Toshio Hisatomi

Tumor Necrosis Factor-α Mediates Oligodendrocyte Death and Delayed Retinal Ganglion Cell Loss in a Mouse Model of Glaucoma

Glaucoma is a widespread ocular disease characterized by a progressive loss of retinal ganglion cells (RGCs). Previous studies suggest that the cytokine tumor necrosis factor-α (TNF-α) may contribute to the disease process, although its role in vivo and its mechanism of action are unclear. To investigate pathophysiological mechanisms in glaucoma, we induced ocular hypertension (OH) in mice by angle closure via laser irradiation. This treatment resulted in a rapid upregulation of TNF-α, followed sequentially by microglial activation, loss of optic nerve oligodendrocytes, and delayed loss of RGCs. Intravitreal TNF-α injections in normal mice mimicked these effects. Conversely, an anti-TNF-α-neutralizing antibody or deleting the genes encoding TNF-α or its receptor, TNFR2, blocked the deleterious effects of OH. Deleting the CD11b/CD18 gene prevented microglial activation and also blocked the pathophysiological effects of OH. Thus TNF-α provides an essential, although indirect, link between OH and RGC loss in vivo. Blocking TNF-α signaling or inflammation, therefore, may be helpful in treating glaucoma.

The critical role of ocular-infiltrating macrophages in the development of choroidal neovascularization

Choroidal neovascularization (CNV) is directly related to visual loss in some eye diseases, such as age‐related macular degeneration. Although several human histological studies have suggested the participation of macrophages in CNV formation, the precise mechanisms are still not fully understood. In this study, we elucidated the role of ocular‐infiltrating macrophages in experimental CNV using CCR2 knockout (KO) mice, wild‐type mice, and C57BL/6 (B6) mice. CCR2 is the receptor of monocyte chemoattractant protein‐1, and the number of infiltrating macrophage and the area of CNV were significantly reduced in CCR2 KO mice. Enriched ocular‐infiltrating macrophages from B6 mice actually showed angiogenic ability in a dorsal air sac assay. Moreover, their expression of class II, CD40, B7‐1 and B7‐2 molecules, and the mRNA for potential angiogenic factors, such as vascular endothelial growth factor, basic fibroblast growth factor, and tumor necrosis factor α, was also observed. Collectively, we conclude that ocular‐infiltrating macrophages play an important role in CNV generation.

Monocyte chemoattractant protein 1 mediates retinal detachment-induced photoreceptor apoptosis.

Photoreceptor apoptosis is a major cause of visual loss in retinal detachment (RD) and several other visual disorders, but the underlying mechanisms remain elusive. Recently, increased expression of monocyte chemoattractant protein 1 (MCP-1) was reported in vitreous humor samples of patients with RD and diabetic retinopathy as well as in the brain tissues of patients with neurodegenerative diseases, including Alzheimers disease and multiple sclerosis. Here we report that MCP-1 plays a critical role in mediating photoreceptor apoptosis in an experimental model of RD. RD led to increased MCP-1 expression in the Müller glia and increased CD11b+ macrophage/microglia in the detached retina. An MCP-1 blocking antibody greatly reduced macrophage/microglia infiltration and RD-induced photoreceptor apoptosis. Confirming these results, MCP-1 gene-deficient mice showed significantly reduced macrophage/microglia infiltration after RD and very little photoreceptor apoptosis. In primary retinal mixed cultures, MCP-1 was cytotoxic for recoverin+ photoreceptors, and this toxicity was eliminated through immunodepleting macrophage/microglia from the culture. In vivo, deletion of the gene encoding CD11b/CD18 nearly eliminated macrophage/microglia infiltration to the retina after RD and the loss of photoreceptors. Thus, MCP-1 expression and subsequent macrophage/microglia infiltration and activation are critical for RD-induced photoreceptor apoptosis. This pathway may be an important therapeutic target for preventing photoreceptor apoptosis in RD and other CNS diseases that share a common etiology.

Brilliant Blue G Selectively Stains The Internal Limiting Membrane/brilliant Blue G–assisted Membrane Peeling

Purpose: To report the use of the dye brilliant blue G (BBG) for staining of the internal limiting membrane (ILM) during macular hole (MH) and epiretinal membrane (ERM) surgery. Methods: This study was designed as an interventional, noncomparative, prospective, clinical case series. Twenty eyes from 20 consecutive patients with MH or ERM underwent BBG-assisted ILM and ERM removal. In MH cases, a posterior vitreous detachment was created, followed by the injection of 0.25 mg/mL BBG solution into the vitreous cavity and immediate washout of the BBG. This technique improved visualization of the ILM, enabling peeling and surgery to be performed successfully. However, in ERM cases, staining of the ERM could not be confirmed at this concentration. Finally, the ILM including the ERM was removed in all cases. Preoperative and postoperative ophthalmic examinations were performed. Results: Postoperatively, 17 patients (85%) had visual acuity improved by at least 2 Snellen lines. No adverse effects were observed postoperatively during the observation period (mean follow-up ± SD, 7.3 ± 1.0 months). Conclusions: BBG selectively stains the ILM. This technique can facilitate the management of MH and ERM surgery without any adverse effects, as was shown in this short-term study.

Relocalization of Apoptosis-Inducing Factor in Photoreceptor Apoptosis Induced by Retinal Detachment in Vivo

Apoptosis-inducing factor (AIF) is a novel mediator in apoptosis. AIF is a flavoprotein that is normally confined to the mitochondrial intermembrane space, yet translocates to the nucleus in several in vitro models of apoptosis. To investigate the role of AIF in the apoptotic process in vivo, we induced retinal detachment (RD) by subretinal injection of sodium hyaluronate, either in Brown Norway rats or in C3H mice. Apoptotic DNA fragmentation, as determined by terminal nick-end labeling, was most prominent 3 days after RD. The subcellular localization of AIF was examined by immunohistochemistry and immunoelectron microscopy. In normal photoreceptor cells, AIF was present in the mitochondrion-rich inner segment. However, AIF was found in the nucleus after RD. Photoreceptor apoptosis developed similarly in C3H control mice, and in mice bearing the gld or lpr mutations, indicating that cell death occurs independently from the CD95/CD95 ligand system. Both the mitochondrio-nuclear transition of AIF localization and the nuclear DNA fragmentation were inhibited by subretinal application of brain-derived neurotrophic factor. To our knowledge, this is the first description of AIF relocalization occurring in a clinically relevant, in vivo model of apoptosis.

Preclinical investigation of internal limiting membrane staining and peeling using intravitreal brilliant blue G.

Purpose: To investigate the effects of intravitreal brilliant blue G (BBG) on the morphology and functions of the retina and its possible use for staining and peeling of the internal limiting membrane (ILM). Methods: Rat eyes (n = 78) underwent gas compression vitrectomy. BBG solution was then injected into the vitreous cavity. The eyes were enucleated at 2 weeks and 2 months. Light as well as electron microscopy, terminal nick-end labeling staining, and electroretinography (ERG) were used to investigate retinal damage and function. To test the clinical potential of BBG, ILM staining was evaluated in primate eyes after pars plana vitrectomy followed by ILM peeling. Results: In the rat eyes, no pathologic changes were observed with light microscopy. Electron microscopy revealed that high doses of BBG induced vacuolization in the inner retinal cells, but apoptosis was not detected. There was no reduction in the amplitude of the ERG waves. In the primate eyes, the ILM was clearly visualized after the intravitreous injection of BBG and was peeled off easily from the retina. Conclusions: These results demonstrate that BBG, which has low potential for toxicity, high staining ability, and ease of handling, is a good candidate dye for ILM peeling.

Critical role of photoreceptor apoptosis in functional damage after retinal detachment

Purpose. Although apoptosis is assumed to play a pivotal role in retinal function loss, its mechanism and real influence on retinal function are still unclear. To investigate the relation between retinal function and apoptosis, we studied photoreceptor apoptosis in experimental retinal detachment (RD). Methods. We induced RD by subretinal injection of sodium hyaluronate in Brown Norway rats. Apoptotic photoreceptors were detected by TdT-dUTP Terminal Nick-End Labeling (TUNEL). To evaluate the function of the detached retina, electroretinograms (ERGs) were taken on day 1, 3 with corneal electrodes and full-field stimulation. Results. Apoptotic DNA fragmentation appeared 12 hours after RD, was most prominent on day 3, and decreased thereafter. The ERGs showed that the amplitudes of dark-adapted a-waves and light adapted 2Hz b-waves decreased immediately after RD and continued to decrease over time. The administration of Fas/Fc chimera recombinant protein or a caspase inhibitor, Z-VAD.fmk, failed to prevent either photoreceptor apoptosis or retinal functional damage. In contrast, brain derived neurotrophic factor (BDNF) and basic fibroblast growth factor (bFGF) significantly impeded both apoptosis and dysfunction. The ERGs recognized the functional changes sensitively, and these ERG changes correlated well to the amount of photoreceptor apoptosis. Immunohistochemical study showed that apoptosis-inducing factor (AIF), a novel caspase-independent apoptotic factor, was relocalized from mitochondria to the nucleus in this process. Conclusions. The present results showed that apoptosis was a key phenomenon in the retinal dysfunction in RD and that this process was transmitted mainly by mitochondria-dependent pathways rather than Fas/Fas-L or downstream caspase dependent pathways.

Receptor interacting protein kinase mediates necrotic cone but not rod cell death in a mouse model of inherited degeneration

Retinitis pigmentosa comprises a group of inherited retinal photoreceptor degenerations that lead to progressive loss of vision. Although in most cases rods, but not cones, harbor the deleterious gene mutations, cones do die in this disease, usually after the main phase of rod cell loss. Rod photoreceptor death is characterized by apoptotic features. In contrast, the mechanisms and features of subsequent nonautonomous cone cell death remain largely unknown. In this study, we show that receptor-interacting protein (RIP) kinase mediates necrotic cone cell death in rd10 mice, a mouse model of retinitis pigmentosa caused by a mutation in a rod-specific gene. The expression of RIP3, a key regulator of programmed necrosis, was elevated in rd10 mouse retinas in the phase of cone but not rod degeneration. Although rd10 mice lacking Rip3 developed comparable rod degeneration to control rd10 mice, they displayed a significant preservation of cone cells. Ultrastructural analysis of rd10 mouse retinas revealed that a substantial fraction of dying cones exhibited necrotic morphology, which was rescued by Rip3 deficiency. Additionally, pharmacologic treatment with a RIP kinase inhibitor attenuated histological and functional deficits of cones in rd10 mice. Thus, necrotic mechanisms involving RIP kinase are crucial in cone cell death in inherited retinal degeneration, suggesting the RIP kinase pathway as a potential target to protect cone-mediated central and peripheral vision loss in patients with retinitis pigementosa.

Programmed necrosis, not apoptosis, is a key mediator of cell loss and DAMP-mediated inflammation in dsRNA-induced retinal degeneration

There is no known treatment for the dry form of an age-related macular degeneration (AMD). Cell death and inflammation are important biological processes thought to have central role in AMD. Here we show that receptor-interacting protein (RIP) kinase mediates necrosis and enhances inflammation in a mouse model of retinal degeneration induced by dsRNA, a component of drusen in AMD. In contrast to photoreceptor-induced apoptosis, subretinal injection of the dsRNA analog poly(I : C) caused necrosis of the retinal pigment epithelium (RPE), as well as macrophage infiltration into the outer retinas. In Rip3−/− mice, both necrosis and inflammation were prevented, providing substantial protection against poly(I : C)-induced retinal degeneration. Moreover, after poly(I : C) injection, Rip3−/− mice displayed decreased levels of pro-inflammatory cytokines (such as TNF-α and IL-6) in the retina, and attenuated intravitreal release of high-mobility group box-1 (HMGB1), a major damage-associated molecular pattern (DAMP). In vitro, poly(I : C)-induced necrosis were inhibited in Rip3-deficient RPE cells, which in turn suppressed HMGB1 release and dampened TNF-α and IL-6 induction evoked by necrotic supernatants. On the other hand, Rip3 deficiency did not modulate directly TNF-α and IL-6 production after poly(I : C) stimulation in RPE cells or macrophages. Therefore, programmed necrosis is crucial in dsRNA-induced retinal degeneration and may promote inflammation by regulating the release of intracellular DAMPs, suggesting novel therapeutic targets for diseases such as AMD.

Laboratory Evidence of Sustained Chronic Inflammatory Reaction in Retinitis Pigmentosa

PURPOSE To study the nature of retinal inflammatory response in rd10 mice, an animal model of retinitis pigmentosa (RP), and to investigate the effect of an antioxidant on retinal inflammation and photoreceptor apoptosis. DESIGN Experimental study. PARTICIPANTS AND CONTROLS This study included 42 untreated rd10 mice, 30 N-acetylcysteine (NAC)-treated rd10 mice, and 20 C57BL/6 mice as controls. METHODS Real-time polymerase chain reaction (PCR) was performed to evaluate the expression levels of inflammatory factors (proinflammatory cytokines and chemokines) in rd10 mouse retinas. Rd10 mice were treated with an antioxidant NAC, and its effect on retinal inflammation and photoreceptor apoptosis were examined by immunohistochemistry. MAIN OUTCOME MEASURES Real-time PCR and immunohistochemistry. RESULTS We demonstrated sequential events involving increased expression of proinflammatory cytokines and chemokines, activation of microglia, and photoreceptor apoptosis during retinal degeneration of rd10 mice. Furthermore, antioxidant treatment with NAC prevented the photoreceptor cell death along with suppression of inflammatory factors and microglial activation. CONCLUSIONS Sustained chronic inflammatory reaction may contribute to the pathogenesis of retinal degeneration in rd10 mice, suggesting interventions for ocular inflammatory reaction using antioxidants as a potential treatment for patients with RP. FINANCIAL DISCLOSURE(S) The authors have no proprietary or commercial interest in any of the materials discussed in this article.