Pharmaceutical therapies targeting autophagy for the treatment of age-related macular degeneration.

Age-related macular degeneration (AMD) is a major cause of irreversible vision loss in the elderly. Although new therapies have recently emerged, there are currently no ways of preventing the development of the disease. Changes in intracellular recycling processes. Changes in intracellular recycling processes, called autophagy, lead to debris accumulation and cellular dysfunction in AMD models and AMD patients. Drugs that enhance autophagy hold promise as therapies for slowing AMD progression in preclinical models; however, more studies in humans are required. While a definitive cure for AMD will likely hinge on a personalized medicine approach, treatments that enhance autophagy hold promise for slowing vision loss.

Aging induces cell loss and a decline in phagosome processing in the mouse retinal pigment epithelium.

Age-related macular degeneration (AMD) is a leading cause of irreversible vision loss and dysfunction in the retinal pigment epithelium (RPE) with age is known to contribute to disease development. The aim of this study was to investigate how the C57BL/6J mouse RPE changes with age. RPE structure was found to change with age and eccentricity, with cell size increasing, nuclei lost, and tight junctions altered in the peripheral retina. Phagocytosis of photoreceptor outer segments (POS) by the RPE was investigated using gene expression analysis and histology. RNA-Seq transcriptomic gene profiling of the RPE showed a downregulation of genes involved in phagosome processing and histological analysis showed a decline in phagosome-lysosome association in the aged tissue. In addition, failures in the autophagy pathway that modulates intracellular waste degradation were observed in the aged RPE tissue. These findings highlight that RPE cell loss and slowing of POS processing contribute to RPE dysfunction with age and may predispose the aging eye to AMD development.

Anomalies in neurovascular coupling during early diabetes: A review.

Diabetic retinopathy is the most feared complication for those with diabetes. Although visible vascular pathology traditionally defines the management of this condition, it is now recognised that a range of cellular changes occur in the retina from an early stage of diabetes. One of the most significant functional changes that occurs in those with diabetes is a loss of vasoregulation in response to changes in neural activity. There are several retinal cell types that are critical for mediating so-called neurovascular coupling, including Müller cells, microglia and pericytes. Although there is a great deal of evidence that suggests that Müller cells are integral to regulating the vasculature, they only modulate part of the vascular tree, highlighting the complexity of vasoregulation within the retina. Recent studies suggest that retinal immune cells, microglia, play an important role in mediating vasoconstriction. Importantly, retinal microglia contact both the vasculature and neural synapses and induce vasoconstriction in response to neurally expressed chemokines such as fractalkine. This microglial-dependent regulation occurs via the vasomediator angiotensinogen. Diabetes alters the way microglia regulate the retinal vasculature, by increasing angiotensinogen expression, causing capillary vasoconstriction and contributing to a loss of vascular reactivity to physiological signals. This article summarises recent studies showing changes in vascular regulation during diabetes, the potential mechanisms by which this occurs and the significance of these early changes to the progression of diabetic retinopathy.

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.

Treatments targeting autophagy ameliorate the age-related macular degeneration phenotype in mice lacking APOE (apolipoprotein E).

Age-related macular degeneration (AMD) is a leading cause of vision loss with recent evidence indicating an important role for macroautophagy/autophagy in disease progression. In this study we investigate the efficacy of targeting autophagy for slowing dysfunction in a mouse model with features of early AMD. Mice lacking APOE (apolipoprotein E; B6.129P2-Apoetm1UncJ/Arc) and C57BL/6 J- (wild-type, WT) mice were treated with metformin or trehalose in the drinking water from 5 months of age and the ocular phenotype investigated at 13 months. Control mice received normal drinking water. APOE-control mice had reduced retinal function and thickening of Bruch's membrane consistent with an early AMD phenotype. Immunohistochemical labeling showed reductions in MAP1LC3B/LC3 (microtubule-associated protein 1 light chain 3 beta) and LAMP1 (lysosomal-associated membrane protein 1) labeling in the photoreceptors and retinal pigment epithelium (RPE). This correlated with increased LC3-II:LC3-I ratio and alterations in protein expression in multiple autophagy pathways measured by reverse phase protein array, suggesting autophagy was slowed. Treatment of APOE-mice with metformin or trehalose ameliorated the loss of retinal function and reduced Bruch's membrane thickening, enhancing LC3 and LAMP1 labeling in the ocular tissues and restoring LC3-II:LC3-I ratio to WT levels. Protein analysis indicated that both treatments boost ATM-AMPK driven autophagy. Additionally, trehalose increased p-MAPK14/p38 to enhance autophagy. Our study shows that treatments targeting pathways to enhance autophagy have the potential for treating early AMD and provide support for the use of metformin, which has been found to reduce the risk of AMD development in human patients.Abbreviations:AMD: age-related macular degeneration; AMPK: 5' adenosine monophosphate-activated protein kinase APOE: apolipoprotein E; ATM: ataxia telangiectasia mutated; BCL2L1/Bcl-xL: BCL2-like 1; DAPI: 4'-6-diamidino-2-phenylindole; ERG: electroretinogram; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GCL: ganglion cell layer; INL: inner nuclear layer; IPL: inner plexiform layer; IS/OS: inner and outer photoreceptor segments; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; OCT: optical coherence tomography; ONL: outer nuclear layer; OPs: oscillatory potentials; p-EIF4EBP1: phosphorylated eukaryotic translation initiation factor 4E binding protein 1; p-MAPK14/p38: phosphorylated mitogen-activated protein kinase 14; RPE: retinal pigment epithelium; RPS6KB/p70 S6 kinase: ribosomal protein S6 kinase; SQSTM1/p62: sequestosome 1; TP53/TRP53/p53: tumor related protein 53; TSC2: TSC complex subunit 2; WT: wild type.

Fractalkine-induced microglial vasoregulation occurs within the retina and is altered early in diabetic retinopathy.

Local blood flow control within the central nervous system (CNS) is critical to proper function and is dependent on coordination between neurons, glia, and blood vessels. Macroglia, such as astrocytes and Müller cells, contribute to this neurovascular unit within the brain and retina, respectively. This study explored the role of microglia, the innate immune cell of the CNS, in retinal vasoregulation, and highlights changes during early diabetes. Structurally, microglia were found to contact retinal capillaries and neuronal synapses. In the brain and retinal explants, the addition of fractalkine, the sole ligand for monocyte receptor Cx3cr1, resulted in capillary constriction at regions of microglial contact. This vascular regulation was dependent on microglial Cx3cr1 involvement, since genetic and pharmacological inhibition of Cx3cr1 abolished fractalkine-induced constriction. Analysis of the microglial transcriptome identified several vasoactive genes, including angiotensinogen, a constituent of the renin-angiotensin system (RAS). Subsequent functional analysis showed that RAS blockade via candesartan abolished microglial-induced capillary constriction. Microglial regulation was explored in a rat streptozotocin (STZ) model of diabetic retinopathy. Retinal blood flow was reduced after 4 wk due to reduced capillary diameter and this was coincident with increased microglial association. Functional assessment showed loss of microglial-capillary response in STZ-treated animals and transcriptome analysis showed evidence of RAS pathway dysregulation in microglia. While candesartan treatment reversed capillary constriction in STZ-treated animals, blood flow remained decreased likely due to dilation of larger vessels. This work shows microglia actively participate in the neurovascular unit, with aberrant microglial-vascular function possibly contributing to the early vascular compromise during diabetic retinopathy.

Piezo2 Knockdown Inhibits Noxious Mechanical Stimulation and NGF-Induced Sensitization in A-Delta Bone Afferent Neurons.

Piezo2 is a mechanically gated ion-channel that has a well-defined role in innocuous mechanical sensitivity, but recently has also been suggested to play a role in mechanically induced pain. Here we have explored a role for Piezo2 in mechanically evoked bone nociception in Sprague Dawley rats. We have used an in vivo electrophysiological bone-nerve preparation to record the activity of single Aδ bone afferent neurons in response to noxious mechanical stimulation, after Piezo2 knockdown in the dorsal root ganglia with intrathecal injections of Piezo2 antisense oligodeoxynucleotides, or in control animals that received mismatch oligodeoxynucleotides. There were no differences in the number of Aδ bone afferent neurons responding to the mechanical stimulus, or their threshold for mechanical activation, in Piezo2 knockdown animals compared to mismatch control animals. However, bone afferent neurons in Piezo2 knockdown animals had reduced discharge frequencies and took longer to recover from stimulus-evoked fatigue than those in mismatch control animals. Piezo2 knockdown also prevented nerve growth factor (NGF)-induced sensitization of bone afferent neurons, and retrograde labeled bone afferent neurons that expressed Piezo2 co-expressed TrkA, the high affinity receptor for NGF. Our findings demonstrate that Piezo2 contributes to the response of bone afferent neurons to noxious mechanical stimulation, and plays a role in processes that sensitize them to mechanical stimulation.

The Contribution of Microglia to the Development and Maturation of the Visual System.

Microglia, the resident immune cells of the central nervous system (CNS), were once considered quiescent cells that sat in readiness for reacting to disease and injury. Over the last decade, however, it has become clear that microglia play essential roles in maintaining the normal nervous system. The retina is an easily accessible part of the central nervous system and therefore much has been learned about the function of microglia from studies in the retina and visual system. Anatomically, microglia have processes that contact all synapses within the retina, as well as blood vessels in the major vascular plexuses. Microglia contribute to development of the visual system by contributing to neurogenesis, maturation of cone photoreceptors, as well as refining synaptic contacts. They can respond to neural signals and in turn release a range of cytokines and neurotrophic factors that have downstream consequences on neural function. Moreover, in light of their extensive contact with blood vessels, they are also essential for regulation of vascular development and integrity. This review article summarizes what we have learned about the role of microglia in maintaining the normal visual system and how this has helped in understanding their role in the central nervous system more broadly.

Subthreshold Nano-Second Laser Treatment and Age-Related Macular Degeneration.

The presence of drusen is an important hallmark of age-related macular degeneration (AMD). Laser-induced regression of drusen, first observed over four decades ago, has led to much interest in the potential role of lasers in slowing the progression of the disease. In this article, we summarise the key insights from pre-clinical studies into the possible mechanisms of action of various laser interventions that result in beneficial changes in the retinal pigment epithelium/Bruch's membrane/choriocapillaris interface. Key learnings from clinical trials of laser treatment in AMD are also summarised, concentrating on the evolution of laser technology towards short pulse, non-thermal delivery such as the nanosecond laser. The evolution in our understanding of AMD, through advances in multimodal imaging and functional testing, as well as ongoing investigation of key pathological mechanisms, have all helped to set the scene for further well-conducted randomised trials to further explore potential utility of the nanosecond and other subthreshold short pulse lasers in AMD.

Photoreceptor Degeneration in Pro23His Transgenic Rats (Line 3) Involves Autophagic and Necroptotic Mechanisms.

Photoreceptor death contributes to 50% of irreversible vision loss in the western world. Pro23His (P23H) transgenic albino rat strains are widely used models for the most common rhodopsin gene mutation associated with the autosomal dominant form of retinitis pigmentosa. However, the mechanism(s) by which photoreceptor death occurs are not well understood and were the principal aim of this study. We first used electroretinogram recording and optical coherence tomography to confirm the time course of functional and structural loss. Electroretinogram analyses revealed significantly decreased rod photoreceptor (a-wave), bipolar cell (b-wave) and amacrine cell responses (oscillatory potentials) from P30 onward. The cone-mediated b-wave was also decreased from P30. TUNEL analysis showed extensive cell death at P18, with continued labeling detected until P30. Focused gene expression arrays indicated activation of, apoptosis, autophagy and necroptosis in whole retina from P14-18. However, analysis of mitochondrial permeability changes (ΔΨm) using JC-1 dye, combined with immunofluorescence markers for caspase-dependent (cleaved caspase-3) and caspase-independent (AIF) cell death pathways, indicated mitochondrial-mediated cell death was not a major contributor to photoreceptor death. By contrast, reverse-phase protein array data combined with RIPK3 and phospho-MLKL immunofluorescence indicated widespread necroptosis as the predominant mechanism of photoreceptor death. These findings highlight the complexity of mechanisms involved in photoreceptor death in the Pro23His rat model of degeneration and suggest therapies that target necroptosis should be considered for their potential to reduce photoreceptor death.

Fluorescent Labeling and Quantification of Vesicular ATP Release Using Live Cell Imaging.

Adenosine triphosphate (ATP) is actively transported into vesicles for purinergic neurotransmission by the vesicular nucleotide transporter, VNUT, encoded by the gene, solute carrier 17, member 9 (SLC17A9). In this chapter, methods are described for fluorescent labeling of VNUT positive cells and quantification of vesicular ATP release using live cell imaging. Directions for preparation of viable dissociated neurons and cellular labeling with an antibody against VNUT and for ATP containing synaptic vesicles with fluorescent ATP markers, quinacrine or MANT-ATP, are detailed. Using confocal microscope live cell imaging, cells positive for VNUT can be observed colocalized with fluorescent ATP vesicular markers, which occur as discrete puncta near the cell membrane. Vesicular release, stimulated with a depolarizing, high potassium physiological saline solution induces ATP marker fluorescence reduction at the cell membrane and this can be quantified over time to assess ATP release. Pretreatment with the voltage gated calcium channel blocker, cadmium, blocks depolarization-induced membrane fluorescence changes, suggesting that VNUT-positive neurons release ATP via calcium-dependent exocytosis. This technique may be applied for quantifying vesicular ATP release across the peripheral and central nervous system and is useful for unveiling the intricacies of purinergic neurotransmission.

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.

Reversibility of Retinal Ganglion Cell Dysfunction From Chronic IOP Elevation.

Purpose: To test the hypothesis that the capacity for retinal ganglion cells to functionally recover from chronic IOP elevation is dependent on the duration of IOP elevation. Methods: IOP elevation was induced in one eye in anesthetized (isoflurane) adult C57BL6/J mice using a circumlimbal suture. Sutures were left in place for 8 and 16 weeks (n = 30 and 28). In two other groups the suture was cut after 8 and 12 weeks (n = 30 and 28), and ganglion cell function (electroretinography) and retinal structure (optical coherence tomography) were assessed 4 weeks later. Ganglion cell density was quantified by counting RBPMS (RNA-binding protein with multiple splicing)-stained cells. Results: With IOP elevation (∼10 mm Hg above baseline), ganglion cell function declined to 75% ± 8% at 8 weeks and 59% ± 4% at 16 weeks relative to contralateral control eyes. The retinal nerve fiber layer was thinner at 8 (84% ± 4%) and 16 weeks (83% ± 3%), without a significant difference in total retinal thickness. Ganglion cell function recovered with IOP normalization (suture removal) at week 8 (97% ± 7%), but not at week 12 (73% ± 6%). Ganglion cell loss was found in all groups (-8% to -13%). Conclusions: In the mouse circumlimbal suture model, 12 weeks of IOP elevation resulted in irreversible ganglion cell dysfunction, whereas retinal dysfunction was fully reversible after 8 weeks of IOP elevation.

The renin-angiotensin system and the retinal neurovascular unit: A role in vascular regulation and disease.

The retina is known to have a local renin-angiotensin system (RAS) and dysfunction in the RAS is often associated with diseases of the retinal vasculature that cause irreversible vision loss. Regulation of the retinal vasculature to meet the metabolic needs of the tissues occurs through a mechanism called neurovascular coupling, which is critical for maintaining homeostatic function and support for neurons. Neurovascular coupling is the process by which support cells, including glia, regulate blood vessel calibre and blood flow in response to neural activity. In retinal vascular diseases, this coupling mechanism is often disrupted. However, the role that angiotensin II (Ang II), the main effector peptide of the RAS, has in regulating both the retinal vasculature and neurovascular coupling is not fully understood. As components of the RAS are located on the principal neurons, glia and blood vessels of the retina, it is possible that Ang II has a role in regulating communication and function between these three cell types, and therefore the capacity to regulate neurovascular coupling. This review focuses on components of the RAS located on the retinal neurovascular unit, and the potential of this system to contribute to blood flow modulation in the healthy and compromised retina.

Targeting P2X7 receptors as a means for treating retinal disease.

Age-related macular degeneration and glaucoma are the commonest causes of irreversible vision loss in industrialized countries. The purine ATP is known to regulate a range of cellular functions in the retina via its action on P2 receptors, especially the P2X7 receptor. Although agents that attenuate P2X7 receptor function have been in development for many years, no compound is currently approved for the treatment of eye disease. However, newer compounds that cross the blood-brain barrier could have potential to reduce vision loss. This review will outline recent information relating to the role of P2X7 in age-related macular degeneration and glaucoma and, subsequently, we will discuss recent developments for attenuating P2X7 receptor function.

Rod Photoreceptor Activation Alone Defines the Release of Dopamine in the Retina.

Retinal dopamine is released by a specialized subset of amacrine cells in response to light and has a potent influence on how the retina responds to, and encodes, visual information. Here, we address the critical question of which retinal photoreceptor is responsible for coordinating the release of this neuromodulator. Although all three photoreceptor classes-rods, cones, and melanopsin-containing retinal ganglion cells (mRGCs)-have been shown to provide electrophysiological inputs to dopaminergic amacrine cells (DACs), we show here that the release of dopamine is defined only by rod photoreceptors. Remarkably, this rod signal coordinates both a suppressive signal at low intensities and drives dopamine release at very bright light intensities. These data further reveal that dopamine release does not necessarily correlate with electrophysiological activity of DACs and add to a growing body of evidence that rods define aspects of retinal function at very bright light levels.

Failure of Autophagy-Lysosomal Pathways in Rod Photoreceptors Causes the Early Retinal Degeneration Phenotype Observed in Cln6nclf Mice.

Purpose: Vision loss caused by photoreceptor death represents one of the first symptoms in neuronal ceroid lipofuscinosis, a condition characterized by accumulation of intracellular waste. Cln6nclf mice have a naturally occurring mutation in ceroid-lipofuscinosis neuronal (CLN) protein 6 and are a model of this disorder. In order to identify the effect intracellular waste (lipofuscin) accumulation plays in driving retinal degeneration, the time course of degeneration was carefully characterized functionally using the electroretinogram and structurally using histology. Methods: Cln6nclf and C57BL/6J, wild-type, mice were studied at postnatal day 18 (P18), P30, P60, P120, and P240, and retinal degeneration was correlated with changes in the retinal pigment epithelial (RPE) and neuronal autophagy-lysosomal pathways using super-resolution microscopy. Results: In Cln6nclf mice there was significant loss of rod photoreceptor function at P18, prior to photoreceptor nuclei loss at P60. In contrast, cone pathway function was not affected until P240. The loss of rod photoreceptor function correlated with significant disruption of the autophagy-lysosomal degradation pathways within photoreceptors, but not in the RPE or other retinal neurons. Additionally, there was cytosolic accumulation of P62 and undigested mitochondrial-derived, ATP synthase subunit C in the photoreceptor layers of Cln6nclf mice at P30. Conclusions: These results suggest that rod photoreceptors have an increased sensitivity to disturbances in the autophagy-lysosomal pathway and the subsequent failure of mitochondrial turnover, relative to other retinal cells. It is likely that primary failure of the rod photoreceptors rather than the RPE or other retinal neurons underlies the early visual dysfunction that occurs in the Cln6nclf mouse model.

The Role of the Microglial Cx3cr1 Pathway in the Postnatal Maturation of Retinal Photoreceptors.

Microglia are the resident immune cells of the CNS, and their response to infection, injury and disease is well documented. More recently, microglia have been shown to play a role in normal CNS development, with the fractalkine-Cx3cr1 signaling pathway of particular importance. This work describes the interaction between the light-sensitive photoreceptors and microglia during eye opening, a time of postnatal photoreceptor maturation. Genetic removal of Cx3cr1 (Cx3cr1GFP/GFP ) led to an early retinal dysfunction soon after eye opening [postnatal day 17 (P17)] and cone photoreceptor loss (P30 onward) in mice of either sex. This dysfunction occurred at a time when fractalkine expression was predominantly outer retinal, when there was an increased microglial presence near the photoreceptor layer and increased microglial-cone photoreceptor contacts. Photoreceptor maturation and outer segment elongation was coincident with increased opsin photopigment expression in wild-type retina, while this was aberrant in the Cx3cr1GFP/GFP retina and outer segment length was reduced. A beadchip array highlighted Cx3cr1 regulation of genes involved in the photoreceptor cilium, a key structure that is important for outer segment elongation. This was confirmed with quantitative PCR with specific cilium-related genes, Rpgr and Rpgrip1, downregulated at eye opening (P14). While the overall cilium structure was unaffected, expression of Rpgr, Rpgrip1, and centrin were restricted to more proximal regions of the transitional zone. This study highlighted a novel role for microglia in postnatal neuronal development within the retina, with loss of fractalkine-Cx3cr1 signaling leading to an altered distribution of cilium proteins, failure of outer segment elongation and ultimately cone photoreceptor loss.SIGNIFICANCE STATEMENT Microglia are involved in CNS development and disease. This work highlights the role of microglia in postnatal development of the light-detecting photoreceptor neurons within the mouse retina. Loss of the microglial Cx3cr1 signaling pathway resulted in specific alterations in the cilium, a key structure in photoreceptor outer segment elongation. The distribution of key components of the cilium transitional zone, Rpgr, Rpgrip1, and centrin, were altered in retinae lacking Cx3cr1 with reduced outer segment length and cone photoreceptor death observed at later postnatal ages. This work identifies a novel role for microglia in the postnatal maturation of retinal photoreceptors.

Prophylactic laser in age-related macular degeneration: the past, the present and the future.

The presence of drusen in the posterior eye is a hallmark feature of the early stages of age-related macular degeneration and their size is an indicator of risk of progression to vision-threatening forms of the disease. Since the initial observations that laser treatment can resolve drusen, there has been great interest in whether laser treatment can be used to reduce the progression of age-related macular degeneration. In this article, we review the development of lasers for the treatment of those with age-related macular degeneration. We provide an overview of the clinical trial results that demonstrated drusen resolution but that had mixed effects on progression of disease. In addition, we provide a summary of the recent developments in pulsed lasers that are designed to reduce the energy applied to the posterior eye to provide the therapeutic effects of conventional continuous wave lasers while reducing the secondary tissue effects.

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.