and their intercellular junctions (IJ) in inner nuclear layer were intact. However, vacuolar changes were seen in one of the cells (uranyl acetate-lead citrate; magnification x6200).
inner nuclear layer showed cytoplasmic edema; however, most of the intercellular adhesions were intact at this layer (Fig. 2). Mononuclear cells were observed in the inner nuclear layer. INTERPRETATION
Retinal ischemia is thought to contribute to the pathophysiology of several retinal disorders, including glaucoma, retinal vascular occlusion and diabetic retinopathy. 11 Hence, several experimental models of ocular ischemia-reperfusion injury have been developed to investigate the histologic and biochemical changes in the retina. 2•9•10•12 The exact mechanisms of ischemic preconditioning are not fully understood. Recently, the role of adenosine has attracted considerable attention as a possible mediator of ischemic preconditioning. 2•6•13•14 Roth and colleagues 14 observed a significant increase in the adenosine concentration in the rat retina following ischemia. Moreover, it has been shown that adenosine, acting through Al and A2a receptors, is a critical component in the induction of ischemic tolerance after ischemic preconditioning. 6 Leukocytes are known to play an important role in ischemia-reperfusion injury. Activated leukocytes damage the endothelial and parenchymal cells through the production of cytotoxic mediators, including oxygen free radicals and inflammatory cytokines. 1 Recently, Nonaka and associates 2 demonstrated inhibitory
effects of ischemic preconditioning on leukocyte rolling and subsequent leukocyte accumulation during retinal ischemia-reperfusion injury. They also investigated the possible role of adenosine and found that administration of adenosine Al receptor antagonist immediately after ischemic preconditioning suppressed the beneficial effects of preconditioning on leukocyte behaviour. Administration of selective adenosine Al receptor agonist 1 hour before ischemia without preceding ischemic preconditioning had an effect on leukocyte rolling similar to that of ischemic preconditioning. These findings suggest that the inhibitory effects of ischemic preconditioning on leukocyte accumulation during retinal ischemia-reperfusion injury are mediated through the adenosine AI receptor. Adenosine is known to inhibit neutrophil aggregation, neutrophil adherence to endothelial cells and production of cytokine from monocytes. 15 In our study, mononuclear cell counts indicated that ischemic preconditioning significantly attenuated leukocyte accumulation. Roth and colleagues4 found that inhibition of protein synthesis by cycloheximide attenuated the protective effect of preconditioning in the rat retina and suggested that a transient alteration in protein expression was a factor in ischemic preconditioning. Recently, induction of heat shock protein 27 was reported to be associated with ischemic preconditioning in the rat retina. 7 Hence, the expression of cytoprotective proteins, such as heat shock protein, may be a neuroprotective event induced by ischemic preconditioning.
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Zhang and coworkers 16 reported that ischemic preconditioning diminished apoptotic cell death in the rat retina. Similarly, in our study, apoptotic cell counts were significantly lower in the ischemic preconditioning group than in the control group. This result indicates an inhibitory effect of the inner plexiform layer on leukocyte infiltration and apoptotic cell death. Casson and colleagues 17 recently reported that preexisting hyperglycemia and the intraocular delivery of glucose also attenuate ischemic retinal injury in rats. They proposed that this finding is largely due to the differences in energy metabolism between the brain and the retina: unlike the brain, the retina has the ability to maintain most of its adenosine triphosphate requirement in the absence of oxygen through anaerobic glycolysis. They suggested that elevated glucose levels in the vitreous of the hyperglycemic rats caused relative preservation of retinal adenosine triphosphate levels by anaerobic glycolysis during the ischemia, thereby decreasing the retinal injury. In conclusion, we found that ischemic preconditioning attenuated ischemia-reperfusion injury in the rat retina. Factors involved in ischemic preconditioning may be used as future novel therapeutic approaches for preventing retinal ischemic damage in humans. REFERENCES
1. Kurokawa T, Takagi H. Mechanisms and prevention of ischemia-reperfusion injury. Transplant Proc 1999;31: 1775-6. 2. NonakaA, Kiryu J, TsujikawaA, Yamashiro K, Nishijima K, Miyamoto K, et al. Inhibitory effect of ischemic preconditioning on leukocyte participation in retinal ischemia-reperfusion injury. Invest Ophthalmol Vis Sci 2001;42:2380--5. 3. Ozden S, Klldact B, Miiftiioglu S, Cakar N, Yildirim C. Effect of trimetazidine on retinal ischemia/reperfusion injury in rats. Ophthalmologica 2001;215:309-17. 4. Roth S, Li B, Rosenbaum PS, Gupta H, Goldstein IM, Maxwell KM, et al. Preconditioning provides complete protection against retinal ischemic injury in rats. Invest Ophthalmol Vis Sci 1998;39:777-85. 5. Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic
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myocardium. Circulation 1986;74:1124-36. 6. LiB, RothS. Retinal ischemic preconditioning in the rat: requirement for adenosine and repetitive induction. Invest Ophthalmol Vis Sci 1999;40:1200--16. 7. Li Y, RothS, Laser M, Ma JX, Crosson CE. Retinal preconditioning and the induction of heat-shock protein 27. Invest Ophthalmol Vis Sci 2003;44:1299-304. 8. Sakamoto K, Kuwagata M, Nakahara T, Ishii K. Late preconditioning in rat retina: involvement of adenosine and ATP-sensitive K(+) channel. Eur J Pharmacol2001;418: 89-93. 9. Pulsinelli WA, Brierley JB. A new model of bilateral hemispheric ischemia in the unanesthesized rat. Stroke 1979;10:267-72. 10. Sano Y, Kanematsu EA, Yoshiura M. Uric acid as biochemical marker for retinal and optic nerve damage after occlusion and reperfusion of common carotid and vertebral arteries in rat. Jpn J Ophthalmol1992;36:76-83. 11. Rosenbaum DM, Rosenbaum PS, Singh M, Gupta G, Gupta H, Li B, et al. Functional and morphologic comparison of two methods to produce transient retinal ischemia in the rat. J Neuroophthalmol2001;21:62-8. 12. Szabo ME, Droy-Lefaix MT, Doly M, Carre C, Braquet P. Ischemia and reperfusion-induced histologic changes in the rat retina: demonstration of free radical-mediated mechanism. Invest Ophthalmol Vis Sci 1991;32:1471-8. 13. Li B, Rosenbaum PS, Jennings NM, Maxwell KM, Roth S. Differing roles of adenosine receptor subtypes in retinal ischemia-reperfusion injury in the rat. Exp Eye Res 1999;68:9-17. 14. RothS, RosenbaumPS, Osinski J, Park SS, ToledanoAY, Li B, et al. Ischemia induces significant changes in purine nucleoside concentration in the retina-choroid in rats. Exp Eye Res 1997;65:771-9. 15. Harada N, Okajima K, Murakami K, Usune S, Sato C, Ohshima K, et al. Adenosine and selective A(2A) receptor agonists reduce ischemia/reperfusion injury of rat liver mainly by inhibiting leukocyte activation. J Pharmacal Exp Ther 2000;294:1034-42. 16. Zhang C, Rosenbaum DM, Shaikh AR, Li Q, Rosenbaum PS, Pelliam DJ, et al. Ischemic preconditioning attenuates apoptotic cell death in the rat retina. Invest Ophthalmol Vis Sci 2002;43:3059-66. 17. Casson RJ, Chidlow G, Wood JPM, Osborne NN. The effect of hyperglycemia on experimental retinal ischemia. Arch Ophthalmol2004;122:361-6.
Key words: retinal ischemia, ischemia-reperfusion injury, ischemic preconditioning, apoptosis, rat
Protective effect of ischemic preconditioning on retinal ischemia-reperfusion injury in rats Davut Ozbay, * MD; Serap Ozden, * MD; Sevda Mtifttioglu, t MD; Figen Kaymaz,t MD; Volkan Yaylah,* MD; Cern Yildmm,* MD; Sinan Tathpmar,* MD ABSTRACT • RESUME Background: A short period of ischemia can induce remarkable tissue resistance to the deleterious effects of subsequent ischemia and reperfusion. We performed a study to investigate the effect of ischemic preconditioning on retinal ischemiareperfusion injury in rats. Methods: Ten Wistar albino rats were divided into two groups of five animals (I 0 eyes): one group underwent 5 minutes of ischemic preconditioning (achieved by clamping the common carotid arteries at the time of vertebral artery cauterization), and the other did not (control group). In both groups, the vertebral arteries were occluded bilaterally with an electric needle coagulator under an operating microscope. Forty-eight hours later the rats were reanesthesized, and both common carotid arteries were clamped to interrupt blood flow. The duration of ischemia was 30 minutes. The clamp was then removed to enable reperfusion for 4 hours. The animals were killed by decapitation, and retinal sections were evaluated under light and electron microscopy. The signs of ischemia-reperfusion injury (cellular degeneration, vacuolization between retinal layers, increase in retinal thickness due to edema, mononuclear cell infiltration and apoptotic cell count) were recorded. Results: Light microscopy of retinal sections from rats in the ischemic preconditioning group showed a well-preserved retinal structure. The mean thickness values (and standard deviation [SD]) for the inner nuclear layer (I 04.0 IJm [2.54 IJm] vs. 49.0 IJm [I 0.83 IJm]) and inner plexiform layer ( 134.8 IJm [I 0.13 IJm] vs. 88.5 IJm [17.46 IJm]) were significantly higher in the control group than in the preconditioning group (p =0.009), indicating increased retinal thickness in the former group due to tissue edema resulting from ischemia-reperfusion injury. The mean mononuclear cell count (6.67 [SD 1.97] vs. 2.5 [SD 1.0]) and apoptotic cell count (18.2 [SD 5.7] vs. 5.3 [SD 1.0]) were significantly higher in the control group than in the preconditioning group (p = 0.002), indicating an inhibitory effect of ischemic preconditioning on leukocyte infiltration and apoptotic cell death.
Interpretation: Ischemic preconditioning attenuated ischemia-reperfusion injury in the rat retina. From *the Department of Ophthalmology, Pamukkale University Medical Faculty, Denizli, Turkey, and tthe Department of Histology and Embryology, Hacettepe University Medical Faculty, Ankara, Turkey
Presented at the 4th International Glaucoma Symposium, Barcelona, Mar. 19-22, 2003 Correspondence to: Prof. Dr. Serap Ozden, P.K. 185, 20003 Denizli, Turkey; fax +90 258 2410040, [email protected]
Originally received Oct. 23, 2003
This article has been peer-reviewed.
Accepted for publication July 6, 2004
Can j Ophthalmol 2004;39:727-32
Ischemic preconditioning-Ozbay et al
727
Ischemic preconditioning-O zbay et al
Contexte : Une breve periode d'ischemie peut induire une remarquable resistance tissulaire aux effets nocifs d'une ischemie et d'une reperfusion subsequentes. Nous avons etudie les effets du preconditionnement ischemique sur Ia blessure de l'ischemie-reperfusion retinienne chez les rats. Methodes : Dix rats albinos Wistar ont ete repartis en deux groupes de cinq (dix yeux) : un groupe a ete Soumis a 5 minutes de preconditionnement ischemique (par clampage des arteres carotides communes lors d'une cauterisation de l'artere vertebrale); l'autre, laisse intact, a servi de temoin. Chez les deux groupes, les arteres vertebrales ont ete obliterees bilateralement avec un coagulateur electrique (aiguille), sous microscope operatoire. Quarante-huit heures apres, les rats ont ete anesthesies de nouveau et les deux arteres carotides communes ont ete clampees pour interrompre le flot sanguin. L'ischemie a dure 30 minutes. On a ensuite retire Ia clampe pour permettre Ia reperfusion pendant 4 heures. Les animaux ont ete decapites et les sections de Ia retine, evaluees sous microscopie classique et electronique. Les signes de blessure de l'ischemie-reperfusion (degenerescence cellulaire, vacuolisation entre les couches retiniennes, accroissement de l'epaisseur de Ia retine dG a l'redeme, infiltration des cellules mononucleaires et decompte des cellules apoptotiques) ont ete notes. Resultats : La microscopie classique des sections retiniennes chez les rats du groupe de preconditionnement ischemique a montre une structure retinienne bien conservee. L'epaisseur moyenne (et ecart type [ET]) de Ia couche nucleaire interne (104,0 IJm [2,54 IJm] c. 49,0 IJm [10,83 IJm]) et celle de Ia couche plexiforme interne (134,8 IJm [10,13 IJm] c. 88,5 IJm [17,46 1Jm]) etaient beaucoup plus grandes chez le groupe temoin que chez le groupe de preconditionnement (p = 0,009), ce qui indique une plus forte augmentation de l'epaisseur de Ia retine chez le premier groupe due a l'redeme tissulaire consecutif a Ia blessure causee par l'ischemie-reperfusion. Lecompte moyen des cellules mononucleaires (6,67 [ET I,97] c. 2,5 [ET I ,0]) et celui des cellules apoptotiques ( 18,2 [ET 5,7] c. 5,3 [ET I ,0]) etaient significativement superieurs dans le groupe temoin (p = 0,002), ce qui indique l'effet inhibiteur du preconditionnement ischemique sur !'infiltration des leukocytes et Ia mort des cellules apoptotiques. Interpretation : Le preconditionneme nt ischemique a attenue Ia blessure de l'ischemie-reperfu sion dans Ia retine des rats.
I
schemia is an important cause of tissue injury. Inadequate blood supply leads to tissue hypoxia and accumulation of metabolic waste products, which may cause cellular damage. The phenomenon of worsening tissue injury after perfusion is called reperfusion injury. 1 Leukocytes are thought to play a major role in ischemia-reperfusion injury. 1•2 Production of oxygen free radicals has also been reported to be an important factor in the pathophysiology of this condition. 1•3 A short period of ischemia can induce remarkable tissue resistance to the deleterious effects of subsequent prolonged ischemia and reperfusion. This phenomenon, known as ischemic preconditioning,4 was first demonstrated in canine myocardium. 5 Although the exact mechanisms underlying ischemic preconditioning are unknown, release of adenosine, de novo
728
CAN J OPHTHALMOL-VOL. 39, NO.7, 2004
protein synthesis and opening of adenosine triphosphate-sensitive potassium channels have been proposed as possible factors. 4·6-8 The purpose of this study was to investigate the effect of ischemic preconditioning on retinal ischemiaperfusion injury in rats. METHODS
The study was carried out on 10 Wistar albino rats (20 eyes) weighing 250-350 g each, obtained from the Hifzisihha Institute Animal Laboratory, Ankara, Turkey. The rats were housed in wire-bottomed cages at room temperature with a 12-hour light/dark cycle. All animals were fed standard rat chow and were given only water for 12 hours before surgery. The care
Ischemic preconditioning-Ozbay et al
and handling .of the animals were in accordance with the guidelines of the US National Institutes of Health. The rats were divided into two groups of five animals (10 eyes): one group underwent 5 minutes of ischemic preconditioning (achieved by clamping the common carotid arteries at the time of vertebral artery cauterization), and the other did not (control group). The surgical procedure was identical in the two groups. Surgical procedure We used the four-vessel occlusion method9•10 to induce retinal ischemia. The rats were anesthesized with an intramuscular injection of ketamine hydrochloride, 50 mg/kg (Ketalar, Pfizer Canada Inc., Kirkland, Que.), and xylazine, 10 mglk:g (Rompun, Bayer, Leverkusen, Germany). Following skin shaving and preparation with 10% povidone-iodine solution, a 2-cm incision was made behind the occipital bone directly overlying the first two cervical vertebrae. The vertebral arteries were occluded bilaterally by coagulation of the first cervical vertebra with an electric needle coagulator through the alar foramina under an operating microscope. The incision was closed with interrupted 4-0 silk sutures. Forty-eight hours after the initial procedure, the rats were anesthesized as previously, and a 3-cm midline cervical incision was made. Both common carotid arteries were exposed, and a nontraumatic microvascular clamp was placed around either artery to interrupt the blood flow. The absence of blood flow in the retina was monitored by direct ophthalmoscopy. The carotid artery clipping time (i.e., duration of ischemia) was 30 minutes. The clamp was then removed and the eye allowed to reperfuse for 4 hours. Retinal reperfusion was confirmed by direct ophthalmoscopy. The cervical incision was closed with interrupted 4-0 silk sutures. At the end of the reperfusion period the animals were killed by decapitation, and the eyeballs were enucleated and fixed in 2.5% glutaraldehyde solution for histologic study. Histopathological evaluation Tissue samples were fixed by immersion in 2.5% glutaraldehyde in phosphate buffer and postfixed in 1% osmium tetroxide in the same buffer. After dehydration in ethanol gradients at room temperature, the tissue samples were embedded in araldite. Semithin
(1 J.iffi) and thin (70 nm) sections were cut, examined and photographed under a light microscope (Olympus BH2, Olympus America Inc., Melville, NY) and electron microscope (Zeiss EM9-S2, Carl Zeiss, Inc., Thornwood, NY) respectively. Semithin sections were stained with methylene blue-azure II, and ultrathin sections were stained with uranyl acetate and lead citrate. We recorded the signs of ischemia-reperfusion injury at the microscopic level (i.e., cellular degeneration, vacuolization between retinal layers, increase in retinal thickness due to edema, mononuclear cell infiltration and cell apoptosis) in the two groups. The thickness of the inner plexiform layer, inner nuclear layer and ganglion cell layer was measured by means of a compass on the photographs obtained with the light microscope. We calculated mononuclear and apoptotic cell counts as the mean value for three different regions under 40x magnification. Statistical analysis We performed statistical analysis using the MannWhitney U test. A p value less than 0.05 was accepted as statistically significant. RESULTS
In retinal tissue samples obtained from rats in the control group, light microscopy showed vacuolization due to edema in the outer nuclear layer and between the inner and outer segments of photoreceptors (Fig. 1). Prominent erythrocyte infiltration was also noted between the nuclei of the outer nuclear layer. Increased thickness of the outer plexiform layer due to edema and vacuolization in some ganglion cells within the ganglion cell layer were also observed. Mononuclear cell infiltration was present and was prominent in the ganglion cell layer. In the ischemic preconditioning group, light microscopy of semithin retinal sections showed a well-preserved retinal structure (Fig. 1). The retinal layer most affected was the ganglion cell layer. Vacuolar degeneration within the cytoplasm of ganglion cells was noted in some areas. Within the inner nuclear layer, an increase in the distance between nuclei was observed in some parts of the retina, indicating edema and mononuclear cell infiltration. Some vacuolization was noted in the inner plexiform layer; however, an increase in the thickness of this layer and mononuclear
CAN J OPHTHALMOL-VOL. 39, NO.7, 2004
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Ischemic preconditioning-Ozbay et al
'
Fig. 1-Light photomicrographs of retinal sections from rats that received retinal ischemia-reperfusion injury with (preconditioning group) or without (control group) ischemic preconditioning. Left: Control group. Edema in retinal layers and infiltrative cells in ganglion cell layer, inner plexiform layer (IPL) and inner nuclear layer (INL) were prominent. Vacuoles (V) were also seen in ganglion cells and INL. Right: Preconditioning group. Note less edema compared to control group. Thickness of outer plexiform layer (OPL) was almost normal (methylene blue-azure II; magnification x20).
Table 1-Mean thickness of retinal layers and mean cell counts in rats that received ischemia-reperfusion injury with (preconditioning group) or without (control group) ischemic preconditioning Mean (and standard deviation)
Variable Inner nuclear layer thickness, !Jill Inner plexiform layer thickness, 1-1m Ganglion cell layer thickness, !Jill Mononuclear cell count Apoptotic cell count
Control group (n = 10)
Preconditioning group (n = 10)
p value
I04.0 (2.54)
49.0 (I 0.83)
0.009
134.8 (10.13)
88.5 ( 17.46)
0.009
151.4 (20.7) 6.7 ( 1.97) 18.2 (5.7)
122.0 (36.15) 2.5 (1.0) 5.3 ( 1.0)
0.295 0.002 0.002
infiltration were not prominent. The cells in the outer nuclear layer were relatively well preserved, and there were considerably fewer erythrocytes than in the control group. The inner nuclear and inner plexiform layers (but not the ganglion cell layer) were significantly thicker in the control group than in the preconditioning group (p =0.009) (Table 1). These results indicate increased retinal thickness in the control group due to tissue edema resulting from ischemia-reperfusion injury. The mean mononuclear cell count and apoptotic cell count were significantly higher in the control
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group than in the ischemic preconditioning group (p 0.002) (Table 1). In the control group, electron microscopy showed edema between the axons of the inner plexiform layer. Vacuolization within cells, disruption between the intercellular adhesions and mononuclear cells were observed in the inner nuclear layer (Fig. 2). There was also vacuolization around the nuclei of the photoreceptors that form the outer nuclear layer as well as apoptotic cells. In the preconditioning group, electron microscopic evaluation revealed vacuolar swelling in the axons of the inner plexiform layer. Some of the cells within the
=
Ischemic preconditioning-Ozbay et al
Fig. 2-Eiectron photomicrographs. Left: Control group. Infiltrative lymphocytes (L) were seen near apoptotic cells (a) in inner nuclear layer. Vacuolar degeneration was prominent in amacrine cells. Right: Ischemic preconditioning group. Most cells and their intercellular junctions (IJ) in inner nuclear layer were intact. However, vacuolar changes were seen in one of the cells (uranyl acetate-lead citrate; magnification x6200).
inner nuclear layer showed cytoplasmic edema; however, most of the intercellular adhesions were intact at this layer (Fig. 2). Mononuclear cells were observed in the inner nuclear layer. INTERPRETATION
Retinal ischemia is thought to contribute to the pathophysiology of several retinal disorders, including glaucoma, retinal vascular occlusion and diabetic retinopathy. 11 Hence, several experimental models of ocular ischemia-reperfusion injury have been developed to investigate the histologic and biochemical changes in the retina. 2•9•10•12 The exact mechanisms of ischemic preconditioning are not fully understood. Recently, the role of adenosine has attracted considerable attention as a possible mediator of ischemic preconditioning. 2•6•13•14 Roth and colleagues 14 observed a significant increase in the adenosine concentration in the rat retina following ischemia. Moreover, it has been shown that adenosine, acting through Al and A2a receptors, is a critical component in the induction of ischemic tolerance after ischemic preconditioning. 6 Leukocytes are known to play an important role in ischemia-reperfusion injury. Activated leukocytes damage the endothelial and parenchymal cells through the production of cytotoxic mediators, including oxygen free radicals and inflammatory cytokines. 1 Recently, Nonaka and associates 2 demonstrated inhibitory
effects of ischemic preconditioning on leukocyte rolling and subsequent leukocyte accumulation during retinal ischemia-reperfusion injury. They also investigated the possible role of adenosine and found that administration of adenosine Al receptor antagonist immediately after ischemic preconditioning suppressed the beneficial effects of preconditioning on leukocyte behaviour. Administration of selective adenosine Al receptor agonist 1 hour before ischemia without preceding ischemic preconditioning had an effect on leukocyte rolling similar to that of ischemic preconditioning. These findings suggest that the inhibitory effects of ischemic preconditioning on leukocyte accumulation during retinal ischemia-reperfusion injury are mediated through the adenosine AI receptor. Adenosine is known to inhibit neutrophil aggregation, neutrophil adherence to endothelial cells and production of cytokine from monocytes. 15 In our study, mononuclear cell counts indicated that ischemic preconditioning significantly attenuated leukocyte accumulation. Roth and colleagues4 found that inhibition of protein synthesis by cycloheximide attenuated the protective effect of preconditioning in the rat retina and suggested that a transient alteration in protein expression was a factor in ischemic preconditioning. Recently, induction of heat shock protein 27 was reported to be associated with ischemic preconditioning in the rat retina. 7 Hence, the expression of cytoprotective proteins, such as heat shock protein, may be a neuroprotective event induced by ischemic preconditioning.
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Zhang and coworkers 16 reported that ischemic preconditioning diminished apoptotic cell death in the rat retina. Similarly, in our study, apoptotic cell counts were significantly lower in the ischemic preconditioning group than in the control group. This result indicates an inhibitory effect of the inner plexiform layer on leukocyte infiltration and apoptotic cell death. Casson and colleagues 17 recently reported that preexisting hyperglycemia and the intraocular delivery of glucose also attenuate ischemic retinal injury in rats. They proposed that this finding is largely due to the differences in energy metabolism between the brain and the retina: unlike the brain, the retina has the ability to maintain most of its adenosine triphosphate requirement in the absence of oxygen through anaerobic glycolysis. They suggested that elevated glucose levels in the vitreous of the hyperglycemic rats caused relative preservation of retinal adenosine triphosphate levels by anaerobic glycolysis during the ischemia, thereby decreasing the retinal injury. In conclusion, we found that ischemic preconditioning attenuated ischemia-reperfusion injury in the rat retina. Factors involved in ischemic preconditioning may be used as future novel therapeutic approaches for preventing retinal ischemic damage in humans. REFERENCES
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myocardium. Circulation 1986;74:1124-36. 6. LiB, RothS. Retinal ischemic preconditioning in the rat: requirement for adenosine and repetitive induction. Invest Ophthalmol Vis Sci 1999;40:1200--16. 7. Li Y, RothS, Laser M, Ma JX, Crosson CE. Retinal preconditioning and the induction of heat-shock protein 27. Invest Ophthalmol Vis Sci 2003;44:1299-304. 8. Sakamoto K, Kuwagata M, Nakahara T, Ishii K. Late preconditioning in rat retina: involvement of adenosine and ATP-sensitive K(+) channel. Eur J Pharmacol2001;418: 89-93. 9. Pulsinelli WA, Brierley JB. A new model of bilateral hemispheric ischemia in the unanesthesized rat. Stroke 1979;10:267-72. 10. Sano Y, Kanematsu EA, Yoshiura M. Uric acid as biochemical marker for retinal and optic nerve damage after occlusion and reperfusion of common carotid and vertebral arteries in rat. Jpn J Ophthalmol1992;36:76-83. 11. Rosenbaum DM, Rosenbaum PS, Singh M, Gupta G, Gupta H, Li B, et al. Functional and morphologic comparison of two methods to produce transient retinal ischemia in the rat. J Neuroophthalmol2001;21:62-8. 12. Szabo ME, Droy-Lefaix MT, Doly M, Carre C, Braquet P. Ischemia and reperfusion-induced histologic changes in the rat retina: demonstration of free radical-mediated mechanism. Invest Ophthalmol Vis Sci 1991;32:1471-8. 13. Li B, Rosenbaum PS, Jennings NM, Maxwell KM, Roth S. Differing roles of adenosine receptor subtypes in retinal ischemia-reperfusion injury in the rat. Exp Eye Res 1999;68:9-17. 14. RothS, RosenbaumPS, Osinski J, Park SS, ToledanoAY, Li B, et al. Ischemia induces significant changes in purine nucleoside concentration in the retina-choroid in rats. Exp Eye Res 1997;65:771-9. 15. Harada N, Okajima K, Murakami K, Usune S, Sato C, Ohshima K, et al. Adenosine and selective A(2A) receptor agonists reduce ischemia/reperfusion injury of rat liver mainly by inhibiting leukocyte activation. J Pharmacal Exp Ther 2000;294:1034-42. 16. Zhang C, Rosenbaum DM, Shaikh AR, Li Q, Rosenbaum PS, Pelliam DJ, et al. Ischemic preconditioning attenuates apoptotic cell death in the rat retina. Invest Ophthalmol Vis Sci 2002;43:3059-66. 17. Casson RJ, Chidlow G, Wood JPM, Osborne NN. The effect of hyperglycemia on experimental retinal ischemia. Arch Ophthalmol2004;122:361-6.
Key words: retinal ischemia, ischemia-reperfusion injury, ischemic preconditioning, apoptosis, rat