Manabu Hirasawa

Retinal dysfunction and progressive retinal cell death in SOD1-deficient mice.

The superoxide dismutase (SOD) family is a major antioxidant system, and deficiency of Cu,Zn-superoxide dismutase (SOD1) in mice leads to many different phenotypes that resemble accelerated aging. The purpose of this study was to examine the morphology and physiology of the sensory retina in Sod1(-/-) mice. The amplitudes of the a- and b-waves of electroretinograms elicited by stimuli of different intensity were reduced in senescent Sod1(-/-) mice, and this reduction in amplitude was more pronounced with increasing age. Retinal morphometric analyses showed a reduced number of nuclei in both the inner nuclear cell layer and outer nuclear cell layer. Electron microscopy revealed swollen cells and degenerated mitochondria in the inner nuclear cell and outer nuclear cell layer of senescent Sod1(-/-) mice indicating necrotic cell death. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling revealed no significant differences in the number of apoptotic cells between Sod1(-/-) and wild-type mice, and activated caspase-3 could not be detected in the retina of Sod1(-/-) mice. In addition to the age-related macular degeneration-like phenotypes previously reported, Sod1(-/-) mice also present progressive retinal degeneration. Our results indicate that Sod1(-/-) mice may be a good model system in which to study the mechanism of reactive oxygen species-mediated retinal degeneration.

Retinal Ganglion Cell Loss in Superoxide Dismutase 1 Deficiency

PURPOSE To investigate the influence of deficiency in superoxide dismutase (SOD) 1, a major antioxidative enzyme, on retinal ganglion cells (RGCs). METHODS In the SOD1 total knockout (SOD1-deficient) mice, the level of superoxide anion was measured using dihydroethidium. The number of RGCs was counted in both the retinal sections and the flat-mount retinas after retrograde labeling. Thickness of nerve fiber layer (NFL) was measured in the sections, and the amount of neurofilament protein was measured by immunoblot analysis. Pattern electroretinogram (ERG), which reflects the function of retinal ganglion cells, dark-adapted ERG, and cone ERG were performed. The intraocular pressure (IOP) was measured with an induction-impact tonometer. The levels of SOD-1 and -2 were measured by ELISA, in the serum of 47 newly diagnosed consecutive normal tension glaucoma (NTG) patients and 44 consecutive control subjects. RESULTS The level of superoxide anion in the RGC layer was significantly higher in 24-week-old SOD1-deficient mice than in wild-type mice. The RGC number was significantly reduced in 24-week-old SOD1-deficient mice, although they were not in 8-week-old mice. The NFL thickness and neurofilament protein were reduced in 24-week-old SOD1-deficient mice. The amplitude of pattern ERG was significantly reduced, although dark-adapted and cone ERGs showed no impairment, in 24-week-old SOD1-deficient mice. The IOP level was not changed in the SOD1-deficient mice. The serum level of SOD1, but not SOD2, was significantly lower in the NTG patients than in the healthy controls. CONCLUSIONS SOD1 deficiency causes RGC vulnerability, which may be involved in the underlying condition of NTG.

Disruption of Cell-Cell Junctions and Induction of Pathological Cytokines in the Retinal Pigment Epithelium of Light-Exposed Mice

PURPOSE To elucidate the influences of light exposure on the retinal pigment epithelium (RPE) in vivo that may be involved in the pathogenesis of AMD. METHODS Six- to 7-week-old BALB/c mice were exposed to light at 2000 lux for 3 hours. Flat-mount RPE samples were immunostained with anti-ZO-1 antibody for evaluating tight junction, anti-N-cadherin, and anti-β-catenin antibodies for adherens junction, and stained with phalloidin for actin cytoskeleton. The reactive oxygen species (ROS) level was measured using DCFH-DA; Rho-associated coiled-coil forming kinase (ROCK) activity was by ELISA. Cytokine expression was analyzed by real-time RT-PCR and/or ELISA in the RPE-choroid, and macrophage recruitment was by real-time RT-PCR and immunohistochemistry. Either an antioxidant, N-Acetyl-L-cysteine (NAC), or a ROCK inhibitor, Y-27632, were administered to analyze the roles of ROS and ROCK activation, respectively. RESULTS Light exposure disrupted staining patterns of tight junctions, adherens junctions, and actin cytoskeleton in the RPE, where ROS was elevated. However, NAC treatment avoided the RPE changes, reducing ROS. ROCK activity increased after light exposure was suppressed by NAC, and the structural disruptions were suppressed by Y-27632. The levels of MCP-1, CCL11, and IL-6 increased after light exposure were suppressed by NAC. Light-induced MCP-1 and IL-6 were suppressed by Y-27632. Macrophage recruitment after light exposure was also suppressed either by NAC or Y-27632. CONCLUSIONS Light exposure induced ROS and Rho/ROCK activation, which caused disruption of cell-cell junctions (tight junctions and adherens junctions) and actin cytoskeleton, the RPEs barrier structure, and induced AMD-associated pathological changes in the RPE-choroid.

Neuroprotective response after photodynamic therapy : Role of vascular endothelial growth factor

BackgroundAnti-vascular endothelial growth factor (VEGF) drugs and/or photodynamic therapy (PDT) constitute current treatments targeting pathological vascular tissues in tumors and age-related macular degeneration. Concern that PDT might induce VEGF and exacerbate the disease has led us to current practice of using anti-VEGF drugs with PDT simultaneously. However, the underlying molecular mechanisms of these therapies are not well understood.MethodsWe assessed VEGF levels after PDT of normal mouse retinal tissue, using a laser duration that did not cause obvious tissue damage. To determine the role of PDT-induced VEGF and its downstream signaling, we intravitreally injected a VEGF inhibitor, VEGFR1 Fc, or a PI3K/Akt inhibitor, LY294002, immediately after PDT. Then, histological and biochemical changes of the retinal tissue were analyzed by immunohistochemistry and immunoblot analyses, respectively.ResultsAt both the mRNA and protein levels, VEGF was upregulated immediately and transiently after PDT. VEGF suppression after PDT resulted in apoptotic destruction of the photoreceptor cell layer in only the irradiated area during PDT. Under these conditions, activation of the anti-apoptotic molecule Akt was suppressed in the irradiated area, and levels of the pro-apoptotic protein BAX were increased. Intravitreal injection of a PI3K/Akt inhibitor immediately after PDT increased BAX levels and photoreceptor cell apoptosis.ConclusionCytotoxic stress caused by PDT, at levels that do not cause overt tissue damage, induces VEGF and activates Akt to rescue the neural tissue, suppressing BAX. Thus, the immediate and transient induction of VEGF after PDT is neuroprotective.

Angiopoietin-like Protein 2 Is a Multistep Regulator of Inflammatory Neovascularization in a Murine Model of Age-related Macular Degeneration

Choroidal neovascularization (CNV) is a pathogenic process of age-related macular degeneration, a vision-threatening disease. The retinal pigment epithelium and macrophages both influence CNV development. However, the underlying mechanisms remain obscure. Here, we focus on Angptl2 (angiopoietin-like protein 2), a cytokine involved in age-related systemic diseases. Angptl2 was originally identified as an adipocytokine and is also expressed in the eye. Using a laser-induced CNV model, we found that Angptl2 KO mice exhibited suppressed CNV development with reduced macrophage recruitment and inflammatory mediator induction. The mediators monocyte chemotactic protein-1, interleukin-1β (Il-1β), Il-6, matrix metalloprotease-9 (Mmp-9), and transforming growth factor-β1 (Tgf-β1) that were up-regulated during CNV development were all suppressed in the retinal pigment epithelium-choroid of CNV models generated in the Angptl2 KO mice. Bone marrow transplantation using wild-type and KO mice suggested that both bone marrow-derived and host-derived Angptl2 were responsible for macrophage recruitment and CNV development. Peritoneal macrophages derived from Angptl2 KO mice expressed lower levels of the inflammatory mediators. In the wild-type peritoneal macrophages and RAW264.7 cells, Angptl2 induced the mediators via integrins α4 and β2, followed by the downstream activation of NF-κB and ERK. The activation of NF-κB and ERK by Angptl2 also promoted macrophage migration. Therefore, Angptl2 from focal tissue might trigger macrophage recruitment, and that from recruited macrophages might promote expression of inflammatory mediators including Angptl2 in an autocrine and/or paracrine fashion to facilitate CNV development. Angptl2 might therefore represent a multistep regulator of CNV pathogenesis and serve as a new therapeutic target for age-related macular degeneration.

Secondary macular hole associated with central retinal vein occlusion treated with corticosteroid injection

Many investigations into the development of the fovea suggest that ROP and prematurity itself alter the development of the central retina. Central retinal thickness is signifi cantly higher in preterm children than in full-term children, which may be attributable mainly to ROP in the former. In a population-based study of 6-year-old children in Australia, Huynh et al. reported that mean central foveal thickness in the normal eye was 161 μm, whereas Franco and Cynthia reported that in ROP eyes, it is 229 μm. The foveas of subjects with a history of mild ROP have structural abnormalities that are probably a consequence of perturbations of neurovascular development. The alteration of normal development of the central fovea also explains why children with a history of mild ROP have mild defi cits in letter acuity that cannot be corrected by careful refraction. In the current case, there was no history of adverse events beyond the mild ROP that resolved spontaneously, and there were no cicatricial or vascular sequelae. To our knowledge, we are the fi rst to report a congenital retinal macrovessel with foveal dysplasia of ROP.

Cyclooxygenase inhibitor improved an exudative lesion of choroidal neovascularization in age-related macular degeneration.

Purpose To report a case in which a nonselective cyclooxygenase (COX) inhibitor improved an exudative lesion in age-related macular degeneration. Methods/Results A 64-year-old man had a complaint of metamorphopsia in the left eye. Visual acuity was 0.5 in the left eye. Fluorescein angiography and indocyanine angiography showed juxtafoveal occult choroidal neovascularization (CNV) in the left eye, and pegaptanib sodium 0.3 mg was administered once every 6 weeks. After 4 months, visual acuity improved to 0.8. After 8 months follow-up, optical coherence tomography (OCT) showed subretinal fluid (SRF) and retinal pigment epithelium (RPE) irregularity. Visual acuity was 0.4. We recommended further pegaptanib sodium injections, but the patient did not consent to the treatment. Onset of cellulitis of the left toe occurred 1 week before the scheduled visit after 9 months. The patient was treated with loxoprofen sodium (nonselective COX inhibitor) and cefdinir for 7 days. At 2 weeks after onset of cellulitis, SRF had disappeared and OCT showed improvement of RPE irregularity. Conclusions A COX inhibitor had an effect on vascular permeability in CNV and may have improved exudative changes.