Lipid Hydroperoxide Stimulates Leukocyte±Endothelium Interaction in the Retinal Microcirculation KAZUSHI TAMAI a *,AKIHISA MATSUBARA a ,KAZUYUKI TOMIDA a , YOSHITO MATSUDA a ,HIROSHI MORITA a ,DONALD ARMSTRONG b AND YUICHIRO OGURA a a Department of Ophthalmology, Nagoya City University Medical School, Nagoya, Japan and b Department of Small Animal Clinical Sciences, University of Florida, College of Veterinary Medicine, Gainesville, FL, U.S.A. (Received St. Louis 14 September 2001 and accepted in revised form 18 January 2002) Leukocyte dynamics were evaluatyed in vivo in rat retinal microcirculation following exposure to lipid hydroperoxide (LHP) in the vitreous. Various amounts (1, 5, 10 or 100 mg) of LHP (18:2) dissolved in 5 ml of sodium borate buffer (SBB, 0.02M) were injected into the vitreous of Brown-Norway rats. As a comparative study, 10 mg of linoleic acid (LA) dissolved in 5 ml of SBB was injected in the same way. Rats that did not undergo injection were evaluated as un-treated. At 2 to 48 hr after LHP exposure, the following were examined: (1) the ¯ux of rolling leukocytes along the major retinal veins, (2) the number of leukocytes that accumulated in the retinal microvasculature using acridine orange digital ¯uorography and (3) the diameter of major retinal vessels. In the LHP-treated eyes, leukocyte rolling along the major retinal veins was observed and the number increased in a dose-dependent manner ( 1 to 10 mg). The ¯ux of rolling leukocytes peaked at 6 hr after LHP (10±100 mg) injection. No rolling leukocytes were observed in LA-treated or un-treated eyes. The number of accumulated leukocytes started to increase at 4 hr and peaked at 24 hr after LHP (10 mg) injection. This number was signi®cantly higher than that in LA-treated and un-treated eyes. Venous dilation was seen from 4 hr after LHP (10 mg) injection and became signi®cant at 6 and 24 hr as compared with LA-treated and un-treated eyes.. The results indicate that increased LHP levels in the vitreous due to oxidative stress enhance leukocyte±enothelium interaction in the retinal microcirculation. # 2002 Elsevier Science Ltd. Key words: lipid hydroperoxides; leukocyte; endothelium; retina; microcirculation; acridine orange. 1. Introduction Lipid hydroperoxides (LHPs) are intermediate products that are generated by peroxidative reactions of unsaturated fatty acids, and are associated with a variety of disorders such as aging (Miquel et al., 1998), atherosclerosis (El Kossi and Zakhary, 2000), throm- bosis (Jennings et al., 1991), retinal degeneration (Anderson et al., 1984), and radiation-induced dis- orders (Hruza and Pentland, 1993). Propagation reaction of invasive LHP may lead to apoptosis or necrosis (Forrest et al., 1994). The eye, especially the retina, is rich in a large amount of poly-unsaturated fatty acids with double bonds that are oxidized by reactive oxygen and/or LHP, and LHPs are subsequently accumulated. Accordingly, LHPs are closely related to various ocular disorders such as diabetic retinopathy (Armstrong et al., 1992; Augustin et al., 1993; Tanaka, 1998), Eales' disease (Bhooma et al., 1996), proliferative vitreoretinopathy (Boker et al., 1994), retinopathy of prematurity (Lakatos et al., 1982), age-related macular degener- ation (Spaide et al., 1999), and cataract (Bhuyan et al., 1986). In fact, serum LHP levels are elevated in diabetic patients (Nishigaki et al., 1981; Armstrong et al., 1992). LHPs are also increased in the vitreous of eyes with proliferative diabetic retinopathy (Augustin et al., 1993; Tanaka, 1998; Verdejo et al., 1999). Increased levels of LHP generate oxidative damage to the cell membrane of vessel endothelium. Recently, oxygen-free radicals and oxidants have been shown to induce up-regulation of adhesion molecules and alter adhesive interaction between leukocytes and endo- thelial cells (Patel et al., 1991; Fraticelli et al., 1996). It is certain that leukocyte±endothelial interaction plays an important role in retinal microcirculatory disorders (Nishiwaki et al., 1996b; Tsujikawa et al., 1998; Miyamoto et al., 1998). The relationship of high levels of LHP in the vitreous to leukocyte± endothelium interaction in the retinal circulation, however, has not been characterized. In this study, LHP was injected into the vitreous of rats and leukocyte±endothelium reactivity was eval- uated using acridine orange digital ¯uorography. With this method, leukocyte dynamics in the retinal Exp. Eye Res. (2002) 75, 69±75 doi:10.1006/exer.2002.1178, available online at http://www.idealibrary.com on 0014-4835/02/ $35.00/0 # 2002 Elsevier Science Ltd. * Address correspondence to: Kazushi Tamai, Department of Ophthalmology, Nagoya Higashi-Shimin Hospital, 1-2-23 Waka- mizu, Chikusa-ku, Nagoya 464-0071, Japan. E-mail: kazuta- mai@aol.com