Protective Effects of Selectin Ligands/Inhibitor (SKK-60060) against Retinal Ischemia-Reperfusion Injury AKIHISA MATSUBARA a *, KAZUYUKI TOMIDA a , YOSHITO MATSUDA a , KAZUSHI TAMAI a , AKIRA TASHITA b , TAKAHITO JOMORI b , AKITAKA TSUJIKAWA c AND YUICHIRO OGURA a a Department of Ophthalmology, Nagoya City University Medical School, Nagoya, Japan, b Drug Discovery Research Department, Sanwa Kagaku Kenkyusho Co. Ltd., Mie, Japan and c Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto, Japan (Received Cleveland 16 December 1999 and accepted in revised form 23 May 2000) A newly developed selectin ligand inhibitor (SKK-60060) has been shown to block P- and L-selectins in vitro. We examined its inhibition of leukocyte±endothelial interactions in vivo against retinal ischemia-reperfusion injury and protective effects on ischemia-induced retinal damage. Retinal ischemia was induced by temporary ligation of the optic sheath for 60 min in anesthetized pigmented rats. SKK-60060 was administered 5 min before reperfusion and 4, 12, 24 and 48 hr thereafter, and leukocyte dynamics in the retinal microcirculation were evaluated using acridine orange digital ¯uorography. After 7 days of reperfusion, ischemia-induced retinal damage was also assessed histologically. SKK-60060 treatment suppressed leukocyte rolling during the reperfusion period; their numbers in the SKK-60060-treated rats were reduced by 67 . 0% (P 5 0 . 01) and 53 . 2% (P 5 0 . 01) at 12 and 24 hr, respectively. The subsequent leukocyte accumulation was also inhibited in SKK-60060-treated rats; accumulated leukocytes in the SKK-60060-treated rats were reduced by 72 . 8% (P 5 0 . 01) and 53 . 4%(P 5 0 . 01) at 12 and 24 hr, respectively. Retinal venous vasodilation in SKK-60060-treated rats were signi®cantly suppressed at each time point (P 5 0 . 05). Histological examination demonstrated protective effects of SKK-60060 on ischemia-induced retinal damage, which were more substantial in the inner retina (P 5 0 . 01). SKK-60060 signi®cantly inhibits the leukocyte rolling along the major retinal veins and their accumulation during the reperfusion period. These results suggest therapeutic potential of SKK-60060 for ischemia-reperfusion injury. # 2000 Academic Press Key words: leukocytes; selectin; ischemia-reperfusion injury; microcirculation; retina; selectin inhibitor. 1. Introduction Leukocytes are important players in infection defense and in healing processes, but also contribute to negative aspects of in¯ammation. Leukocytes are thought to play a central role in ischemia reperfusion injury (Suzuki et al., 1993), because their accumu- lation causes tissue injury by impeding blood ¯ow or by producing superoxide radicals (Werns, Shea and Lucchesi, 1985) and in¯ammatory cytokines (Ghezzi et al., 1991). Many experimental studies have shown improvement in ischemia-reperfusion injury with reduction of leukocyte participation. Thus immuno- suppression by sublethal whole body X-irradiation (Strachan et al., 1992), and leukopenia produced by leukocyte ®ltration or anticancer drugs (Heinel et al., 1994) successfully reduces leukocyte accumulation and tissue damage during ischemia reperfusion injury. Leukocyte±endothelial interactions are regulated by multistep processes (Osborn, 1990), with each step mediated by distinct adhesion molecules (Lawrence and Springer, 1991). Leukocyte rolling is the ®rst step in a cascade of events that lead to ®rm adhesion and transmigration through the endothelium. It is estab- lished that selectins can rapidly arrest freely ¯owing leukocytes and mediate their rolling along the endothelium of blood vessels (Butcher, 1991; Springer, 1994). Anti-adhesion molecule treatments that inhibit selectin and control in¯ammation are therefore attracting attention. Inhibition of selectin has been suggested as a possible treatment for ischemia- reperfusion injury (Winn et al., 1993) and many experimental studies have shown inhibitory effects on myocardial ischemic damage and leukocyte accumu- lation with monoclonal antibodies against adhesion molecules (Weyrich et al., 1993; Chen et al., 1994), oligosaccharides like sialyl Lewis X (sLe x )(Buerke et al., 1994; Yamada et al., 1998) or sulfatides (Yamada et al., 1998). Our previous study (Tsujikawa et al., 1999) demonstrated that P-selectin or intercellular adhesion molecule-1 (ICAM-1) monoclonal antibodies (mAb) attenuate leukocyte accumulation and subsequent tissue injury during retinal ischemia- reperfusion injury. Exp. Eye Res. (2000) 71, 283±293 doi:10.1006/exer.2000.0880, available online at http://www.idealibrary.com on 0014-4835/00/09028311 $35.00/0 # 2000 Academic Press * Address correspondence to: Akihisa Matsubara, Department of Ophthalmology, Nagoya City University Medical School, 1-Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan. E-mail: matubara@med.nagoya-cu.ac.jp