The contribution of pattern recognition receptor signalling in the development of age related macular degeneration: the role of toll-like-receptors and the NLRP3-inflammasome.

Age-related macular degeneration (AMD) is a leading cause of irreversible vision loss, characterised by the dysfunction and death of the photoreceptors and retinal pigment epithelium (RPE). Innate immune cell activation and accompanying para-inflammation have been suggested to contribute to the pathogenesis of AMD, although the exact mechanism(s) and signalling pathways remain elusive. Pattern recognition receptors (PRRs) are essential activators of the innate immune system and drivers of para-inflammation. Of these PRRs, the two most prominent are (1) Toll-like receptors (TLR) and (2) NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3)-inflammasome have been found to modulate the progression of AMD. Mutations in TLR2 have been found to be associated with an increased risk of developing AMD. In animal models of AMD, inhibition of TLR and NLRP3 has been shown to reduce RPE cell death, inflammation and angiogenesis signalling, offering potential novel treatments for advanced AMD. Here, we examine the evidence for PRRs, TLRs2/3/4, and NLRP3-inflammasome pathways in macular degeneration pathogenesis.

Exploring the pathogenesis of age-related macular degeneration: A review of the interplay between retinal pigment epithelium dysfunction and the innate immune system.

Age-related macular degeneration (AMD) is a leading cause of irreversible vision loss in the older population. Classical hallmarks of early and intermediate AMD are accumulation of drusen, a waste deposit formed under the retina, and pigmentary abnormalities in the retinal pigment epithelium (RPE). When the disease progresses into late AMD, vision is affected due to death of the RPE and the light-sensitive photoreceptors. The RPE is essential to the health of the retina as it forms the outer blood retinal barrier, which establishes ocular immune regulation, and provides support for the photoreceptors. Due to its unique anatomical position, the RPE can communicate with the retinal environment and the systemic immune environment. In AMD, RPE dysfunction and the accumulation of drusen drive the infiltration of retinal and systemic innate immune cells into the outer retina. While recruited endogenous or systemic mononuclear phagocytes (MPs) contribute to the removal of noxious debris, the accumulation of MPs can also result in chronic inflammation and contribute to AMD progression. In addition, direct communication and indirect molecular signaling between MPs and the RPE may promote RPE cell death, choroidal neovascularization and fibrotic scarring that occur in late AMD. In this review, we explore how the RPE and innate immune cells maintain retinal homeostasis, and detail how RPE dysfunction and aberrant immune cell recruitment contribute to AMD pathogenesis. Evidence from AMD patients will be discussed in conjunction with data from preclinical models, to shed light on future therapeutic targets for the treatment of AMD.

Transcriptomic analysis of choroidal neovascularization reveals dysregulation of immune and fibrosis pathways that are attenuated by a novel anti-fibrotic treatment.

Neovascular AMD (nAMD) leads to vision loss and is a leading cause of visual impairment in the industrialised world. Current treatments that target blood vessel growth have not been able to treat subretinal fibrosis and nAMD patients continue to lose vision. The molecular mechanisms involved in the development of fibrotic lesions in nAMD are not well understood. The aim of this study was to further understand subretinal fibrosis in the laser photocoagulation model of choroidal neovascularization (CNV) by studying the whole transcriptome of the RPE/choroid following CNV and the application of an anti-fibrotic following CNV. Seven days after laser induced CNV, RPE and choroid tissue was separated and underwent RNAseq. Differential expression analysis and pathway analysis revealed an over representation of immune signalling and fibrotic associated pathways in CNV compared to control RPE/choroid tissue. Comparisons between the mouse CNV model to human CNV revealed an overlap in upregulated expression for immune genes (Ccl2, Ccl8 and Cxcl9) and extracellular matrix remodeling genes (Comp, Lrcc15, Fndc1 and Thbs2). Comparisons between the CNV model and other fibrosis models showed an overlap of over 60% of genes upregulated in either lung or kidney mouse models of fibrosis. Treatment of CNV using a novel cinnamoyl anthranilate anti-fibrotic (OCX063) in the laser induced CNV model was selected as this class of drugs have previously been shown to target fibrosis. CNV lesion leakage and fibrosis was found to be reduced using OCX063 and gene expression of genes within the TGF-beta signalling pathway. Our findings show the presence of fibrosis gene expression pathways present in the laser induced CNV mouse model and that anti-fibrotic treatments offer the potential to reduce subretinal fibrosis in AMD.

Anti-inflammatory and neuroprotective properties of the corticosteroid fludrocortisone in retinal degeneration.

The pathogenesis of outer retinal degenerations has been linked to the elevation of cytokines that orchestrate pro-inflammatory responses within the retinal milieu, and which are thought to play a role in diseases such as geographic atrophy (GA), an advanced form of AMD. Here we sought investigate the anti-inflammatory and mechanistic properties of fludrocortisone (FA), as well as triamcinolone acetonide (TA), on Müller cell-mediated cytokine expression in response to inflammatory challenge. In addition, we investigated the neuroprotective efficacy of FA and TA in a photo-oxidative damage (PD), a model of outer retinal degeneration. Expression of CCL2, IL-6, and IL-8 with respect to FA and TA were assessed in Müller cells in vitro, following simulation with IL-1ß or TNF-α. The dependency of this effect on mineralocorticoid and glucocorticoid signaling was also interrogated for both TA and TA via co-incubation with steroid receptor antagonists. For the PD model, C57BL/6 mice were intravitreally injected with FA or TA, and changes in retinal pathology were assessed via electroretinogram (ERG) and optical coherence tomography (OCT). FA and TA were found to dramatically reduce the expression of CCL2, IL-6, and IL-8 in Müller glia in vitro after inflammatory challenge with IL-1ß or TNF-α (P < 0.05). Though FA acts as both a mineralocorticoid and glucocorticoid receptor agonist, co-incubation with selective steroid antagonists revealed that the suppressive effect of FA on CCL2, IL-6, and IL-8 expression is mediated by glucocorticoid signaling (P < 0.05). In PD, intravitreal FA was found to ameliorate outer-retinal atrophy as measured by ERG and OCT (P < 0.05), while TA had no significant effect (P > 0.05). Our data indicate potent anti-inflammatory and mechanistic properties of corticosteroids, specifically FA, in suppressing inflammation and neurodegeneration degeneration associated with outer retinal atrophy. Taken together, our findings indicate that corticosteroids such as FA may have value as a potential therapeutic for outer retinal degenerations where such pro-inflammatory factors are implicated, including AMD.

Photoreceptor ablation following ATP induced injury triggers Müller glia driven regeneration in zebrafish.

Retinal regeneration research offers hope to people affected by visual impairment due to disease and injury. Ongoing research has explored many avenues towards retinal regeneration, including those that utilizes implantation of devices, cells or targeted viral-mediated gene therapy. These results have so far been limited, as gene therapy only has applications for rare single-gene mutations and implantations are invasive and in the case of cell transplantation donor cells often fail to integrate with adult neurons. An alternative mode of retinal regeneration utilizes a stem cell population unique to vertebrate retina - Müller glia (MG). Endogenous MG can readily regenerate lost neurons spontaneously in zebrafish and to a very limited extent in mammalian retina. The use of adenosine triphosphate (ATP) has been shown to induce retinal degeneration and activation of the MG in mammals, but whether this is conserved to other vertebrate species including those with higher regenerative capacity remains unknown. In our study, we injected a single dose of ATP intravitreal in zebrafish to characterize the cell death and MG induced regeneration. We used TUNEL labelling on retinal sections to show that ATP caused localised death of photoreceptors and ganglion cells within 24 h. Histology of GFP-transgenic zebrafish and BrdU injected fish demonstrated that MG proliferation peaked at days 3 and 4 post-ATP injection. Using BrdU labelling and photoreceptor markers (Zpr1) we observed regeneration of lost rod photoreceptors at day 14. This study has been undertaken to allow for comparative studies between mammals and zebrafish that use the same specific induction method of injury, i.e. ATP induced injury to allow for direct comparison of across species to narrow down resulting differences that might reflect the differing regenerative capacity. The ultimate aim of this work is to recapitulate pro-neurogenesis Müller glia signaling in mammals to produce new neurons that integrate with the existing retinal circuit to restore vision.

Potential mechanisms of retinal ganglion cell type-specific vulnerability in glaucoma.

Glaucoma is a neurodegenerative disease characterised by progressive damage to the retinal ganglion cells (RGCs), the output neurons of the retina. RGCs are a heterogenous class of retinal neurons which can be classified into multiple types based on morphological, functional and genetic characteristics. This review examines the body of evidence supporting type-specific vulnerability of RGCs in glaucoma and explores potential mechanisms by which this might come about. Studies of donor tissue from glaucoma patients have generally noted greater vulnerability of larger RGC types. Models of glaucoma induced in primates, cats and mice also show selective effects on RGC types - particularly OFF RGCs. Several mechanisms may contribute to type-specific vulnerability, including differences in the expression of calcium-permeable receptors (for example pannexin-1, P2X7, AMPA and transient receptor potential vanilloid receptors), the relative proximity of RGCs and their dendrites to blood supply in the inner plexiform layer, as well as differing metabolic requirements of RGC types. Such differences may make certain RGCs more sensitive to intraocular pressure elevation and its associated biomechanical and vascular stress. A greater understanding of selective RGC vulnerability and its underlying causes will likely reveal a rich area of investigation for potential treatment targets.

Electrical Field Shaping Techniques in a Feline Model of Retinal Degeneration.

The majority of preclinical studies investigating multi-electrode field shaping stimulation strategies for retinal prostheses, have been conducted in normally-sighted animals. This study aimed to reassess the effectiveness of two electrical field shaping techniques that have been shown to work in healthy retinae, in a more clinically relevant animal model of photoreceptor degeneration. Four cats were unilaterally blinded via intravitreal injections of adenosine triphosphate. Cortical responses to traditional monopolar (MP) stimulation, focused multipolar (FMP) stimulation and two-dimensional current steering were recorded. Contrary to our previous work, we found no significant difference between the spread of cortical activation elicited by FMP and MP stimulation, and we were not able to reproduce cortical responses to singleelectrode retinal stimulation using two-dimensional current steering. These findings suggest that while shown to be effective in normally-sighted animals, these techniques may not be readily translatable to patients with retinal degeneration and require further optimization.

Safety Studies for a 44-Channel Suprachoroidal Retinal Prosthesis: A Chronic Passive Study.

Purpose: Following successful clinical outcomes of the prototype suprachoroidal retinal prosthesis, Bionic Vision Australia has developed an upgraded 44-channel suprachoroidal retinal prosthesis to provide a wider field of view and more phosphenes. The aim was to evaluate the preclinical passive safety characteristics of the upgraded electrode array. Methods: Ten normal-sighted felines were unilaterally implanted with an array containing platinum electrodes (44 stimulating and 2 returns) on a silicone carrier near the area centralis. Clinical assessments (color fundus photos, optical coherence tomography, full-field electroretinography, intraocular pressure) were performed under anesthesia prior to surgery, and longitudinally for up to 20 weeks. Histopathology grading of fibrosis and inflammation was performed in two animals at 13 to 15 weeks. Results: Eight animals showed safe electrode array insertion (good retinal health) and good conformability of the array to the retinal curvature. Eight animals demonstrated good mechanical stability of the array with only minor (<2 disc diameters) lateral movement. Four cases of surgical or stability complications occurred due to (1) bulged choroid during surgery, (2) hemorrhage from a systemic bleeding disorder, (3) infection, and (4) partial erosion of thin posterior sclera. There was no change in retinal structure or function (other than that seen at surgery) at endpoint. Histopathology showed a mild foreign body response. Electrodes were intact on electrode array removal. Conclusions: The 44-channel suprachoroidal electrode array has an acceptable passive safety profile to proceed to clinical trial. The safety profile is expected to improve in human studies, as the complications seen are specific to limitations (anatomic differences) with the feline model.

Nanosecond Laser Treatment for Age-Related Macular Degeneration Does Not Induce Focal Vision Loss or New Vessel Growth in the Retina.

Purpose: Subthreshold, nanosecond pulsed laser treatment shows promise as a treatment for age-related macular degeneration (AMD); however, the safety profile needs to be robustly examined. The aim of this study was to investigate the effects of laser treatment in humans and mice. Methods: Patients with AMD were treated with nanosecond pulsed laser at subthreshold (no visible retinal effect) energy doses (0.15-0.45 mJ) and retinal sensitivity was assessed with microperimetry. Adult C57BL6J mice were treated at subthreshold (0.065 mJ) and suprathreshold (photoreceptor loss, 0.5 mJ) energy settings. The retinal and vascular responses were analyzed by fundus imaging, histologic assessment, and quantitative PCR. Results: Microperimetry analysis showed laser treatment had no effect on retinal sensitivity under treated areas in patients 6 months to 7 years after treatment. In mice, subthreshold laser treatment induced RPE loss at 5 hours, and by 7 days the RPE had retiled. Fundus imaging showed reduced RPE pigmentation but no change in retinal thickness up to 3 months. Electron microscopy revealed changes in melanosomes in the RPE, but Bruch's membrane was intact across the laser regions. Histologic analysis showed normal vasculature and no neovascularization. Suprathreshold laser treatment did not induce changes in angiogenic genes associated with neovascularization. Instead pigment epithelium-derived factor, an antiangiogenic factor, was upregulated. Conclusions: In humans, low-energy, nanosecond pulsed laser treatment is not damaging to local retinal sensitivity. In mice, treatment does not damage Bruch's membrane or induce neovascularization, highlighting a reduced side effect profile of this nanosecond laser when used in a subthreshold manner.