Search

The formation of fibrovascular membranes (FVMs) is a serious sight-threatening complication of proliferative diabetic retinopathy (PDR) that may result in retinal detachment and eventual blindness. During the formation of these membranes, neurite/process outgrowth occurs in retinal neurons and glial cells, which may both serve as a scaffold and have guiding or regulatory roles. To further understand this process, we investigated whether previously identified candidate proteins, from vitreous of PDR patients with FVMs, could induce neurite outgrowth in an experimental setting. Retinal explants of C57BL6/N mouse pups on postnatal day 3 (P3) were cultured in poly-L-lysine- and laminin-coated dishes. Outgrowth stimulation experiments were performed with the addition of potential inducers of neurite outgrowth. Automated analysis of neurite outgrowth was performed by measuring β–tubulin-immunopositive neurites using Image J. Expression of PDGF receptors was quantified by RT-PCR in FVMs of PDR patients. Platelet-derived growth factor (PDGF) induced neurite outgrowth in a concentration-dependent manner, whilst neuregulin 1 (NRG1) and connective tissue growth factor (CTGF) did not. When comparing three different PDGF dimers, treatment with PDGF-AB resulted in the highest neurite induction, followed by PDGF-AA and -BB. In addition, incubation of retinal explants with vitreous from PDR patients resulted in a significant induction of neurite outgrowth as compared to non-diabetic control vitreous from patients with macular holes, which could be prevented by addition of CP673451, a potent PDGF receptor (PDGFR) inhibitor. Abundant expression of PDGF receptors was detected in FVMs. Our findings suggest that PDGF may be involved in the retinal neurite outgrowth, which is associated with the formation of FVMs in PDR. [ABSTRACT FROM AUTHOR]

Neurodegenerative diseases and central nervous system (CNS) trauma are highly irreversible, in part because adult mammals lack a robust regenerative capacity. A multifactorial problem underlies the limited axonal regeneration potential. Strikingly, neuroinflammation seems able to induce axonal regrowth in the adult mammalian CNS. It is increasingly clear that both blood-borne and resident inflammatory cells as well as reactivated glial cells affect axonal regeneration. The scope of this review is to give a comprehensive overview of the knowledge that links inflammation (with a focus on the innate immune system) to axonal regeneration and to critically reflect on the controversy that still prevails about the cells, molecules and pathways that are dominating the scene. Also, a brief overview is given of what is already known about the crosstalk between and the heterogeneity of cell types that might play a role in axonal regeneration. Recent research indicates that inflammation-induced axonal regrowth is not solely driven by a single-cell population but probably relies on the crosstalk between multiple cell types and the strong regulation of these cell populations in time and space. Moreover, there is growing evidence that the different cell populations are highly heterogeneous and as such can react differently upon injury. This could explain the controversial results that have been obtained over the past years. The primary focus of this manuscript is the retinofugal system of adult mammals, however, when relevant, insights or examples of the spontaneous regenerating zebrafish model and spinal cord research are added. [ABSTRACT FROM AUTHOR]

Purpose: Diabetic retinopathy (DR) is characterized by an early stage of inflammation and vessel leakage, and an advanced vasoproliferative stage. Also, neurodegeneration might play an important role in disease pathogenesis. The aim of this study was to investigate the effect of the Rho kinase (ROCK) inhibitor, AMA0428, on these processes. Methods: The response to ROCK inhibition by AMA0428 (1 µg) was studiedin vivousing the murine model for streptozotocin (STZ)-induced diabetes, focusing on early non-proliferative DR features and the oxygen-induced retinopathy (OIR) model to investigate proliferative DR. Intravitreal (IVT) administration of AMA0428 was compared with murine anti-VEGF-R2 antibody (DC101, 6.2 µg) and placebo (H2O/PEG; 1C8). Outcome was assessed by analyzing leukostasis using fluorescein isothiocyanate coupled concanavalin A (FITC-ConA) and vessel leakage (bovine serum albumin conjugated with fluorescein isothiocyanate; FITC-BSA)/neovascularization and neurodegeneration by immunohistological approaches (hematoxylin and eosin (H&E), terminal deoxynucleotidyl transferase–mediated biotinylated UTP nick end labeling (TUNEL), Brn3a). ELISA and Western blotting were employed to unravel the consequences of ROCK inhibition (1 µM AMA0428) on myosin phosphatase target protein (MYPT)-1 phosphorylation, endothelial nitric oxide synthase (eNOS) phosphorylation, and vascular endothelial growth factor (VEGF) levels in retinas of diabetic mice, on NF-κβ activity and ICAM-1 expression in endothelial cells (ECs). Results: In vivo, AMA0428 significantly reduced vessel leakage and neovascularization, respectively, in the STZ and OIR model, comparable to DC101 therapy. Additionally, the ROCK inhibitor decreased neurodegeneration in both models and inhibited leukostasis by 30% (p< 0.05) in the STZ model (p< 0.05), while DC101 had no positive effect on the outcome of these latter processes. ROCK activity was upregulated in the diabetic retina and AMA0428 administration resulted in decreased phospho-MYPT-1, enhanced phospho-eNOS, and reduced VEGF levels.In vitro, AMA0428 interfered with NF-κβ activity, thereby inhibiting ICAM-1 expression in ECs. Conclusions: Targeting ROCK with AMA0428 effectively attenuated outcome in an early DR model (STZ) and a late vasoproliferative retinopathy model (OIR). These findings make AMA0428 a promising candidate with an additional anti-inflammatory and neuroprotective benefit for DR patients, as compared with anti-VEGF treatment. [ABSTRACT FROM PUBLISHER]

Purpose: Previous reports have yielded conflicting data on the activity of bevacizumab (Avastin), a recombinant humanized monoclonal antibody against VEGF-A, in the mouse. The current study was designed to further explore the use of this VEGF inhibitor in various murine models of ocular diseases and compare it to the widely used murine anti-VEGF-R2 neutralizing antibody (DC101). Methods: Murine models of laser-induced choroidal neovascularization (CNV), oxygen-induced retinopathy (OIR) and glaucoma filtration surgery (GFS) were used to investigate the effect of bevacizumab. Mice either received an intravitreal (CNV-OIR) or subconjunctival (GFS) injection. In all models, they were divided in two groups ( n = 10 per group). In the first group, one eye was injected with bevacizumab (1 µl; 25 µg) and the other eye was used as a negative control and received an injection of NaCl (1 µl; 0.9%). In the second group, one eye was injected with DC101 (1 µl; 6.2 µg), whereas an isotype-matched control antibody (1C8; 4.8 µg) was administered in the contralateral eye. Treatment outcome was studied by clinical investigation (GFS) and immunohistological analysis of angiogenesis (CD31/FITC-dextran/H&E) and fibrosis (Sirius Red). Results: Analysis of blood vessel density (CNV) and blood vessel growth (OIR) showed a comparable decrease after intravitreal administration of bevacizumab or DC101. Furthermore, in the mouse model of GFS, clinical investigation of the bleb and a CD31 staining on sections demonstrated that subconjunctival injection of both antibodies similarly improved the surgical outcome (bleb area and survival) by reducing angiogenesis. Moreover, morphometric analysis after Sirius Red staining showed a comparable reduction in collagen deposition after administration of the inhibitors. Conclusion: Our findings consistently demonstrate that bevacizumab is as effective as the murine anti-VEGF-R2 antibody (DC101) in mouse models of CNV, OIR and GFS, thus confirming its suitability for translational ophthalmological research. [ABSTRACT FROM AUTHOR]