論文

移植片対宿主病(GVHD)によるドライアイ(DE)発症に伴い近視化を伴う3症例を経験したので報告する。症例1は32歳、男性。Fanconi貧血(先天性再生不良性貧血)に対し非血縁者骨髄移植を行った。移植前の自覚的視力検査による等価球面度数(SE)は右眼-2.75Dと左眼-3.00Dであった。移植後4ヵ月時GVHDによるDEを発症し、同時期のSEは右眼-2.75Dと左眼-4.50Dで左眼の近視化を認めた。移植後7ヵ月時のSEは右眼-3.00Dと左眼-4.625Dで時期の差があるが両眼の近視化を認めた。症例2は34歳、女性。急性リンパ性白血病に対し非血縁骨髄移植を行った。移植前の自覚的視力検査によるSEは右眼+3.375Dと左眼+0.50D、移植後13ヵ月時にGVHDによるDEを発症し、同時期のSEは右眼+2.50Dと左眼0.00Dと両眼の近視化を認めた。症例3は40歳、女性。急性骨髄性白血病に対し血縁末梢血幹細胞移植を行った。移植前の自覚的視力検査のSEは右眼-1.50Dと左眼0.00D。移植後9ヵ月時にDEを発症し、同時期のSEは右眼-1.50Dと左眼-0.625Dで左眼の近視化を認めた。他覚的検査では移植前後のSEの差が右眼1.125D、左眼1.75Dと両眼の近視化を認めた。3症例ともに白内障は認めず、眼底に異常は認められなかった。炎症性疾患であるGVHDによるDE発症時期に、近視化する3症例を認めた。GVHDと近視化との関連に関する因子について今後さらなる検討が必要である。(著者抄録)

Degeneration or dysfunction of the retinal pigment epithelium (RPE) can induce secondary photoreceptor atrophy and catastrophic vision loss in patients with agerelated macular degeneration (AMD). AMD is the leading cause of vision loss in the elderly in industrialized countries and no cure exists for the "dry" or atrophic form to date. However, recent pre-clinical data from several groups suggests that embryonic stem cell-derived RPE cell transplantation may prevent photoreceptor degeneration in animal models of RPE degeneration. Another approach may be to derive RPE cells from autologous induced pluripotent stem cells (iPSCs) reprogrammed from dermal tissue. However, the safety of this approach has been questioned on several levels. In this chapter we will summarize work reported by several groups, including our own, that clearly demonstrate that transplanted RPE cells can provide anatomical and functional photoreceptor rescue in animal models of retinal degeneration. We will also discuss some of the prevailing concerns and challenges associated with this technique.

Vascular networks develop from a growing vascular front that responds to VEGF and other guidance cues. Angiogenesis is required for normal tissue function, but, under conditions of stress, inappropriate vascularization can lead to disease. Therefore, inhibition of angiogenic sprouting may prevent neovascularization in patients with blinding neovascular eye diseases, including macular degeneration. VEGF antagonists have therapeutic benefits but also can elicit off-target effects. Here, we found that the Ras pathway, which functions downstream of a wide range of cytokines including VEGF, is active in the growing vascular front of developing and pathological vascular networks. The endogenous Ras inhibitor p120RasGAP was expressed predominately in quiescent VEGF-insensitive endothelial cells and was ectopically downregulated in multiple neovascular models. MicroRNA-132 negatively regulated p120RasGAP expression. Experimental delivery of alpha-miR-132 to developing mouse eyes disrupted tip cell Ras activity and prevented angiogenic sprouting. This strategy prevented ocular neovascularization in multiple rodent models even more potently than the VEGF antagonist, VEGF-trap. Targeting microRNA-132 as a therapeutic strategy may prove useful for treating multiple neovascular diseases of the eye and for preventing vision loss regardless of the neovascular stimulus.

Current therapies directed at controlling vascular abnormalities in cancers and neovascular eye diseases target VEGF and can slow the progression of these diseases. While the critical role of VEGF in development has been well described, the function of locally synthesized VEGF in the adult eye is incompletely understood. Here, we show that conditionally knocking out Vegfa in adult mouse retinal pigmented epithelial (RPE) cells, which regulate retinal homeostasis, rapidly leads to vision loss and ablation of the choriocapillaris, the major blood supply for the outer retina and photoreceptor cells. This deletion also caused rapid dysfunction of cone photoreceptors, the cells responsible for fine visual acuity and color vision. Furthermore, Vegfa deletion showed significant downregulation of multiple angiogenic genes in both physiological and pathological states, whereas the deletion of the upstream regulatory transcriptional factors HIFs did not affect the physiological expressions of angiogenic genes. These results suggest that endogenous VEGF provides critical trophic support necessary for retinal function. Targeting factors upstream of VEGF, such as HIFs, may be therapeutically advantageous compared with more potent and selective VEGF antagonists, which may have more off-target inhibitory trophic effects.

PURPOSE. Retinal pigment epithelium (RPE) autologous grafts can be readily derived from induced pluripotent stem (iPS) cells. It is critical to stringently characterize iPS-RPE using standardized and quantifiable methods to be confident that they are safe and adequate replacements for diseased RPE before utilizing them in clinical settings. One important and required function is that the iPS-RPE phagocytose photoreceptor outer segments (POS). METHODS. We developed a flow cytometry-based assay to monitor binding and internalization of FITC labeled POS by ARPE-19, human fetal RPE (hfRPE), and two types of iPS-RPE. Expression and density of alpha(v)beta(5) integrin, CD36, and MerTK receptors, which are required for phagocytosis, were compared. RESULTS. Trypsinization of treated RPE cells results in the release of bound POS. The number of freed POS, the percentage of cells that internalized POS, the brightness of the FITC signal from the cells, and the surface density of the phagocytosis receptors on single RPE cells were measured using flow cytometry. These assays reveal that receptor density is dynamic during differentiation and this can affect the binding and internalization dynamics of the RPE cells. Highly differentiated iPS-RPE phagocytose POS more efficiently than hfRPE. CONCLUSIONS. Caution should be exercised to not use RPE grafts until demonstrating that they are fully functional. The density of the phagocytosis receptors is dynamic and may be used as a predictor for how well the iPS-RPE cells will function in vivo. The phagocytosis dynamics observed between iPS-RPE and primary RPE is very encouraging and adds to mounting evidence that iPS-RPE may be a viable replacement for dysfunctional or dying RPE in human patients. (Invest Ophthalmol Vis Sci. 2012;53:6282-6290) DOI:10.1167/iovs.12-9721

The vascular and nervous systems display a high degree of cross-talk and depend on each other functionally. In the vascularization of the central nervous system, astrocytes have been thought to sense tissue oxygen levels in hypoxia-inducible factors (HIFs)-dependent manner and control the vascular growth into the hypoxic area by secreting VEGF. However, recent genetic evidences demonstrate that not only astrocyte HIFs but also astrocyte VEGF expression is dispensable for developmental angiogenesis of the retina. This study demonstrates that hypoxia-inducible factor 1 alpha subunit (HIF-1 alpha), a key transcription factor involved in cellular responses to hypoxia, is most abundantly expressed in the neuroretina, especially retinal progenitor cells (RPCs). A neuroretina-specific knockout of HIF-1 alpha (alpha Cre(+)Hif1 alpha(flox/flox)) showed impaired vascular development characterized by decreased tip cell filopodia and reduced vessel branching. The astrocyte network was hypoplastic in alpha Cre(+)Hif1 alpha(flox/flox) mice. Mechanistically, platelet-derived growth factor A (PDGF-A), a mitogen for astrocytes, was downregulated in the neuroretina of alpha Cre(+)Hif1 alpha(flox/flox) mice. Supplementing PDGF-A restored reduced astrocytic and vascular density in alpha Cre(+)Hif1 alpha(flox/flox) mice. Our data demonstrates that the neuroretina but not astrocytes acts as a primary oxygen sensor which ultimately controls the retinal vascular development by regulating an angiogenic astrocyte template. (C) 2011 Elsevier Inc. All rights reserved.

Understanding pathogenesis at the molecular level is the first step toward developing new therapeutic approaches. Here, we review the molecular mechanisms of visual dysfunction in two common diseases, innate chorioretinal inflammation and diabetic retinopathy, and the role of the ubiquitin-proteasome system (UPS) in both processes. In innate chorioretinal inflammation, interleukin-6 family ligands induce STAT3 activation in photoreceptors, which causes UPS-mediated excessive degradation of the visual substance, rhodopsin. In diabetic retinopathy, angiotensin II type 1 receptor (AT1R) signaling activates ERK in the inner layers of the retina, causing UPS-mediated excessive degradation of the synaptic vesicle protein, synaptophysin. This latter effect may decrease synaptic activity, in turn adversely affecting neuronal survival. Both mechanisms involve increased UPS activity and the subsequent excessive degradation of a protein required for visual function. Finally, we review the therapeutic potential of regulating the UPS to protect tissue function, citing examples from clinical applications in other medical fields.

PURPOSE. AMP-activated protein kinase (AMPK) is a sensor of cellular energy status. The purpose of the present study was to elucidate the roles of AMPK in the pathogenesis of diabetic retinopathy using the known AMPK activators resveratrol and AICAR (5-aminoimidazole-4-carboxamide ribonucleoside) in a mouse model. METHODS. C57BL/6 mice with streptozotocin-induced diabetes were treated with resveratrol orally at 50 mg/kg for 7 days or with AICAR intraperitoneally at 100 mg/kg 24 hours before death. Retinal protein levels of phosphorylated and total AMPK, phosphorylated nuclear factor (NF)-kappa B p65, intercellular adhesion molecule (ICAM)-1, and vascular endothelial growth factor (VEGF) were evaluated by Western blot analysis or enzyme-linked immunosorbent assay. Retinal activity of sirtuin (SIRT) 1 was measured by deacetylase fluorometric assay. Leukocyte adhesion to the retinal vasculature was examined with a concanavalin A lectin perfusion-labeling technique. RESULTS. Induction of diabetes in mice led to retinal AMPK dephosphorylation, which was significantly reversed by either resveratrol or AICAR. Either resveratrol or AICAR significantly reversed SIRT1 deactivation and NF-kappa B phosphorylation, both of which were induced in the diabetic retina. Administration of resveratrol to diabetic mice significantly reduced diabetes-induced retinal leukocyte adhesion, together with retinal expression of ICAM-1 and VEGF. CONCLUSIONS. The present findings reveal that diabetes-induced retinal inflammation stems from downregulation of the AMPK pathway, leading subsequently to SIRT1 deactivation and NF-kappa B activation. The data also suggest the potential use of the AMPK activator resveratrol as a therapeutic agent for diabetic retinopathy. (Invest Ophthalmol Vis Sci. 2011;52:9142-9148) DOI: 10.1167/iovs.11-8041

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. (Invest Ophthalmol Vis Sci. 2011;52:4143-4150) DOI:10.1167/iovs.106294

Light damage to the retina accelerates retinal degeneration in human diseases and rodent models. Recently, the polyphenolic phytoalexin resveratrol has been shown to exert various bioactivities in addition to its classical antioxidant property. In the present study, we investigated the effect of resveratrol on light-induced retinal degeneration together with its underlying molecular mechanisms. BALB/c mice with light exposure (5000-lux white light for 3 hours) were orally pretreated with resveratrol at a dose of 50 mg/kg for 5 days. Retinal damage was evaluated by TdT-mediated dUTP nick-end labeling, outer nuclear layer morphometry, and electroretinography. Administration of resveratrol to mice with light exposure led to a significant suppression of light-induced pathological parameters, including TdT-mediated dUTP nick-end labeling-positive retinal cells, outer nuclear layer thinning, and electroretinography changes. To clarify the underlying molecular mechanisms, the nuclear translocation of activator protein-1 subunit c-fos was evaluated by enzyme-linked immunosorbent assay, and the retinal activity of sirtuin 1 was measured by deacetylase fluorometric assay. Retinal activator protein-1 activation, up-regulated following light exposure, was significantly reduced by application of resveratrol. In parallel, retinal sirtuin 1 activity, reduced in animals with light damage, was significantly augmented by resveratrol treatment. Our data suggest the potential use of resveratrol as a therapeutic agent to prevent retinal degeneration related to light damage. (Am J Pathol 2010, 1 77:1725-1731; DOI: 10.2353/ajpath.2010.100098)

Purpose: To establish a novel murine intraocular lens (IOL) implantation model to study the protective effects of colored-IOLs against retinal phototoxicity. Methods: Two-millimeter diameter IOL buttons were created from IOLs for clinical use. Extra-capsular crystalline lens extraction and IOL implantation were performed in BALB/c mice using a technique similar to human cataract surgery. For light exposure experiments, mice were exposed to 5,000 LUX of white light for 24 h on the day after surgery. To investigate the protective effects of yellow IOL against light exposure, ERG measurements were conducted in vivo, followed by TdT-mediated dUTP Nick-End Labeling (TUNEL) and outer nuclear layer (ONL) thickness measurement of retinal tissue in yellow or clear IOL-implanted mice and control mice without surgery. Results: IOLs were successfully implanted in all animals, and IOL buttons without haptics were well stabilized in the capsular bag. Murine eyes developed posterior capsule opacification (PCO) after IOL implantation by postoperative day 5 at the latest. In contrast to the clear IOL-implanted animals stimulated by light exposure, the yellow IOL-implanted animals had significantly reduced numbers of TUNEL-positive cells and retained thickness of the ONL. The ERG showed that yellow IOL implantation prevents a decrease of amplitude in both the a-wave and b-wave compared with clear IOL implantation. Conclusions: We established a new animal model of IOL implantation and demonstrated the protective effects of colored-IOL against retinal phototoxicity after cataract surgery.

PURPOSE. Resveratrol is known as one of the antioxidant polyphenols contained in red wine and grape skin. The purpose of the present study was to investigate the role of resveratrol in ocular inflammation in endotoxin-induced uveitis (EIU). METHODS. EIU was induced in male C57/B6 mice at the age of 6 weeks by a single intraperitoneal injection of lipopolysaccharide (LPS). Animals had received oral supplementation of resveratrol at the doses of 5, 50, 100, or 200 mg/kg for 5 days until LPS injection. Twenty-four hours after LPS administration, leukocyte adhesion to the retinal vasculature was examined with a concanavalin A lectin perfusion-labeling technique. Retinal and retinal pigment epithelium (RPE)-choroidal levels of intercellular adhesion molecule (ICAM)-1, monocyte chemotactic protein (MCP)-1, and 8-hydroxy-2'-deoxyguanosine (8-OHdG) and nuclear translocation of nuclear factor (NF)-kappa B p65 were evaluated by enzyme-linked immunosorbent assay. Retinal and RPE-choroidal activities of silent information regulator two ortholog (SIRT) 1 were measured by deacetylase fluorometric assay. RESULTS. Resveratrol pretreatment led to significant and dose-dependent suppression of leukocyte adhesion to retinal vessels of EIU mice compared with vehicle application. Protein levels of MCP-1 and ICAM-1 in the retina and the RPE-choroid of EIU animals were significantly reduced by resveratrol administration. Importantly, resveratrol-treated animals showed significant decline of retinal 8-OHdG generation and nuclear NF-kappa B P65 translocation, both of which were upregulated after EIU induction. RPE-choroidal SIRT1 activity, reduced in EIU animals, was significantly augmented by treatment with resveratrol. CONCLUSIONS. Resveratrol prevented EIU-associated cellular and molecular inflammatory responses by inhibiting oxidative damage and redox-sensitive NF-kappa B activation. (Invest Ophthalmol Vis Sci. 2009; 50: 3512-3519) DOI: 10.1167/iovs.08-2666

Inflammatory cytokines cause tissue dysfunction. We previously reported that retinal inflammation down-regulates rhodopsin expression and impairs visual function by an unknown mechanism. Here, we demonstrate that rhodopsin levels were preserved by suppressor of cytokine signaling 3 (SOCS3), a negative feedback regulator of STAT3 activation. SOCS3 was expressed mainly in photoreceptor cells in the retina. In the SOCS3-deficient retinas, rhodopsin protein levels dropped sooner, and the reduction was more profound than in the wild type. Visual dysfunction, measured by electroretinogram, was prolonged in retina-specific SOCS3 conditional knockout mice. Visual dysfunction and decreased rhodopsin levels both correlated with increased STAT3 activation enhanced by SOCS3 deficiency. Interleukin 6, one of the inflammatory cytokines found during retinal inflammation, activated STAT3 and decreased rhodopsin protein in adult retinal explants. This was enhanced by inhibiting SOCS3 function in vitro, indicating that rhodopsin reduction was not a secondary effect in the mutant mice. Interestingly, in the inflamed SOCS3-deficient adult retina, rhodopsin decreased post-transcriptionally at least partly through ubiquitin-proteasome-dependent degradation accelerated by STAT3 activation and not transcriptionally as in the developing retina, on which we reported previously. A STAT3-dependent E3 ubiquitin ligase, Ubr1, was responsible for rhodopsin degradation and was up-regulated in the inflamed SOCS3-deficient retinas. These results indicate that in wild-type animals, a decrease in rhodopsin during inflammation is minimized by endogenous SOCS3. However, when STAT3 activation exceeds some threshold beyond the compensatory activity of endogenous SOCS3, rhodopsin levels decrease. These findings suggest SOCS3 as a potential therapeutic target molecule for protecting photoreceptor cell function during inflammation.

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.

PURPOSE. To investigate whether the induction of cellular immunity against vascular endothelial growth factor receptor ( VEGFR) 2 inhibits the development of choroidal neovascularization (CNV). METHODS. H-2Db-restricted peptide corresponding to amino acids 400 to 408 of VEGFR2 was used as an epitope peptide. Dendritic cells (DCs) were harvested from bone marrow progenitors of C57BL/6 mice. Six-week-old C57BL/6 mice received subcutaneous injections of the epitope peptide-pulsed mature DCs three times at 6-day intervals. After the third immunization, laser photocoagulation was performed to induce CNV. One week after photocoagulation, mice were killed to harvest the choroid and splenocytes. CNV volume was evaluated by volumetric measurements. To confirm the specific immunogenicity of the epitope peptides in C57BL/6 mice, CD8 T cells isolated from harvested splenocytes were restimulated to measure interferon (IFN)-gamma and tumor necrosis factor (TNF)-gamma production through enzyme-linked immunospot assay and ELISA. To determine the T-cell subset responsible for the immunotherapy, mice were intraperitoneally injected with an anti-CD4 or anti-CD8 depletion antibody. RESULTS. CNV volume was significantly lower in mice immunized with the VEGFR2 epitope peptide than in those not immunized or immunized with a control peptide gp70. Cytokine assays showed the peptide-specific production of IFN-gamma and TNF-alpha from the CD8 T cells in a dose-dependent manner. In vivo depletion of CD8, but not CD4, T cells significantly reversed the suppressive effect of the VEGFR2 peptide-pulsed DC vaccination on CNV to the level observed in nonimmunized or gp70-immunized animals. CONCLUSIONS. These results indicate that the VEGFR2 peptide-specific induction of cellular immunity inhibits CNV through the cytotoxicity of CD8 T cells. Results of the present study suggested the possibility of DC vaccination targeting VEGFR2 as a novel therapeutic strategy for CNV.

Suppressor of cytokine signaling 3 (SOCS3) is an intracellular, ligand-induced negative feedback modulator of STAT3 activation that acts during inflammation. Here, we dernonstrate that SOCS3 expression is important for normal retinal development in the perinatal period. STAT3 is highly activated in the late-embryonic retina, then downregulated at postnatal day 0 (PO), presumably by the depletion of upstream ligands. We found that SOCS3 was required after PO to shut down the residual STAT3 activation; this loss of activated STAT3 leads to Rhodopsin expression and rod photoreceptor cell differentiation. SOCS3 deficiency failed to terminate STAT3 activation, thereby delaying expression of Rhodopsin and its upstream transcription factor, crx. Development subsequently continued, but its course was temporally erratic, probably because of faulty compensation. Interestingly, SOCS3 protein expression was first detected postnatally, after STAT3 activation was mostly downregulated. It initially appeared in some of the presumptive photoreceptor cells and gradually spread. SOCS3 mRNA level was constant from the late-embryonic to early-postnatal period. Post-transcriptional inhibition of SOCS3 protein expression maintains a high STAT3 activation during late embryogenesis, and after PO, releasing from the inhibition promptly terminates STAT3 activation. Thus, SOCS3 can act as a temporal fine-tuner of STAT3 activation during photoreceptor cell differentiation. (c) 2006 Elsevier Inc. All rights reserved.

Purpose The determination of the molecular mechanism underlying retinal pathogenesis and visual dysfunction during innate inflammation, and the treatment effect of rapamycin thereon. Methods The endotoxin-induced uveitis and retinitis mouse model was established by injecting lipopolysaccharide. The mice were subsequently treated with rapamycin, a mammalian target of rapamycin (mTOR) inhibitor. The rhodopsin mRNA and protein expression level in the retina and the photoreceptor outer segment (OS) length in immunohistochemical stainings were measured, and visual function was recorded by electroretinography. Inflammatory cytokines, their related molecules, mTOR, and LC3 levels were measured by real-time PCR and/or immunoblotting. Leukocyte adhesion during inflammation was analyzed using concanavalin A lectin. Results The post-transcriptional reduction in the visual pigment of rod photoreceptor cells, rhodopsin, OS shortening, and rod photoreceptor cell dysfunction during inflammation were suppressed by rapamycin. Activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappa B) and induction of inflammatory cytokines, such as interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1), and the activation of the downstream signaling protein, signal transducer and activator of transcription 3 (STAT3), which reduces rhodopsin in the retina during inflammation, were attenuated by rapamycin. Increased leukocyte adhesion was also attenuated by rapamycin. Interestingly, although mTOR activation was observed after NF-kappa B activation, mTOR inhibition suppressed NF-kappa B activation at the early phase, indicating that the basal level of activated mTOR was sufficient to activate NF-kappa B in the retina. In addition, the inhibition of NF-kappa B suppressed mTOR activation, suggesting a positive feedback loop of mTOR and NF-kappa B during inflammation. The ratio of LC3II to LC3I, which reflects autophagy induction, was not changed by inflammation but was increased by rapamycin. Conclusions Our results propose the potential use of rapamycin as a neuroprotective therapy to suppress local activated mTOR levels, related inflammatory molecules, and the subsequent visual dysfunction during retinal inflammation.

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment epithelium (RPE) cells. RPE provide critical support for photoreceptors and death or dysfunction of RPE cells is characteristic of age-related macular degeneration (AMD), the leading cause of permanent vision loss in people age 55 and older. While no cure for AMD has been identified, implantation of healthy RPE in diseased eyes may prove to be an effective treatment, and large numbers of RPE cells can be readily generated from pluripotent stem cells. Several interesting questions regarding the safety and efficacy of RPE cell delivery can still be examined in animal models, and well-accepted protocols used to inject RPE have been developed. The technique described here has been used by multiple groups in various studies and involves first creating a hole in the eye with a sharp needle. Then a syringe with a blunt needle loaded with cells is inserted through the hole and passed through the vitreous until it gently touches the RPE. Using this injection method, which is relatively simple and requires minimal equipment, we achieve consistent and efficient integration of stem cell-derived RPE cells in between the host RPE that prevents significant amount of photoreceptor degeneration in animal models. While not part of the actual protocol, we also describe how to determine the extent of the trauma induced by the injection, and how to verify that the cells were injected into the subretinal space using in vivo imaging modalities. Finally, the use of this protocol is not limited to RPE cells; it may be used to inject any compound or cell into the subretinal space.