The objective of this study was to determine the role of individual NFAT isoforms in TNFα-induced retinal leukostasis. To this end, human retinal microvascular endothelial cells (HRMEC) transfected with siRNA targeting individual NFAT isoforms were treated with TNFα, and qRT-PCR was used to examine the contribution of each isoform to the TNFα-induced upregulation of leukocyte adhesion proteins. This showed that NFATc1 siRNA increased ICAM1 expression, NFATc2 siRNA reduced CX3CL1, VCAM1, SELE, and ICAM1 expression, NFATc3 siRNA increased CX3CL1 and SELE expression, and NFATc4 siRNA reduced SELE expression. Transfected HRMEC monolayers were also treated with TNFα and assayed using a parallel plate flow chamber, and both NFATc2 and NFATc4 knockdown reduced TNFα-induced cell adhesion. The effect of isoform-specific knockdown on TNFα-induced cytokine production was also measured using protein ELISAs and conditioned cell culture medium, and showed that NFATc4 siRNA reduced CXCL10, CXCL11, and MCP-1 protein levels. Lastly, the CN/NFAT-signaling inhibitor INCA-6 was shown to reduce TNFα-induced retinal leukostasis in vivo. Together, these studies show a clear role for NFAT-signaling in TNFα-induced retinal leukostasis, and identify NFATc2 and NFATc4 as potentially valuable therapeutic targets for treating retinopathies in which TNFα plays a pathogenic role.

Ischemia-induced hypoxia elicits retinal neovascularization and is a major component of several blinding retinopathies such as retinopathy of prematurity (ROP), diabetic retinopathy (DR) and retinal vein occlusion (RVO). Currently, noninvasive imaging techniques capable of detecting and monitoring retinal hypoxia in living systems do not exist. Such techniques would greatly clarify the role of hypoxia in experimental and human retinal neovascular pathogenesis. In this study, we developed and characterized HYPOX-4, a fluorescence-imaging probe capable of detecting retinal-hypoxia in living animals. HYPOX-4 dependent in vivo and ex vivo imaging of hypoxia was tested in a mouse model of oxygen-induced retinopathy (OIR). Predicted patterns of retinal hypoxia were imaged by HYPOX-4 dependent fluorescence activity in this animal model. In retinal cells and mouse retinal tissue, pimonidazole-adduct immunostaining confirmed the hypoxia selectivity of HYPOX-4. HYPOX-4 had no effect on retinal cell proliferation as indicated by BrdU assay and exhibited no acute toxicity in retinal tissue as indicated by TUNEL assay and electroretinography (ERG) analysis. Therefore, HYPOX-4 could potentially serve as the basis for in vivo fluorescence-based hypoxia-imaging techniques, providing a tool for investigators to understand the pathogenesis of ischemic retinopathies and for physicians to address unmet clinical needs.

To demonstrate the utility of a novel in vivo molecular imaging probe, HYPOX-4, to detect and image retinal hypoxia in real time, in a mouse model of retinal vein occlusion (RVO).

Optical coherence tomography (OCT) has become a standard-of-care in retinal imaging. OCT allows non-invasive imaging of the tissue structure but lacks specificity to contrast agents that could be used for in vivo molecular imaging. Photothermal OCT (PT-OCT) is a functional OCT-based technique that has been developed to detect absorbers in a sample. We demonstrate in vivo PT-OCT in the eye for the first time on both endogenous (melanin) and exogenous (gold nanorods) absorbers. Pigmented mice and albino mice (n = 6 eyes) were used to isolate the photothermal signal from the melanin in the retina. Pigmented mice with laser-induced choroidal neovascularization lesions (n = 7 eyes) were also imaged after a systemic injection of gold nanorods to observe their passive accumulation in the retina. This experiment demonstrates the feasibility of PT-OCT to image the distribution of both endogenous and exogenous absorbers in the mouse retina.

Chronic hyperglycemia is thought to be the major stimulator of retinal dysfunction in diabetic retinopathy (DR). Thus, many diabetes-related systemic factors have been overlooked as inducers of DR pathology. Cell culture models of retinal cell types are frequently used to mechanistically study DR, but appropriate stimulators of DR-like factors are difficult to identify. Furthermore, elevated glucose, a gold standard for cell culture treatments, yields little to no response from many primary human retinal cells. Thus, the goal of this project was to demonstrate the effectiveness of the free fatty acid, palmitic acid and compare its use alone and in combination with elevated glucose as a stimulus for human Müller cells, a retinal glial cell type that is activated early in DR pathogenesis and uniquely responsive to fatty acids. Using RNA sequencing, we identified a variety of DR-relevant pathways, including NFκB signaling and inflammation, intracellular lipid signaling, angiogenesis, and MAPK signaling, that were stimulated by palmitic acid, while elevated glucose alone did not significantly alter any diabetes-relevant pathways. Co-treatment of high glucose with palmitic acid potentiated the expression of several DR-relevant angiogenic and inflammatory targets, including PTGS2 (COX-2) and CXCL8 (IL-8).

The peroxisome proliferator-activated receptor beta/delta (PPARβ/δ) is a transcription factor with roles in metabolism, angiogenesis, and inflammation. It has yet undefined roles in retinal inflammation and diabetic retinopathy (DR). We used RNA-seq to better understand the role of the antagonist and inverse agonist of PPARβ/δ, GSK0660, in TNFα-induced inflammation. Understanding the underlying mechanisms of vascular inflammation could lead to new treatments for DR.

Free fatty acid dysregulation in diabetics may elicit the release of inflammatory cytokines from Müller cells (MC), promoting the onset and progression of diabetic retinopathy (DR). Palmitic acid (PA) is elevated in the sera of diabetics and stimulates the production of the DR-relevant cytokines by MC, including IL-1β, which induces the production of itself and other inflammatory cytokines in the retina as well. In this study we propose that experimental elevation of cytochrome P450 epoxygenase (CYP)-derived epoxygenated fatty acids, epoxyeicosatrienoic acid (EET) and epoxydocosapentaenoic acid (EDP), will reduce PA- and IL-1β-induced MC inflammation. Broad-spectrum CYP inhibition by SKF-525a increased MC expression of inflammatory cytokines. Exogenous 11,12-EET and 19,20-EDP significantly decreased PA- and IL-1β-induced MC expression of IL-1β and IL-6. Both epoxygenated fatty acids significantly decreased IL-8 expression in IL-1β-induced MC and TNFα in PA-induced MC. Interestingly, 11,12-EET and 19,20-EDP significantly increased TNFα in IL-1β-treated MC. GSK2256294, a soluble epoxide hydrolase (sEH) inhibitor, significantly reduced PA- and IL-1β-stimulated MC cytokine expression. 11,12-EET and 19,20-EDP were also found to decrease PA- and IL-1β-induced NFκB-dependent transcriptional activity. These data suggest that experimental elevation of 11,12-EET and 19,20-EDP decreases MC inflammation in part by blocking NFκB-dependent transcription and may represent a viable therapeutic strategy for inhibition of early retinal inflammation in DR.

Diabetic retinopathy (DR) is triggered by retinal cell damage stimulated by the diabetic milieu, including increased levels of intraocular free fatty acids. Free fatty acids may serve as an initiator of inflammatory cytokine release from Müller cells, and the resulting cytokines are potent stimulators of retinal endothelial pathology, such as leukostasis, vascular permeability, and basement membrane thickening. Our previous studies have elucidated a role for peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) in promoting several steps in the pathologic cascade in DR, including angiogenesis and expression of inflammatory mediators. Furthermore, PPARβ/δ is a known target of lipid signaling, suggesting a potential role for this transcription factor in fatty acid-induced retinal inflammation. Therefore, we hypothesized that PPARβ/δ stimulates both the induction of inflammatory mediators by Müller cells as well the paracrine induction of leukostasis in endothelial cells (EC) by Müller cell inflammatory products. To test this, we used the PPARβ/δ inhibitor, GSK0660, in primary human Müller cells (HMC), human retinal microvascular endothelial cells (HRMEC) and mouse retina. We found that palmitic acid (PA) activation of PPARβ/δ in HMC leads to the production of pro-angiogenic and/or inflammatory cytokines that may constitute DR-relevant upstream paracrine inflammatory signals to EC and other retinal cells. Downstream, EC transduce these signals and increase their synthesis and release of chemokines such as CCL8 and CXCL10 that regulate leukostasis and other cellular events related to vascular inflammation in DR. Our results indicate that PPARβ/δ inhibition mitigates these upstream (MC) as well as downstream (EC) inflammatory signaling events elicited by metabolic stimuli and inflammatory cytokines. Therefore, our data suggest that PPARβ/δ inhibition is a potential therapeutic strategy against early DR pathology.

Chronic low-grade retinal inflammation is an essential contributor to the pathogenesis of diabetic retinopathy (DR). It is characterized by increased retinal cell expression and secretion of a variety of inflammatory cytokines; among these, IL-1β has the reputation of being a major driver of cytokine-induced inflammation. IL-1β and other cytokines drive inflammatory changes that cause damage to retinal cells, leading to the hallmark vascular lesions of DR; these include increased leukocyte adherence, vascular permeability, and capillary cell death. Nuclear factor of activated T-cells (NFAT) is a transcriptional regulator of inflammatory cytokines and adhesion molecules and is expressed in retinal cells. Consequently, it may influence multiple pathogenic steps early in DR. We investigated the NFAT-dependency of IL-1β-induced inflammation in human Müller cells (hMC) and human retinal microvascular endothelial cells (hRMEC). Our results show that an NFAT inhibitor, Inhibitor of NFAT-Calcineurin Association-6 (INCA-6), decreased IL-1β-induced expression of IL-1β and TNFα in hMC, while having no effect on VEGF, CCL2, or CCL5 expression. We also demonstrate that INCA-6 attenuated IL-1β-induced increases of IL-1β, TNFα, IL-6, CCL2, and CCL5 (inflammatory cytokines and chemokines), and ICAM-1 and E-selectin (leukocyte adhesion molecules) expression in hRMEC. INCA-6 similarly inhibited IL-1β-induced increases in leukocyte adhesion in both hRMEC monolayers in vitro and an acute model of retinal inflammation in vivo. Finally, INCA-6 rescued IL-1β-induced permeability in both hRMEC monolayers in vitro and an acute model of retinal inflammation in vivo. Taken together, these data demonstrate the potential of NFAT inhibition to mitigate retinal inflammation secondary to diabetes.

Though the pathogenesis of choroidal neovascularization (CNV) is largely unknown in age-related macular degeneration (AMD), inflammasomes may contribute to CNV development and progression. To understand the role NLRP3 inflammasomes in CNV, we used Ccr2RFPCx3cr1GFP dual-reporter mice to characterize migration of Ccr2RFP positive monocytes and Cx3cr1GFP positive microglial cells into CNV lesions after laser-induced rupture of Bruch's membrane. MCC950 was used as NLRP3 inhibitor. Immunostaining was used to confirm localization of NLRP3 inflammasomes in the LCNV lesions. Confocal microscopy was used to image and quantify LCNV volumes. ELISA and qRT-PCR were used to confirm the activation of NLRP3 by monitoring the expression of IL-1β protein and mRNA in choroidal tissues from LCNV mice. In addition, NLRP3 (-/-) LCNV mice were used to investigate whether NLRP3 inflammasomes contribute to the development of LCNV lesions. We observed that RFP positive monocyte-derived macrophages and GFP positive microglia-derived macrophages, in addition to other cell types, were localized in LCNV lesions at day 7 post-laser injury. In addition, NLRP3 inflammasomes are associated with LCNV lesions. Inhibition of NLRP3 inflammasomes, using MCC950, caused an increased Ccr2RFP positive macrophages, Cx3cr1GFP positive microglia, and other cells resulting in an increase in total lesion size. NLRP3 (-/-) LCNV mice, showed significantly increased lesion size compared to age-matched controls. Inhibition of NLRP3, resulted in decreased IL-1β mRNA and protein expression in the choroidal tissues, suggesting that increased lesion size may not be directly related to IL-1β.

TNFα has been identified as playing an important role in pathologic complications associated with diabetic retinopathy and retinal inflammation, such as retinal leukostasis. However, the transcriptional effects of TNFα on retinal microvascular endothelial cells and the different signaling pathways involved are not yet fully understood. In the present study, RNA-seq was used to profile the transcriptome of human retinal microvascular endothelial cells (HRMEC) treated for 4 hours with TNFα in the presence or absence of the NFAT-specific inhibitor INCA-6, in order to gain insight into the specific effects of TNFα on RMEC and identify any involvement of NFAT signaling. Differential expression analysis revealed that TNFα treatment significantly upregulated the expression of 579 genes when compared to vehicle-treated controls, and subsequent pathway analysis revealed a TNFα-induced enrichment of transcripts associated with cytokine-cytokine receptor interactions, cell adhesion molecules, and leukocyte transendothelial migration. Differential expression analysis comparing TNFα-treated cells to those co-treated with INCA-6 revealed 10 genes whose expression was significantly reduced by the NFAT inhibitor, including those encoding the proteins VCAM1 and CX3CL1 and cytokines CXCL10 and CXCL11. This study identifies the transcriptional effects of TNFα on HRMEC, highlighting its involvement in multiple pathways that contribute to retinal leukostasis, and identifying a previously unknown role for NFAT-signaling downstream of TNFα.

Cell therapy demonstrates great potential for the treatment of neurological disorders. Human umbilical tissue-derived cells (hUTCs) were previously shown to have protective and regenerative effects in animal models of stroke and retinal degeneration, but the underlying therapeutic mechanisms are unknown. Because synaptic dysfunction, synapse loss, degeneration of neuronal processes, and neuronal death are hallmarks of neurological diseases and retinal degenerations, we tested whether hUTCs contribute to tissue repair and regeneration by stimulating synapse formation, neurite outgrowth, and neuronal survival. To do so, we used a purified rat retinal ganglion cell culture system and found that hUTCs secrete factors that strongly promote excitatory synaptic connectivity and enhance neuronal survival. Additionally, we demonstrated that hUTCs support neurite outgrowth under normal culture conditions and in the presence of the growth-inhibitory proteins chondroitin sulfate proteoglycan, myelin basic protein, or Nogo-A (reticulon 4). Furthermore, through biochemical fractionation and pharmacology, we identified the major hUTC-secreted synaptogenic factors as the thrombospondin family proteins (TSPs), TSP1, TSP2, and TSP4. Silencing TSP expression in hUTCs, using small RNA interference, eliminated both the synaptogenic function of these cells and their ability to promote neurite outgrowth. However, the majority of the prosurvival functions of hUTC-conditioned media was spared after TSP knockdown, indicating that hUTCs secrete additional neurotrophic factors. Together, our findings demonstrate that hUTCs affect multiple aspects of neuronal health and connectivity through secreted factors, and each of these paracrine effects may individually contribute to the therapeutic function of these cells.

Age-related macular degeneration (AMD) is a leading cause of blindness among the elderly characterized by retinal pigment epithelium (RPE) degeneration with accumulation of abnormal intracellular deposits (lipofuscin) and photoreceptor death. RPE is vital for the retina and integrity of photoreceptors through its phagocytic function which is closely linked to formation of lipofuscin through daily phagocytosis of discarded photoreceptor outer segments (POS). Although phagocytosis has been implicated in AMD, it has not been directly shown to be altered in AMD. RPE phagocytic defect was previously shown to be rescued by subretinal injection of human umbilical tissue derived cells (hUTC) in a rodent model of retinal degeneration (RCS rat) through receptor tyrosine kinase (RTK) ligands and bridge molecules. Here, we examined RPE phagocytic function directly in the RPE from AMD patients and the ability and mechanisms of hUTC to affect phagocytosis in the human RPE.

Human umbilical tissue-derived cells (hUTC or palucorcel) are currently under clinical investigation for the treatment of geographic atrophy, a late stage of macular degeneration, but how hUTC transplantation mediates vision recovery is not fully elucidated. Subretinal administration of hUTC preserves visual function in the Royal College of Surgeons (RCS) rat, a genetic model of retinal degeneration caused by Mertk loss of function. hUTC secrete synaptogenic and neurotrophic factors that improve the health and connectivity of the neural retina. Therefore, we investigated the progression of synapse and photoreceptor loss and whether hUTC treatment preserves photoreceptors and synaptic connectivity in the RCS rats of both sexes. We found that RCS retinas display significant deficits in synaptic development already by postnatal day 21 (P21), before the onset of photoreceptor degeneration. Subretinal transplantation of hUTC at P21 is necessary to rescue visual function in RCS rats, and the therapeutic effect is enhanced with repeated injections. Synaptic development defects occurred concurrently with morphological changes in Müller glia, the major perisynaptic glia in the retina. hUTC transplantation strongly diminished Müller glia reactivity and specifically protected the α2δ-1-containing retinal synapses, which are responsive to thrombospondin family synaptogenic proteins secreted by Müller glia. Müller glial reactivity and reduced synaptogenesis observed in RCS retinas could be recapitulated by CRISPR/Cas9-mediated loss-of-Mertk in Müller glia in wild-type rats. Together, our results show that hUTC transplantation supports the health of retina at least in part by preserving the functions of Müller glial cells, revealing a previously unknown aspect of hUTC transplantation-based therapy.SIGNIFICANCE STATEMENT Despite the promising effects observed in clinical trials and preclinical studies, how subretinal human umbilical tissue-derived cell (hUTC) transplantation mediates vision improvements is not fully known. Using a rat model of retinal degeneration, the RCS rat (lacking Mertk), here we provide evidence that hUTC transplantation protects visual function and health by protecting photoreceptors and preserving retinal synaptic connectivity. Furthermore, we find that loss of Mertk function only in Müller glia is sufficient to impair synaptic development and cause activation of Müller glia. hUTC transplantation strongly attenuates the reactivity of Müller glia in RCS rats. These findings highlight novel cellular and molecular mechanisms within the neural retina, which underlie disease mechanisms and pinpoint Müller glia as a novel cellular target for hUTC transplantation.

Retinal capillary quiescence is regulated by a delicate balance between proangiogenic and anti-angiogenic factors. Pathological angiogenesis is the result of a shift in this balance towards proangiogenic influences. Pathological angiogenesis is produced in a rat model of oxygen-induced retinopathy (OIR) by exposing newborn rat pups to alternating periods of hyperoxia and hypoxia. Based upon previous work, two similar exposure paradigms were investigated and compared, exposure of rat pups to alternating periods of 45 and 12.5% oxygen, and to alternating periods of 40 and 15% oxygen. The resulting retinal pathology was assessed by measurement of retinal clock hours with pathological blood vessel growth and the percentage of the retina that is avascular. The 45 and 12.5% exposure produced significantly greater incidence and severity of pathology than the 40 and 15% protocol. To explain the difference in pathology between these two very similar exposure protocols, retinal levels of proangiogenic vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) and anti-angiogenic pigment epithelium-derived factor (PEDF) were measured by ELISA and western blot analysis at 0, 2, and 6 days post-exposure. In whole retinal lysates, there were no significant differences in VEGFR2 and PEDF levels. However, VEGF levels were approximately 48 and 78% higher on post-oxygen exposure day 0 and 2, respectively, in the group treated with alternating periods of 45 and 12.5% oxygen compared to the group treated with alternating periods of 40 and 15% oxygen. There was no significant difference in VEGF levels between these two groups on day 6 post-exposure. Therefore, the difference in pathology observed between these two experimental paradigms is associated with differences in whole retinal VEGF levels, but not changes in whole retinal VEGFR2 or PEDF levels. The results of this study suggest the existence of a threshold in the rat model of OIR, such that a small change in blood oxygen profile triggers a disproportionate increase in subsequent neovascularization, which is accompanied by more dramatic changes of retinal VEGF level than VEGFR2 or PEDF level. If a similar threshold exists for humans, it could explain why some oxygen-treated premature infants develop retinopathy and others do not, despite similar gestational ages, birth weights and clinical courses.

Wet form of age-related macular degeneration (wet AMD) is a progressive vascular disease that mainly affects older adults and causes severe and irreversible vision loss. A key complication of wet AMD is choroidal neovascularization (CNV), which may be driven in part by NLRP3 inflammasomes that are associated with macrophages migration to CNV lesions. Since activated NLRP3 is correlated with CNV, visualizing NLRP3 inflammasomes and their associated macrophages is of great interest to monitor wet AMD progression and develop effective therapies against it. However, to the best of our knowledge, current ophthalmic imaging systems do not permit such targeted imaging. Therefore, in this study, we developed InflammaProbe-1, an optical imaging probe for targeted visualization of NLRP3 inflammasomes in CNV lesions.

Non-steroidal anti-inflammatory drugs (NSAIDs), which inhibit COX activity, reduce the production of retinal VEGF and neovascularization in relevant models of ocular disease. We hypothesized that COX-2 mediates VEGF production in retinal Müller cells, one of its primary sources in retinal neovascular disease. The purpose of this study was to determine the role of COX-2 and its products in VEGF expression and secretion. These studies have more clearly defined the role of COX-2 and COX-2-derived prostanoids in retinal angiogenesis. Müller cells derived from wild-type and COX-2 null mice were exposed to hypoxia for 0-24 h. COX-2 protein and activity were assessed by western blot analysis and GC-MS, respectively. VEGF production was assessed by ELISA. Wild-type mouse Müller cells were treated with vehicle (0.1% DMSO), 10 microM PGE(2), or PGE(2) + 5 microM H-89 (a PKA inhibitor), for 12 h. VEGF production was assessed by ELISA. Hypoxia significantly increased COX-2 protein (p < 0.05) and activity (p < 0.05), and VEGF production (p < 0.0003). COX-2 null Müller cells produced significantly less VEGF in response to hypoxia (p < 0.05). Of the prostanoids, PGE(2) was significantly increased by hypoxia (p < 0.02). Exogenous PGE(2) significantly increased VEGF production by Müller cells (p < 0.0039), and this effect was inhibited by H-89 (p < 0.055). These data demonstrate that hypoxia induces COX-2, prostanoid production, and VEGF synthesis in Müller cells, and that VEGF production is at least partially COX-2-dependent. Our study suggests that PGE(2), signaling through the EP(2) and/or EP(4) receptor and PKA, mediates the VEGF response of Müller cells.

We assessed the effect of topical ketorolac on laser-induced choroidal neovascularization (CNV), measured retinal PGE(2) and VEGF levels after laser treatment, and determined the effect of ketorolac on PGE(2) and VEGF production. Six laser burns were placed in eyes of rats which then received topical ketorolac 0.4% or artificial tears four times daily until sacrifice. Fluorescein angiography (FA) was performed at 2 and 3 weeks and retinal pigment epithelium-choroid-sclera flat mounts were prepared. The retina and vitreous were isolated at 1, 3, 5, 7, and 14 days after laser treatment and tested for VEGF and PGE(2). Additional animals were lasered and treated with topical ketorolac or artificial tears and tested at 3 and 7 days for retinal and vitreous VEGF and PGE(2.) Ketorolac reduced CNV on FA by 27% at 2 weeks (P<0.001) and 25% at 3 weeks (P<0.001). Baseline retina and vitreous PGE(2) levels were 29.4 μg/g and 16.5 μg/g respectively, and reached 51.2 μg/g and 26.9 μg/g respectively, 24h after laser treatment (P<0.05). Retinal VEGF level was 781pg/g 24h after laser treatment and reached 931pg/g by 7 days (P<0.01). Ketorolac reduced retinal PGE(2) by 35% at 3 days (P<0.05) and 29% at 7 days (P<0.001) and retinal VEGF by 31% at 3 days (P=0.10) and 19% at 7 days (P<0.001). Topical ketorolac inhibited CNV and suppressed retinal PGE(2) and VEGF production.

The mechanisms that regulate the complex physiological task of photoreceptor outer segment assembly remain an enigma. One limiting factor in revealing the mechanism(s) by which this process is modulated is that not all of the role players who participate in this process are known. The purpose of this study was to determine some of the retinal proteins that likely play a critical role in regulating photoreceptor outer segment assembly. To do so, we analyzed and compared the proteome map of tadpole Xenopus laevis retinal pigment epithelium (RPE)-supported retinas containing organized outer segments with that of RPE-deprived retinas containing disorganized outer segments. Solubilized proteins were labeled with CyDye fluors followed by multiplexed two-dimensional separation. The intensity of protein spots and comparison of proteome maps was performed using DeCyder software. Identification of differentially regulated proteins was determined using nanoLC-ESI-MS/MS analysis. We found a total of 27 protein spots, 21 of which were unique proteins, which were differentially expressed in retinas with disorganized outer segments. We predict that in the absence of the RPE, oxidative stress initiates an unfolded protein response. Subsequently, downregulation of several candidate Müller glial cell proteins may explain the inability of photoreceptors to properly fold their outer segment membranes. In this study, we have used identification and bioinformatics assessment of proteins that are differentially expressed in retinas with disorganized outer segments as a first step in determining probable key molecules involved in regulating photoreceptor outer segment assembly.

Optical coherence tomography (OCT) is widely used for ocular imaging in clinical and research settings. OCT natively provides structural information based on the reflectivity of the tissues it images. We demonstrate the utility of photothermal OCT (PTOCT) imaging of gold nanorods (GNR) in the mouse retina in vivo in the laser-induced choroidal neovascularization (LCNV) model to provide additional image contrast within the lesion.