Guey-Shuang Wu

Detection of retinal lipid hydroperoxides in experimental uveitis

In our on-going studies of experimental uveitis, we previously obtained a preliminary indication of phagocyte-mediated retinal lipid peroxidation by measuring conjugated dienes (CD), thiobarbituric acid reactive substances (TBARS) and fluorescent chromolipids. Using gas chromatography/mass spectrometry (GC/MS), the current study detected hydroperoxide-derived 10-, 11-, 13-, 14-, and 17-hydroxydocosahexaenoic acid (HDHE) in retinal membranes. Docosahexaenoic acid (22:6) is the major polyunsaturated fatty acid (PUFA) in photoreceptor membranes. Hydroperoxides from other retinal PUFA were found also. Arachidonic acid (20:4) yielded 8-, 9-, 11-, 12-hydroxyeicosatetraenoic acid (HETE) as major products. Since 12-HETE could also arise from lipoxygenase catalyzed oxygenation of free 20:4, the source of 12-HETE could be both peroxidative and lipoxygenase pathways. Concomitantly, peroxidative loss of 22:6 and accumulation of 20:4 were also noted. At the peak of inflammation, loss of 22:6 was close to 50% of the original amount in the control retinas. In the same time period, 20:4 increased more than two-fold. The present data suggest that the oxygen radicals derived from phagocytes initiate the retinal lipid peroxidation, and the resultant formation of hydroperoxides, oxidative loss of 22:6 and accumulation of 20:4 appear to serve as amplification factors in subsequent biochemical events, such as chemotaxis of PMNs and activation of cyclooxygenase.

Isolation and culture of rat retinal microvessel endothelial cells using magnetic beads coated with antibodies to PECAM-1.

PURPOSE To isolate retinal endothelial cells (RECs) from Lewis rats using magnetic beads coated with antibodies to rat platelet-endothelial cell adhesion molecule-1 (PECAM-1) and to characterize the cultured RECs. METHODS Magnetic beads were coated with anti-rat PECAM-1 antibodies. Retinas were obtained from Lewis rat eyes. After the retinas were digested with collagenase, they were incubated with the antibody-coated beads with agitation. RECs that stuck to the beads were collected with a magnetic particle concentrator and cultured in fibronectin coated wells. The characteristics of the RECs were examined by immunohistochemical study utilizing von Willebrands Factor, acetylated low-density lipoprotein uptake and transmission electron microscopy. RESULTS The cells isolated using the PECAM-1-coated magnetic-bead technique formed a contact-inhibited cobblestone monolayer that stained positive for von Willebrands Factor. These cells revealed low-density lipoprotein uptake. Ultrastructurally, the isolated cells exhibited pinocytic vesicles and a high density of intercellular junctions without fenestrations. CONCLUSION These results indicate that the isolated cells were vascular endothelial cells showing both morphologic and functional characteristics of retinal vascular endothelium. The magnetic-bead technique was useful for isolating high purity RECs that can be cultured to study the physiological, immunological and biochemical role of the endothelium in various ocular diseases.

Microglial stability and repopulation in the retina.

Background/aims: Parenchymal central nervous system microglia are repopulated by bone marrow derived monocytes more slowly than any other reticuloendothelial cells. The contribution of bone marrow derived monocytes to the uninflammed retina has not been studied. The present study sought to determine repopulation of retinal microglia in uniflammed retina by bone marrow derived monocytes in bone marrow chimeric rats. Methods: Chimeric (Y→X) Lewis rats were constructed by transplanting 5×107 male bone marrow cells into lethally irradiated female recipient rats. The chimeras were sacrificed 8, 10, 12, 30, and 52 weeks after bone marrow transplant, and retina, brain, lung, and spleen samples were collected. DNA was extracted and quantified. Y positive infiltrating cells in the collected samples were detected by polymerase chain reaction amplification of a Y chromosome specific 104 bp fragment. Results: There was a rapid repopulation of haematopoietic tissues in the spleen (at 8 weeks), confirming the establishment of chimerism, and to a lesser extent, of lung (at 30 weeks). This repopulation was absent in the brain parenchyma and retina until 52 weeks after transplantation. Conclusions: These data indicate that resident microglia in the retina, much like those in the brain, are stable in number in the retinal compartment (up to 1 year), and repopulation by bone marrow derived cells may be delayed for a year.

Mitochondrial Oxidative DNA Damage in Experimental Autoimmune Uveitis

PURPOSE In experimental autoimmune uveitis (EAU), recent work has demonstrated that retinal damage involves oxidative stress early in uveitis, before macrophage cellular infiltration. The purpose of this study was to determine whether oxidative mitochondrial DNA damage occurs early in EAU, before leukocyte infiltration. METHODS Lewis rats were immunized with S-antigen mixed with complete Freund adjuvant (CFA) to induce EAU. Nonimmunized animals and animals injected with CFA served as controls. Animals were killed on days 3, 4, 7, and 12 after immunization. Damage to mitochondrial DNA and nuclear DNA was assessed using a novel long quantitative polymerase chain reaction technique. TUNEL staining to detect apoptosis and immunohistochemical detection of leukocyte infiltration in EAU retinas were also performed at these times. RESULTS Mitochondrial DNA damage occurred early in EAU, from day 4 to day 12. In the early phase of EAU (days 4-7), there was no inflammatory cell infiltration. On day 12 inflammatory cells infiltrated the retina and uvea. Nuclear DNA damage occurred later in EAU at day 12. Neither mitochondrial nor nuclear DNA damage was detected in the controls. TUNEL-positive staining for apoptosis was detected only at day 12 in EAU retina. CONCLUSIONS Oxidative mitochondrial DNA damage begins at day 4 in EAU, supporting the view that oxidative stress selectively occurs in the mitochondria in the early phase of EAU, before leukocyte infiltration. Such oxidative damage in the mitochondria may be the initial event leading to retinal degeneration in EAU.

Lipid peroxidation in experimental uveitis: Sequential studies

Previously we have detected the occurrence of retinal lipid peroxidation initiated by phagocyte-derived oxygen radicals in experimental autoimmune uveitis (EAU). In the current studies, the confirmation of inflammation-mediated lipid peroxidation was proceeded further to include measurement of multiple parameters, including conjugated dienes, ketodienes, thiobarbituric acid reactive substances and fluorescent chromolipids. The assay for myeloperoxidase, a measure for the number of polymorphonuclear leukocytes in the inflammatory sites was also carried out. The levels of all these parameters were followed through the course of EAU development. The sequential evaluation of histologic changes using both light and electron microscopy was also carried out and the results were correlated with lipid peroxidation indices. These data suggest that the retinal lipid peroxidation plays a causative role in the subsequent retinal degeneration.

Animal model for uveitic glaucoma

Abstract• Background: This study was carried out in order to improve the understanding of the pathogenesis of uveitic glaucoma • Methods: Uveitis was induced in 48 Lewis rats by S-antigen injection. Intraocular pressure (10P) was measured by Tono-Pen-2 daily for 24 days in 16 animals. Histopathology was performed sequentially in 14 rats on days 3, 5, 7, 9, 12, 15 and 18 after S-antigen injection. Aqueous dynamics studies were performed on days 0, 3, 7, 14 and 21. Aqueous humor production was measured using an FIT-Calbumin dilution technique; outflow facility was measured using anterior chamber infusion with constant pressure • Results: IOP decreased to a mean of 16.5 +-4.3 mmHg from days 2-5 after S-antigen injections from a mean pre-experimental value of 20.5 +-5.4 mmHg. IOP increased from days 6 to 20 (35.8+-9.1 mmHg; P=0.00001). Histopathologic study revealed inflammation of the anterior and posterior segments from days 9 to 21 after S-antigen injection. Aqueous humor production decreased and outflow facility increased at day 3 after S-antigen injection. At days 7 and 14 after S-antigen injection, acqueous humor production was increased while outflow facility remained normal or was decreased • Conclusion: This model of uveitis glaucoma is characterized by three overlapping phases: (1) ocular hypertension, (2) ocular hypertension associated with clinical and histologic inflammation and (3) anatomic sequelae of uveitis and variable IOP. This model permits in vivo studies of mechanisms of IOP change associated with uveiteis.

Peroxynitrite-induced apoptosis in photoreceptor cells.

Purpose. To study the mechanisms of peroxynitrite-induced photoreceptor cell damage, using retinal cultures and a peroxynitrite donor, 3-morpholinosydnonimine (SIN-1). Methods. Retinal explants obtained from 20-day-old Lewis rat pups, were exposed to SIN-1 for varying lengths of time at varying concentrations. Apoptosis in the photoreceptor cells was detected using the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method and a DNA fragmentation assay. Selected retinal samples were processed for an ultra-structural analysis to confirm apoptosis. The retinas exposed to SIN-1 were tested for the expression of caspase-3 by immunohistochemistry and a Western blot analysis. The retinas were also evaluated for the prevention of apoptosis in the presence of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk). Results. The retinal explants exposed to SIN-1 showed a significant increase in the presence of TUNEL-positive photoreceptor cells. Similarly lineal increases in TUNELpositive cells were seen in the presence of increasing concentrations of SIN-1. DNA ladder formation was seen with the exposure of SIN-1. Ultrastructurally, SIN-1 exposed retinas revealed typical apoptotic changes in the photoreceptor cell nuclei. The retinas preincubated with urate for 6 hours and exposed to SIN-1 for 16 hours showed significantly fewer TUNEL-positive cells compared to the retinas exposed to SIN-1 alone (p < 0.05). Moreover, the retinas exposed to SIN-1 showed the expression of caspase- 3. This expression, as well as the number of apoptotic photoreceptors, significantly decreased in the presence of Z-VAD-fmk. Conclusions. These results show that peroxynitrite induces apoptosis in photoreceptor cells and that such retinal damage appears to be mediated by caspase-3. The apoptotic process can be minimized by peroxynitrite scavenger urate, as well as by the caspase inhibitor Z-VAD-fmk.

Generation of chemiluminescence in experimental autoimmune uveitis.

Using experimental model of uveitis, the inflammation-associated chemiluminescence was measured by luminol amplification method. The induction of uveitis in Lewis rats was carried out by immunizing bovine S-antigen in complete Freunds adjuvant on hind foot-pad. The chemiluminescence activity from six sets of retina and choroid measured at the peak of inflammation was found to be in the range of 300,000-400,000 counts, amounting to nearly 20-fold increase from non-immunizated control animals under the same conditions. The phagocyte-derived superoxide anion and hydroxyl radical appear to be the two major radicals in the chemiluminescent species. This was revealed by in vitro suppression of chemiluminescence by 5,400 units of superoxide dismutase and 10 mM of D-mannitol. The decreases in counts were 21% and 35% for superoxide dismutase and D-mannitol respectively. The presence of hydrogen peroxide was not established since catalase with dose up to 20,000 units did not cause any significant suppression in counts. In the time sequence studies covering the entire course of uveitis development, the level of chemiluminescent species at day 9 postimmunization increased to 1.5-fold that of day 0, continued to increase to a maximum of 18-fold on day 12 and decreased slowly to about 3-fold in day 19. The pattern of increase appears to coincide with our previous findings in the number of polymorphonuclear leukocytes, the extent of membrane lipid peroxidation and the degree of retinal degeneration. Thus, these radicals play an important role in initiation as well as perpetuation of the membrane oxidative processes that lead to retinal degeneration.

Highly selective inhibitor of inducible nitric oxide synthase enhances S-antigen-induced uveitis.

Purpose. Investigated the effect of N-3-aminomethylbenzylacetamidine (1400W), a highly selective inhibitor of inducible nitric oxide synthase (iNOS), on the effector phase of EAU. Methods. Sixteen Lewis rats were sensitized with bovine retinal S-antigen; ten of them injected subcutaneously with 1400W (20mg/kg) three times a day, from day 11 through day 13 following the injection of S-antigen. Five of the ten rats were also injected intraperitoneally with polyethyleneglycol- modified superoxide dismutase (SOD 1000 IU) twice a day from day 7 through day 13. Six rats received intraperitoneal and/or subcutaneous injections of normal saline from day 7 through day 13. The eyes were enucleated on day 14. The intensity of the inflammatory lesion was assessed by a histological score. The thickness of the choroidal and photoreceptor layers was measured. Results. The histological score was higher in the 1400W-treated rats (26 ± 2.1) than in the saline- (20.5 ± 8; p < 0.0001) or 1400 W/SOD-treated rats (20.5 ± 4.9; p < 0.005). The choroid was thicker in the 1400W-treated rats (60.7 ± 16.8µm) than in the saline- (19.2 ± 9.4µm, p < 0.0005) or the 1400 W/SOD-treated rats (29.6 ± 19.3µm, p < 0.05). The photoreceptor layer was thinner in the 1400W-treated rats (8.4 ± 32.1µm) than in the saline- (40 ± 26.7µm; p < 0.05) or 1400 W/SOD-treated rats (60.8 ± 38.1µm; p < 0.05). Conclusion. The data suggests that 1400W exacerbates choroidal inflammation and photoreceptor damage at the effector phase of S-antigen-induced uveitis. This implies that iNOS expressed in the outer retina may have a protective role in EAU.

Role of Peroxynitrite in Photoreceptor Damage in Experimental Uveitis

With inflammation, as in uveitis, the phagocytes undergo respiratory burst resulting in the release of a variety of microbicidal oxygen metabolites. The most important primary species of these metabolites are superoxide and hydrogen peroxide, both of which are potentially injurious to surrounding tissues. The superoxide-mediated toxicity in vivo, however, has been difficult to demonstrate because of the low chemical reactivity of the superoxide anion. It was presumed that the cytotoxicity was exerted through formation of highly reactive hydroxyl radicals via the interaction of superoxide with hydrogen peroxide (Weiss and LoBuglio 1982); but the current understanding of cytotoxicity via the reactivity of hydroxyl radicals does not always offer a satisfactory explanation for the experimental results generated (Babbs and Griffin 1989). Therefore, it appears that there must be another pathway to account for the toxicity of superoxide in inflammatory conditions, including the reaction of superoxide with another abundantly generated species, nitric oxide, to form the potent oxidant peroxynitrite. The reaction between superoxide and nitric oxide proceeds at a nearly diffusion-limited rate. The product generated by this reaction is capable of abstracting hydrogen atoms from lipids and nitrating aromatic amino acid residues of cellular macromolecules.