Exp. Eye Res. (1997) 65, 771–779 Ischemia Induces Significant Changes in Purine Nucleoside Concentration in the Retina-Choroid in Rats† STEVEN ROTH a *, PEARL S. ROSENBAUM b , JOACHIM OSINSKI c , SAMUEL S. PARK a , ALICIA Y. TOLEDANO a, d , BING LI a  ANDREW A. MOSHFEGHI a From the Departments of a Anesthesia and Critical Care, c Clinical Pharmacology, and d Health Studies, University of Chicago, Chicago, Illinois, and the b Departments of Ophthalmology, Visual Sciences and Pathology, Montefiore Medical Center—Albert Einstein College of Medicine, Bronx, New York, U.S.A. (Received 16 January 1997 and accepted in revised form 10 July 1997) Adenosine, produced from the decomposition of adenosine triphosphate, is believed to provide protective effects during ischemia. On the other hand, adenosine metabolites may serve as precursors for oxygen free radicalformation. The time course of formation of adenosine and its purine metabolites was studied during retinalischemia in rats. Concentrations of adenosine and its purine nucleoside metabolites inosine, hypoxanthine, and xanthine in the retina-choroid of ketamine}xylazine-anesthetized rats were measured duringretinalischemia usinghigh performance liquid chromatography. Quantitative measurements were made possible in the small tissue mass through the use of internalstandards. Ischemia was induced by ligation of the central retinal artery. In each rat, one eye was ischemic while the other served as a non-ischemic control. Eyes were frozen in situ at 1, 5, 10, 20, 30, 60, and 120 min of ischemia.The retina-choroid was then removed from the frozen eyes and analysed. Significant increases in the concentrations of adenosine, inosine, and hypoxanthine in ischemic compared to control retina-choroid were detectable within 1 to 5 min of the onsetof ischemia, and within 10 min for xanthine. Increase in adenosine concentration in ischemic relative to control retina-choroid plateaued at 30 min of ischemia,while inosine and hypoxanthine concentrations increased continuously. The increase in xanthine concentration was exponential throughout the measurement period. This study documented the time-related changes in purine nucleoside concentration during ischemia. Prolonged ischemia results in ongoing production of xanthine,which by serving as a precursor for oxygen free radical formation, could be a pathogenic factor in prolonged retinal ischemia. # 1997 Academic Press Limited Key words : adenosine ; ischemia ; purines ; rat ; retina. 1. Introduction Concentration of adenosine, formed from the break- down of AMP via the action of 5«-nucleotidase (Orford and Saggerson, 1996), is increased in brain tissue or in cerebrospinal fluid during cerebral ischemia and hypoxia (Meno, Ngai and Winns, 1993 ; Phillis et al., 1987). It is believed that, under these conditions, it is an endogenousneuromodulator of centralnervous system excitability, and thus servesa ‘ protective ’ function (Schubertet al., 1994). In the cerebral cortex,for example, adenosine increased blood flow and decreased the release of excitatory amino acids (Meno et al., 1991 ; Sciotti et al., 1992). Increases in adenosine concentration in the cerebral cortex were detected within one minute after the onset of ischemia or hypoxia, and increased concentrations were noted with reperfusion (Hsu et al., 1991 ; Winn, Rubio and Berne, 1981). Severallines of evidence now suggest a role of adenosine during blood flow or oxygen deprivation * Address for correspondence and reprints : Dr Roth, Department of Anesthesia and Critical Care, University of Chicago, 5841 South Maryland, Box MC-4028, Chicago, IL 60637, U.S.A. † Presented in partat the AnnualMeeting ofthe Society for Neuroscience, November, 1996, Washington, DC, U.S.A. in the retina.Retinal ischemia of60 min in cats is followed by large increases in retinal blood flow during reperfusion, an effect attenuated by adenosine receptor blockade (Roth, 1995 ; Ostwald et al., 1997). Increases in retinal blood flow in response to hypoxia or hypoglycemia were also attenuated by adenosine receptor blockade (Gidday and Park, 1993; Zhu and Gidday,1996). Following our preliminary finding that an adenosineA1 receptor agonistimproved recovery after 60 min of retinalischemia in rabbits (Rosenbaum, Roth and Mosseri, 1992), recentevi- dence has been presented that combined pre- and post- ischemic administration of the A1 agonist R-PIA led to improvement in the electroretinogram after 45 min ischemia in the rat (Larsen and Osborne, 1996). These findings with exogenous administration of adenosine agonists suggest that the endogenous production of adenosineduring ischemiais likely a protective response. Harnessing the retina’s own intrinsic pro- tective mechanisms is of obvious clinical relevance in the treatment of retinalvascular disease. However, utilization of such a potentially enticing strategy first requiresthe directmeasurement of adenosine con- centration in the intactretina during control and ischemic conditions. While it has been demonstrated in vitro that adenosine is formed in the retina (Perez et 0014–4835}97}12077109 $25.00}0}ey970391 # 1997 Academic Press Limited