Protective effect of polyethylene glycol-superoxide dismutase on leukocyte dynamics in rat retinal microcirculation under lipid hydroperoxide-induced oxidative stress Akihisa Matsubara a, * , Kazushi Tamai a , Yoshito Matsuda a , Yuji Niwa a , Hiroshi Morita a , Kazuyuki Tomida a , Donald Armstrong b , Yuichiro Ogura a a Departments of Ophthalmology and Visual Science, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan b Department of Opthalmology, College of Medicine, University of Florida, Gainesville, FL and Oxidative Stress Associates Inc., Alachua, FL, USA Received 2 November 2004; accepted in revised form 25 January 2005 Available online 2 March 2005 Abstract The levels of lipid hydroperoxide (LHPs) in vitreous are elevated in a variety of retinal disorders. Recently, we have shown that increased levels of LHPs in the vitreous enhanced leukocyte-endothelium interaction in the retina, which should contribute to the initial disturbance of the retinal microcirculation. Based upon the previous work, the purpose of the present study was to investigate the effect of polyethylene glycol-superoxide dismutase (PEG-SOD), one of the important enzyme antioxidants, on leukocyte-endothelial interaction in the retinal microcirculation under LHP-induced oxidative stress. Male Brown-Norway rats weighing approximately 250 g were used. LHP(18:2) was made from linoleic acid (LA) with lipoxygenase and 10 mg of LHP dissolved in 5 ml of sodium borate buffer (SBB, 0.02 M) was slowly injected into the vitreous using a 33-gauge needle. PEG-SOD (5000 units/kg) was given intravenously 5 min before LHP injection. At 2, 4, 6, 12, 24 and 48 hr after the vitreous injections, we evaluated the number of rolling leukocytes along the major retinal veins and the number of leukocytes that accumulated in the retinal microvasculature with acridine orange digital fluorography. In LHP-treated rats, leukocyte rolling along the major retinal veins was maximal at 6 hr after LHP injection. The number of rolling leukocytes in the PEG-SOD-treated rats was decreased to 5.5% of those in the LHP-treated rats at 6 hr after LHP injection (P!0.01). No rolling leukocytes were observed in either control or vehicle-treated eyes. The number of accumulated leukocytes in LHP-treated eyes started to increase at 12 hr, and peaked at 24 hr which was significantly higher than in both control and vehicle-treated eyes (P!0.01). The number of accumulated leukocytes in the PEG- SOD-treated rats was reduced by 88.0% at 24 hr (P!0.01). Intravenous injection of PEG-SOD significantly inhibited the leukocyte rolling and its accumulation under LHP-induced oxidative stress. These results suggest that PEG-SOD might attenuate various retinal microcirculatory disorders associated with LHP. q 2005 Elsevier Ltd. All rights reserved. Keywords: lipid polyethylene-hydroperoxide; leukocyte; glycol-superoxide dismutase; retinal microcirculation 1. Introduction Free radical and lipid peroxide formation, which can cause oxidative stress-induced damage to cell membranes, are initiated by various factors. As the eye is always exposed to initiators such as oxygen, light, ultraviolet ray and x- irradiation, the relationship between free radicals and ocular diseases has attracted much attention. Many studies have suggested important roles of free radicals and lipid peroxides in various ocular diseases including keratitis, cataract, uveitis, retinal degeneration, diabetic retinopathy, retino- pathy of prematurity and retinal ischemic diseases (Lakatos et al., 1982; Sery and Petrillo, 1984; Bhuyan et al., 1986; Armstrong et al., 1992; Alio et al., 1995; Spaide et al., 1999). The retina contains a high proportion of polyunsaturated fatty acids, which are susceptible to lipid peroxidation. Experimental Eye Research 81 (2005) 193–199 www.elsevier.com/locate/yexer 0014-4835/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2005.01.021 * Corresponding author. Present address: Dr Akihisa Matsubara, Ten Emerson Place-20A, Boston, MA 02114, USA. E-mail addresses: akihisa_matsubara@meei.harvard.edu (A. Matsubara), kazutamai@aol.com (K. Tamai), osa@aol.com (D. Armstrong), ogura@med.nagoya-cu.ac.jp (Y. Ogura).