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The effects of brain-derived neurotrophic factor (BDNF) on retinal ganglion cell (RGC) survival and visual function were assessed in rat and mouse models of optic nerve (ON) crush. ONs were crushed on Day 1, followed by intravitreal injections of a vehicle or BDNF on Days 1 and 8. The spatial frequency threshold was measured using optokinetic tracking on Days 7 and 14. On Day 15, ganglion cell complex (GCC) thickness was quantified using optical coherence tomography. Furthermore, all eyes were enucleated for immunohistochemical analysis of the surviving RGC somas and axons. BDNF significantly reduced the RGC soma in mice and increased GCC thickness in intact eyes, with apparent axonal swelling in both species. It displayed significantly greater RGC soma survival in eyes with ON injury, with moderately thicker axonal bundles in both species and a thicker GCC in rats. Visual function was significantly reduced in all ON-crushed animals, regardless of BDNF treatment. Thus, we obtained a comprehensive analysis of the structural and functional impact of BDNF in intact and ON-crushed eyes in two rodent models. Our results provide a foundation for further BDNF evaluation and the design of preclinical studies on neuroprotectants using BDNF as a reference positive control.

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Diabetic retinopathy (DR) is a common neurovascular complication of type 1 diabetes. Current therapeutics target neovascularization characteristic of end-stage disease, but are associated with significant adverse effects. Targeting early events of DR such as neurodegeneration may lead to safer and more effective approaches to treatment. Two independent prospective clinical trials unexpectedly identified that the PPARα agonist fenofibrate had unprecedented therapeutic effects in DR, but gave little insight into the physiological and molecular mechanisms of action. The objective of the present study was to evaluate potential neuroprotective effects of PPARα in DR, and subsequently to identify the responsible mechanism of action. Here we reveal that activation of PPARα had a robust protective effect on retinal function as shown by Optokinetic tracking in a rat model of type 1 diabetes, and also decreased retinal cell death, as demonstrated by a DNA fragmentation ELISA. Further, PPARα ablation exacerbated diabetes-induced decline of visual function as demonstrated by ERG analysis. We further found that PPARα improved mitochondrial efficiency in DR, and decreased ROS production and cell death in cultured retinal neurons. Oxidative stress biomarkers were elevated in diabetic Pparα-/- mice, suggesting increased oxidative stress. Mitochondrially mediated apoptosis and oxidative stress secondary to mitochondrial dysfunction contribute to neurodegeneration in DR. Taken together, these findings identify a robust neuroprotective effect for PPARα in DR, which may be due to improved mitochondrial function and subsequent alleviation of energetic deficits, oxidative stress and mitochondrially mediated apoptosis.

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PURPOSE: To determine if a Microemulsion Drug Ocular Penetration System (MiDROPS) formulation of cyclosporine A (CsA) delivers more drug and is more efficacious for treatment of dry eye disease (DED) than the current clinical formulation. METHODS: Tissue distribution of CsA was quantified by liquid chromatography with tandem mass spectrometry (LC-MS/MS). To assess tolerability, CsA-MiDROPS (0.1%) was applied to the eyes of rabbits twice per day for 14 days and assessed using ophthalmoscopic examinations. Mice were exposed to desiccating stress for 10 days and received daily topical instillation of the vehicle or test agent. Cornea staining was done to quantify corneal permeability. Histologic quantification of goblet cell (GC) density and CD4+ T-cell infiltration in the conjunctiva was performed. RESULTS: Ophthalmic distribution studies indicate significantly increased drug concentration with CsA-MiDROPS compared with Restasis. CsA-MiDROPS is well tolerated with little toxicity in a 2-week tolerability study. In the DED model, both 0.05% and 0.1% CsA-MiDROPS conferred a significant effect and were more effective than Restasis for treating experimental DED when dosed twice per day. As compared with Restasis dosed twice per day, 0.1% CsA-MiDROPS dosed once per day demonstrated superiority. CONCLUSIONS: CsA-MiDROPS showed superior drug delivery and efficacy compared with other clinical formulations. As this product is simple to produce and needs to be only applied once daily, the clinical development of CsA-MiDROPS will help to reduce societal and patient burdens by lowering drug costs and accelerating/improving the activity of CsA. TRANSLATIONAL RELEVANCE: MiDROPS has broad application concerning the ophthalmic development of lipophilic small molecule therapeutics.

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Purpose: Diabetic retinopathy is a leading cause of vision loss. Previous studies have shown signaling pathways mediated by Stat3 (signal transducer and activator of transcription 3) play a primary role in diabetic retinopathy progression. This study tested CLT-005, a small molecule inhibitor of Stat3, for its dose-dependent therapeutic effects on vision loss in a rat model of diabetic retinopathy. Methods: Brown Norway rats were administered streptozotocin (STZ) to induce diabetes. CLT-005 was administered daily by oral gavage for 16 weeks at concentrations of 125, 250, or 500 mg/kg, respectively, beginning 4 days post streptozotocin administration. Systemic and ocular drug concentration was quantified with mass spectrometry. Visual function was monitored at 2-week intervals from 6 to 16 weeks using optokinetic tracking to measure visual acuity and contrast sensitivity. The presence and severity of cataracts was visually monitored and correlated to visual acuity. The transcription and translation of multiple angiogenic factors and inflammatory cytokines were measured by real-time polymerase chain reaction and Multiplex immunoassay. Results: Streptozotocin-diabetic rats sustain progressive vision loss over 16 weeks, and this loss in visual function is rescued in a dose-dependent manner by CLT-005. This positive therapeutic effect correlates to the positive effects of CLT-005 on vascular leakage and the presence of inflammatory cytokines in the retina. Conclusions: The present study indicates that Stat3 inhibition has strong therapeutic potential for the treatment of vision loss in diabetic retinopathy.

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PURPOSE: Elevation of serum retinol-binding protein 4 (RBP4) induces inflammation in primary human retinal microvascular endothelial cells (HRECs) via a retinol-independent mechanism; thus, it may play a causative role in the development and progression of vascular lesions in diabetic retinopathy (DR). Since HRECs do not express the classical RBP4 receptor, stimulated by retinoic acid gene 6 (STRA6), this study focuses on identifying the endothelial cell receptor and signaling that mediate RBP4-induced inflammation. METHODS: HRECs were treated with a toll-like receptor 4 (TLR4) small molecule inhibitor (Cli95, also known as TAK-242), TLR4 neutralizing antibody, or mitogen-activated protein kinase (MAPK) inhibitors before treatment with purified recombinant RBP4. The HREC inflammatory response was quantified by in vitro leukostasis assays, western blotting, and enzyme-linked immunosorbent assay (ELISA). To understand how the serum binding partner for RBP4, transthyretin (TTR), may affect RBP4 activity, we also measured RBP4 and TTR levels in serum and retinal lysates from RBP4-Tg and wild-type mice. RESULTS: TLR4 inhibition significantly reduced RBP4-induced expression of pro-inflammatory proteins and in vitro leukostasis. RBP4 treatment significantly increased phosphoactivation of p38 and c-Jun N-terminal protein kinase (JNK). The p38 inhibitor (SB203580) attenuated RBP4-stimulated vascular cell adhesion molecule 1 (VCAM-1), intracellular adhesion molecule 1 (ICAM-1), monocyte chemoattractant protein (MCP-1), and interleukin 6 (IL-6) production, while the JNK inhibitor (SP600125) reduced RBP4-stimulated sICAM-1, endothelial cell selectin (E-selectin), and MCP-1 production. The MAPK inhibitors only showed partial (50-70%) suppression of the RBP4-stimulated proinflammatory response. Moreover, TLR4 inhibition did not decrease RBP4-induced MAPK phosphoactivation, suggesting that RBP4-mediated MAPK activation is TLR4 independent and occurs through a secondary unknown receptor. We also found that the RBP4/TTR molar ratio was exceptionally high in the retina of RBP4-Tg mice, indicating an abundance of TTR-free RBP4. CONCLUSIONS: RBP4-induced inflammation is largely mediated by TLR4, and in part, through JNK and p38 MAPK signaling. The high TTR/RBP4 molar ratio in serum likely protects the endothelium from the proinflammatory effects of RBP4 in vivo, whereas elevation of serum RBP4 causes a significant increase in TTR-free RBP4 in retinal tissue. This offers insight into how RBP4-Tg mice can develop retinal neurodegeneration without coincident retinal microvascular pathology.

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Serum retinol-binding protein 4 (RBP4) is the sole specific vitamin A (retinol) transporter in blood. Elevation of serum RBP4 in patients has been linked to cardiovascular disease and diabetic retinopathy. However, the significance of RBP4 elevation in the pathogenesis of these vascular diseases is unknown. Here we show that RBP4 induces inflammation in primary human retinal capillary endothelial cells (HRCEC) and human umbilical vein endothelial cells (HUVEC) by stimulating expression of proinflammatory molecules involved in leukocyte recruitment and adherence to endothelium, including vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), E-selectin, monocyte chemoattractant protein 1 (MCP-1), and interleukin-6 (IL-6). We demonstrate that these novel effects of RBP4 are independent of retinol and the RBP4 membrane receptor STRA6 and occur in part via activation of NADPH oxidase and NF-κB. Importantly, retinol-free RBP4 (apo-RBP4) was as potent as retinol-bound RBP4 (holo-RBP4) in inducing proinflammatory molecules in both HRCEC and HUVEC. These studies reveal that RBP4 elevation can directly contribute to endothelial inflammation and therefore may play a causative role in the development or progression of vascular inflammation during cardiovascular disease and microvascular complications of diabetes.

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