135-142.qxd 20/5/2010 10:19 Ì ™ÂÏ›‰·135 INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 26: 135-142, 2010 135 Evaluation of the aldose reductase inhibitor fidarestat on ischemia-reperfusion injury in rat retina IRINA G. OBROSOVA1, YURY MAKSIMCHYK1, PAL PACHER2, ELISABET AGARDH4, MAJ-LIS SMITH4, AZZA B. EL-REMESSY3 and CARL-DAVID AGARDH4 1Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, LA; 2Section on Oxidative Stress Tissue Injury, Laboratory of Physiological Studies, NIH/NIAAA, Bethesda, MD; 3Program in Clinical and Experimental Therapeutics, College of Pharmacy, University of Georgia, Augusta, GA, USA; 4The Department of Clinical Sciences, University Hospital MAS, Lund University, Malmö, Sweden Received February 24, 2010; Accepted March 29, 2010 DOI: 10.3892/ijmm_00000445 Abstract. This study evaluated the effects of retinal ischemia- intermediate accumulation. These changes were prevented or reperfusion (IR) injury and pre-treatment with the potent and alleviated by the AR inhibitor fidarestat. The results identify specific aldose reductase inhibitor fidarestat on apoptosis, AR as an important therapeutic target for diseases involving aldose reductase and sorbitol dehydrogenase expression, sorbitol IR injury, and provide the rationale for development of pathway intermediate concentrations, and oxidative-nitrosative fidarestat and other AR inhibitors. stress. Female Wistar rats were pre-treated with either vehicle (N-methyl-D-glucamine) or fidarestat, 32 mgkg-1d-1 for both, Introduction in the right jugular vein, for 3 consecutive days. A group of vehicle- and fidarestat-treated rats were subjected to 45-min Sorbitol pathway of glucose metabolism consists of two retinal ischemia followed by 24-h reperfusion. Ischemia was reactions, aldose reductase (AR)-catalyzed NADPH-dependent induced 30 min after the last vehicle or fidarestat adminis- reduction of glucose to its sugar alcohol, sorbitol, followed tration. Retinal IR resulted in a remarkable increase in retinal by sorbitol dehydrogenase(SDH)-catalyzed NAD-dependent cell death. The number of TUNEL-positive nuclei increased oxidation of sorbitol to fructose. For almost 50 years after its 48-fold in the IR group compared with non-ischemic controls discovery (1), it has been considered that activation of the (p<0.01), and this increase was partially prevented by fidarestat. sorbitol pathway of glucose metabolism is a direct consequence AR expression (Western blot analysis) increased by 19% in of hyperglycemia or hypergalactosemia. This notion has been the IR group (p<0.05), and this increase was prevented by supported by numerous studies with AR inhibitors in animal fidarestat. Sorbitol dehydrogenase and nitrated protein expres- models of diabetes and galactose feeding which provided sions were similar among all experimental groups. Retinal evidence for the important role of increased sorbitol pathway sorbitol concentrations tended to increase in the IR group activity in diabetic or diabetes-like complications including but the difference with non-ischemic controls did not achieve cataract (2-4), retinopathy (2,5,6), peripheral and autonomic statistical significance (p=0.08). Retinal fructose concen- neuropathy (7-11), and nephropathy (12,13). The important trations were 2.2-fold greater in the IR group than in the non- role for the first enzyme of the sorbitol pathway, AR, in several ischemic controls (p<0.05). Fidarestat pre-treatment of rats diabetic complications has further been confirmed in studies subjected to IR reduced retinal sorbitol concentration to the with AR-overexpressing and AR-knockout mice (14-17). levels in non-ischemic controls. Retinal fructose concentrations Recent reports suggest that increased sorbitol pathway were reduced by 41% in fidarestat-pre-treated IR group vs. activity is also involved in the pathogenesis of diseases that untreated ischemic controls (p=0.0517), but remained 30% are not accompanied by hyperglycemia including atheroscle- higher than in the non-ischemic control group. In conclusion, rosis (18), cancer (19), and a number of inflammatory condi- IR injury to rat retina is associated with a dramatic increase tions and disorders such polymicrobial sepsis (20), asthma (21) in cell death, elevated AR expression and sorbitol pathway and uveitis (22). Evidence for the important role of this mecha- nism in ischemia-reperfusion (IR) injury, and, in particular, _________________________________________ in cardiac (23-25) and, recently, retinal (26,27) IR injury is Correspondence to: Dr Irina G. Obrosova, Pennington Biomedical emerging. Research Center, Louisiana State University System, 6400 Perkins In human disease, retinal IR injury takes place when retinal Road, Baton Rouge, LA 70808, USA blood flow is interrupted for a long period of time and then E-mail: [email protected] restarted. It occurs in transient ischemia-related diseases, such as central retinal artery occlusion, angle-closure glaucoma, Key words: aldose reductase, apoptosis, fidarestat, nitrated proteins, amaurosis fugax, as well as in diabetes, atherosclerosis, and retinal ischemia-reperfusion, sorbitol dehydrogenase hypertension (26-29). Retinal ischemia and diabetic retino- pathy share many pathophysiological and pathological features 135-142.qxd 20/5/2010 10:19 Ì ™ÂÏ›‰·136 136 OBROSOVA et al: FIDARESTAT ALLEVIATES RETINAL ISCHEMIA-REPERFUSION INJURY including but not limited to b-wave abnormalities in the jugular vein was exposed and catheterized to allow repeated electroretinogram (30,31), glial activation (32,33), retinal i.v. injections. The catheter was tunneled under the skin to neuronal and ganglion cell degeneration (26,34), increased appear through a small cut in the neck scruff. After injection, vascular permeability and breakdown of blood-retinal barrier the catheter was closed and replaced under the skin and (35,36), edema (26,37), capillary cell loss (38,39), and accele- wounds were sutured. Injections were repeated on day 2 under rated angiogenesis (40,41). Also note, that whereas retinal 2% isoflurane anesthesia. On day 3, the animals were anes- ischemia plays an important role in the progression of diabetic thetized with fentanyl (300 μg/kg i.p., B. Braun, Melsungen, retinopathy towards its advanced, or proliferative stage (41), Germany) followed by medetomidin (300 μg/kg i.p., Domitor its treatment with scatter laser photocoagulation restoring Vet, Orion Pharma Animal Health, Sollentuna, Sweden), and oxygen supply is associated with the development of sight- a third dose of fidarestat or NMDG was given 30 min before threatening macular edema (42). Sixty percent of eyes treated induction of ischemia. with scatter photocoagulation showed an increase in foveal thickness, detectable by a scanning retinal thickness analyzer, Induction of ischemia and reperfusion. The pupils were dilated after photocoagulation (42). with cyclopentolate 1% (Cyclogyl®, Alcon, Stockholm, A recent study in AR-deficient mouse model implicated Sweden) followed by the application of a local anesthetic, increased AR activity in neuronal loss and edema associated tetracaine 1% (Tetrakain Chauvin, Novartis Ophthalmic, with retinal IR injury (26). In the same report, IR-associated Copenhagen, Denmark) which allowed the direct observation increase in TUNEL positivity in retinal ganglion cell and inner of retinal blood flow under a stereomicroscope. Retinal neuronal layers, retinal thickening, as well as cytoarchitectural ischemia was induced 30 min after the vehicle or fidarestat disorganization were prevented or markedly reduced by the injection, by careful application of a 5-0 silk suture (Ethicon, ARI fidarestat. In our recent experiments in the rat model of Sollentuna, Sweden) around the vessels and the accom- retinal IR injury, pre-treatment with fidarestat alleviated panying optic nerve of the left eye as described (43). The inflammatory response (27). The present study conducted in ligature was tightened until complete cessation of the retinal the same animal model evaluated the effects of IR injury and blood flow occurred. The right eye served as a non-ischemic fidarestat pre-treatment on retinal apoptosis, AR and sorbitol control. During induction of ischemia, rats were placed in dehydrogenase (SDH) protein expressions, sorbitol pathway cages supplied with a heating pad, to avoid a decrease in intermediate concentrations, and oxidative-nitrosative stress. body temperature. After 45 min, the ligature was carefully removed. After confirmation of restoration of retinal blood Materials and methods flow, anesthesia was reversed by bupenorfin (30 μg/kg s.c., Temgesic®, Schering-Plough, Kenilworth, NJ, USA) and Reagents. Unless otherwise stated, all chemicals were of atipamezol (1 mg/kg s.c., Antisedan Vet, Orion Pharma Animal reagent-grade quality, and were purchased from Sigma Health, Sollentuna, Sweden), and reperfusion continued for Chemical Co., St. Louis, MO. Rabbit polyclonal anti- 24 h, after which all animals were sacrificed. nitrotyrosine antibody was purchased from Upstate, Lake Placid, NY, USA. Mouse anti-AR monoclonal antibody was Euthanasia and retinal dissection. The rats were sedated by purchased from Santa Cruz Biotechnology, Santa Cruz, CA, CO2, and were immediately sacrificed by cervical dislocation. USA. Mouse anti-SDH monoclonal antibody was obtained In approximately half of animals in each experimental group, from Abcam, Cambridge, MA, USA. ApopTag® Peroxidase both eyes were rapidly enucleated, the lenses were removed, In Situ