Effects of slit lamp-delivered retinal laser photobiomodulation in a rat model of choroidal neovascularization.

Neovascular age-related macular degeneration, also known as exudative or wet age-related macular degeneration, is the leading cause of blindness in the developed world. Photobiomodulation has the potential to target the up-stream hypoxic and pro-inflammatory drivers of choroidal neovascularization. This study investigated whether photobiomodulation attenuates characteristic pathological features of choroidal neovascularization in a rodent model. Experimental choroidal neovascularization was induced in Brown Norway rats with laser photocoagulation. A custom-designed, slit-lamp-mounted, 670 nm laser was used to administer retinal photobiomodulation every 3 days, beginning 6 days prior to choroidal neovascularization induction and continuing until the animals were killed 14 days later. The effect of photobiomodulation on the size of choroidal neovascular membranes was determined using isolectin-B4 immunohistochemistry and spectral domain-optical coherence tomography. Vascular leakage was determined with fluorescein angiography. The effect of treatment on levels of vascular endothelial growth factor expression was quantified with enzyme-linked immunosorbent assay. Treatment with photobiomodulation was associated with choroidal neovascular membranes that were smaller, had less fluorescein leakage, and a diminished presence of inflammatory cells as compared to sham eyes. These effects were not associated with a statistically significant difference in the level of vascular endothelial growth factor when compared to sham eyes. The data shown herein indicate that photobiomodulation attenuates pathological features of choroidal neovascularization in a rodent model by mechanisms that may be independent of vascular endothelial growth factor.

Disturbed glucose and pyruvate metabolism in glaucoma with neuroprotection by pyruvate or rapamycin.

Intraocular pressure-sensitive retinal ganglion cell degeneration is a hallmark of glaucoma, the leading cause of irreversible blindness. Here, we used RNA-sequencing and metabolomics to examine early glaucoma in DBA/2J mice. We demonstrate gene expression changes that significantly impact pathways mediating the metabolism and transport of glucose and pyruvate. Subsequent metabolic studies characterized an intraocular pressure (IOP)-dependent decline in retinal pyruvate levels coupled to dysregulated glucose metabolism prior to detectable optic nerve degeneration. Remarkably, retinal glucose levels were elevated 50-fold, consistent with decreased glycolysis but possibly including glycogen mobilization and other metabolic changes. Oral supplementation of the glycolytic product pyruvate strongly protected from neurodegeneration in both rat and mouse models of glaucoma. Investigating further, we detected mTOR activation at the mechanistic nexus of neurodegeneration and metabolism. Rapamycin-induced inhibition of mTOR robustly prevented glaucomatous neurodegeneration, supporting a damaging role for IOP-induced mTOR activation in perturbing metabolism and promoting glaucoma. Together, these findings support the use of treatments that limit metabolic disturbances and provide bioenergetic support. Such treatments provide a readily translatable strategy that warrants investigation in clinical trials.

Physiological response of the retinal pigmented epithelium to 3-ns pulse laser application, in vitro and in vivo.

BACKGROUND: To treat healthy retinal pigmented epithelium (RPE) with the 3-ns retinal rejuvenation therapy (2RT) laser and to investigate the subsequent wound-healing response of these cells. METHODS: Primary rat RPE cells were treated with the 2RT laser at a range of energy settings. Treated cells were fixed up to 7 days post-irradiation and assessed for expression of proteins associated with wound-healing. For in vivo treatments, eyes of Dark Agouti rats were exposed to laser and tissues collected up to 7 days post-irradiation. Isolated wholemount RPE preparations were examined for structural and protein expression changes. RESULTS: Cultured RPE cells were ablated by 2RT laser in an energy-dependent manner. In all cases, the RPE cell layer repopulated completely within 7 days. Replenishment of RPE cells was associated with expression of the heat shock protein, Hsp27, the intermediate filament proteins, vimentin and nestin, and the cell cycle-associated protein, cyclin D1. Cellular tight junctions were lost in lased regions but re-expressed when cell replenishment was complete. In vivo, 2RT treatment gave rise to both an energy-dependent localised denudation of the RPE and the subsequent repopulation of lesion sites. Cell replenishment was associated with the increased expression of cyclin D1, vimentin and the heat shock proteins Hsp27 and αB-crystallin. CONCLUSIONS: The 2RT laser was able to target the RPE both in vitro and in vivo, causing debridement of the cells and the consequent stimulation of a wound-healing response leading to layer reformation.

Preclinical and clinical studies of photobiomodulation therapy for macular oedema.

AIMS/HYPOTHESIS: Diabetic macular oedema (DME) is the leading cause of visual impairment in people with diabetes. Intravitreal injections of vascular endothelial growth factor inhibitors or corticosteroids prevent loss of vision by reducing DME, but the injections must be given frequently and usually for years. Here we report laboratory and clinical studies on the safety and efficacy of 670 nm photobiomodulation (PBM) for treatment of centre-involving DME. METHODS: The therapeutic effect of PBM delivered via a light-emitting diode (LED) device was tested in transgenic mice in which induced Müller cell disruption led to photoreceptor degeneration and retinal vascular leakage. We also developed a purpose-built 670 nm retinal laser for PBM to treat DME in humans. The effect of laser-delivered PBM on improving mitochondrial function and protecting against oxidative stress was studied in cultured rat Müller cells and its safety was studied in pigmented and non-pigmented rat eyes. We then used the retinal laser to perform PBM in an open-label, dose-escalation Phase IIa clinical trial involving 21 patients with centre-involving DME. Patients received 12 sessions of PBM over 5 weeks for 90 s per treatment at a setting of 25, 100 or 200 mW/cm2 for the three sequential cohorts of 6-8 patients each. Patients were recruited from the Sydney Eye Hospital, over the age of 18 and had centre-involving DME with central macular thickness (CMT) of >300 µm with visual acuity of 75-35 Log minimum angle of resolution (logMAR) letters (Snellen visual acuity equivalent of 20/30-20/200). The objective of this trial was to assess the safety and efficacy of laser-delivered PBM at 2 and 6 months. The primary efficacy outcome was change in CMT at 2 and 6 months. RESULTS: LED-delivered PBM enhanced photoreceptor mitochondrial membrane potential, protected Müller cells and photoreceptors from damage and reduced retinal vascular leakage resulting from induced Müller cell disruption in transgenic mice. PBM delivered via the retinal laser enhanced mitochondrial function and protected against oxidative stress in cultured Müller cells. Laser-delivered PBM did not damage the retina in pigmented rat eyes at 100 mW/cm2. The completed clinical trial found a significant reduction in CMT at 2 months by 59 ± 46 µm (p = 0.03 at 200 mW/cm2) and significant reduction at all three settings at 6 months (25 mW/cm2: 53 ± 24 µm, p = 0.04; 100 mW/cm2: 129 ± 51 µm, p < 0.01; 200 mW/cm2: 114 ± 60 µm, p < 0.01). Laser-delivered PBM was well tolerated in humans at settings up to 200 mW/cm2 with no significant side effects. CONCLUSIONS/INTERPRETATION: PBM results in anatomical improvement of DME over 6 months and may represent a safe and non-invasive treatment. Further testing is warranted in randomised clinical trials. TRIAL REGISTRATION: ClinicalTrials.gov NCT02181400 Graphical abstract.

Improved immunohistochemical detection of phosphorylated mitogen-activated protein kinases in the injured rat optic nerve head.

The activity of mitogen-activated protein kinases (MAPKs) is largely controlled by addition or removal of phosphate groups, which are carried out by kinase or phosphatase enzymes, respectively. Determining the phosphorylation status of MAPK isoenzymes, therefore, aids elucidation of the physiological and pathological roles of this enzyme. In practical terms, however, end-point procurement of appropriate experimental tissues produces conditions where MAPK phosphorylation status can rapidly alter, thus giving rise to aberrant data. We therefore attempted to instigate a means of stabilising end-point MAPK phosphorylation levels when procuring tissues for analysis. We employed a well-described rat model of ocular hypertension in which MAPK isoenzyme activation occurs in the optic nerve head (ONH), but can vary according to the level of resultant tissue pathology. Animals were appropriately treated and after 3 days were perfused in the presence or absence of a cocktail of phosphatase inhibitors (PIs), immediately prior to tissue fixation, in order to prevent dephosphorylation of phosphorylated MAPKs. Immunohistochemical labelling for phosphorylated MAPKs in untreated ONH sections was unaffected by the presence of PIs in the perfusate. MAPK activation was detected by immunohistochemistry in the treated ONH, but findings varied considerably, particularly in animals with less extensive tissue damage. The presence of PIs in the perfusate, however, significantly reduced this variation and enabled consistent changes to be detected, particularly in the animals with less extensive tissue damage. Thus, the addition of PIs to the perfusate is suggested when studying MAPK activation by immunohistochemistry, especially in the ONH.

Spatio-temporal characterization of S- and M/L-cone degeneration in the Rd1 mouse model of retinitis pigmentosa.

BACKGROUND: The Pde6brd1 (Rd1) mouse is widely used as a murine model for human retinitis pigmentosa. Understanding the spatio-temporal patterns of cone degeneration is important for evaluating potential treatments. In the present study we performed a systematic characterization of the spatio-temporal patterns of S- and M/L-opsin+ cone outer segment and cell body degeneration in Rd1 mice, described the distribution and proportion of dual cones in Rd1 retinas, and examined the kinetics of microglial activation during the period of cone degeneration. RESULTS: Outer segments of S- and M/L-cones degenerated far more rapidly than their somas. Loss of both S- and M/L-opsin+ outer segments was fundamentally complete by P21 in the central retina, and 90% complete by P45 in the peripheral retina. In comparison, degeneration of S- and M/L-opsin+ cell bodies proceeded at a slower rate. There was a marked hemispheric asymmetry in the rate of S-opsin+ and M/L-opsin+ cell body degeneration. M/L-opsin+ cones were more resilient to degeneration in the superior retina, whilst S-opsin+ cones were relatively preserved in the inferior retina. In addition, cone outer segment and cell body degeneration occurred far more rapidly in the central than the peripheral retina. At P14, the superior retina comprised a minority of genuine S-cones with a much greater complement of genuine M/L-opsin cones and dual cones, whilst the other three retinal quadrants had broadly similar numbers of genuine S-cones, genuine M/L-cones and dual cones. At P60, approximately 50% of surviving cones in the superior, nasal and temporal quadrants were dual cones. In contrast, the inferior peripheral retina at P60 contained almost exclusively genuine S-cones with a tiny minority of dual cones. Microglial number and activity were stimulated during rod breakdown, remained relatively high during cone outer segment degeneration and loss of cone somas in the central retina, and decreased thereafter in the period coincident with slow degeneration of cone cell bodies in the peripheral retina. CONCLUSION: The results of the present study provide valuable insights into cone degeneration in the Rd1 mouse, substantiating and extending conclusions drawn from earlier studies.

Expression and activation of mitogen-activated protein kinases in the optic nerve head in a rat model of ocular hypertension.

BACKGROUND: Glaucoma is a leading cause of irreversible blindness manifesting as an age-related, progressive optic neuropathy with associated retinal ganglion cell (RGC) loss. Mitogen-activated protein kinases (MAPKs: p42/44 MAPK, SAPK/JNK, p38 MAPK) are activated in various retinal disease models and likely contribute to the mechanisms of RGC death. Although MAPKs play roles in the development of retinal pathology, their action in the optic nerve head (ONH), where the initial insult to RGC axons likely resides in glaucoma, remains unexplored. METHODS: An experimental paradigm representing glaucoma was established by induction of chronic ocular hypertension (OHT) via laser-induced coagulation of the trabecular meshwork in Sprague-Dawley rats. MAPKs were subsequently investigated over the following days for expression and activity alterations, using RT-PCR, immunohistochemistry and Western immunoblot. RESULTS: p42/44 MAPK expression was unaltered after intraocular pressure (IOP) elevation, but there was a significant activation of this enzyme in ONH astrocytes after 6-24 h. Activated SAPK/JNK isoforms were present throughout healthy RGC axons but after IOP elevation or optic nerve crush, they both accumulated at the ONH, likely due to RGC axon transport disruption, and were subject to additional activation. p38 MAPK was expressed by a population of microglia which were significantly more populous following IOP elevation. However it was only significantly activated in microglia after 3 days, and then only in the ONH and optic nerve; in the retina it was solely activated in RGC perikarya. CONCLUSIONS: In conclusion, each of the MAPKs showed a specific spatio-temporal expression and activation pattern in the retina, ONH and optic nerve as a result of IOP elevation. These findings likely reflect the roles of the individual enzymes, and the cells in which they reside, in the developing pathology following IOP elevation. These data have implications for understanding the mechanisms of ocular pathology in diseases such as glaucoma.

Neuroprotection in glaucoma: recent advances and clinical translation.

Intraocular pressure (IOP) reduction is currently the only evidence-based treatment strategy for glaucoma. However, IOP control in some individuals is challenging. Despite optimal treatment, a significant proportion of individuals will progress, with loss of visual field, loss of driving vision and impaired quality of life. A new modality that could augment current treatment and reduce the rate of neurodegeneration to preserve vision throughout life would be a major breakthrough. A vast number of studies have reported effective neuroprotection in animal models of glaucoma; however, translation to the clinic remains a major hurdle. Herein, we explore the therapeutic advancements in non-IOP-dependent neuroprotection research based upon potential pathogenic mechanisms and propose strategies to improve the clinical translation of neuroprotective research in glaucoma.

Retinal pigment epithelium in the pathogenesis of age-related macular degeneration and photobiomodulation as a potential therapy?

The retinal pigment epithelium (RPE) comprises a monolayer of cells located between the neuroretina and the choriocapillaries. The RPE serves several important functions in the eye: formation of the blood-retinal barrier, protection of the retina from oxidative stress, nutrient delivery and waste disposal, ionic homeostasis, phagocytosis of photoreceptor outer segments, synthesis and release of growth factors, reisomerization of all-trans-retinal during the visual cycle, and establishment of ocular immune privilege. Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries. Dysfunction of the RPE has been associated with the pathogenesis of AMD in relation to increased oxidative stress, mitochondrial destabilization and complement dysregulation. Photobiomodulation or near infrared light therapy which refers to non-invasive irradiation of tissue with light in the far-red to near-infrared light spectrum (630-1000 nm), is an intervention that specifically targets key mechanisms of RPE dysfunction that are implicated in AMD pathogenesis. The current evidence for the efficacy of photobiomodulation in AMD is poor but its safety profile and proposed mechanisms of action motivate further research as a novel therapy for AMD.

Glucose metabolism in mammalian photoreceptor inner and outer segments.

Photoreceptors are the first-order neurons of the visual pathway, converting light into electrical signals. Rods and cones are the two main types of photoreceptors in the mammalian retina. Rods are specialized for sensitivity at the expense of resolution and are responsible for vision in dimly lit conditions. Cones are responsible for high acuity central vision and colour vision. Many human retinal diseases are characterized by a progressive loss of photoreceptors. Photoreceptors consist of four primary regions: outer segments, inner segments, cell bodies and synaptic terminals. Photoreceptors consume large amounts of energy, and therefore, energy metabolism may be a critical juncture that links photoreceptor function and survival. Cones require more energy than rods, and cone degeneration is the main cause of clinically significant vision loss in retinal diseases. Photoreceptor segments are capable of utilizing various energy substrates, including glucose, to meet their large energy demands. The pathways by which photoreceptor segments meet their energy demands remain incompletely understood. Improvements in the understanding of glucose metabolism in photoreceptor segments may provide insight into the reasons why photoreceptors degenerate due to energy failure. This may, in turn, assist in developing bio-energetic therapies aimed at protecting photoreceptors.

Investigations into localized re-treatment of the retina with a 3-nanosecond laser.

BACKGROUND AND OBJECTIVES: Subvisual retinal lasers necessarily cause clinically invisible lesions, hence, they could intentionally or inadvertently be targeted at precisely the same or an overlapping location during repeat laser treatment. Herein, we investigated the structural integrity and cellular responses of localized re-treatment using a nanosecond laser (2RT) currently in trials for early age-related macular degeneration. MATERIALS AND METHODS: Rats were randomly assigned to one of five groups: sham, subvisual 2RT, subvisual 2RT re-treatment, visual effect 2RT, visual effect 2RT re-treatment. Re-treatment groups were lasered on days 0 and 21; single laser groups were only lasered on day 21. All rats were euthanized at day 28 and eyes were then dissected and processed for immunohistochemistry. For re-treatment, the laser was targeted at precisely the same locations on both delivery occasions. Analytical endpoints included monitoring of retinal vascular integrity overlying lesions, investigation into any potential choroidal neovascularization, assessment of the RPE, quantification of collateral injury to photoreceptors or other neuronal classes, and delineation of glial reactivity. RESULTS: Repeat laser administration to rats caused ostensibly identical retinal-RPE-choroid responses to those obtained in age-matched rats that received only a single application. Specifically, 7 days after treatment, RPE cells were re-populating lesion sites. No obvious consistent differences were evident between the single and repeat laser groups. Moreover, repeat laser caused no (measurable) additive injury to photoreceptors or other retinal neuronal classes from single laser treatment. In re-lasered animals, there was no increase in microglial activity overlying and adjacent to lesion sites relative to single lasered rats. Finally, there was no evidence of choroidal neovascularization after repeat laser treatment. CONCLUSIONS: The overall results provide a measure of confidence that re-treatment of patients with 2RT should not provide any additional risk of developing visual scotomas, choroidal neovascularizations, or inflammatory events. Indeed, the collated results indicate that the metabolic and structural disruption to the RPE-retina caused by short pulse duration laser treatment is resolved within a short time frame such that re-treatment elicits a phenotype indistinguishable from single treatment. Lasers Surg. Med. 48:602-615, 2016. © 2016 Wiley Periodicals, Inc.

Short-pulse duration retinal lasers: a review.

The development of lasers for biological use was an important medical advance in the 20th century with numerous evidence-based therapeutic applications to retinal disease, including capillary leakage at the macula. Although the role of photocoagulative laser in the treatment of macular oedema has diminished, there is evidence for a modified role in clinical management, particularly for extrafoveal leakage. Additionally, it may reduce the frequency of required intravitreal injections and assist in visual stabilization when used as an adjunct. The tissue destructive effect of photocoagulative lasers has motivated the development of safer macular lasers and the search for novel therapeutic applications, including treatment of drusen and regeneration of dysfunctional retinal pigment epithelium.

Role of the nucleolus in neurodegenerative diseases with particular reference to the retina: a review.

The nucleolus has emerged as a key regulator of cellular growth and the response to stress, in addition to its traditionally understood function in ribosome biogenesis. The association between nucleolar function and neurodegenerative disease is increasingly being explored. There is also recent evidence indicating that the nucleolus may well be crucial in the development of the eye. In this present review, the role of the nucleolus in retinal development as well as in neurodegeneration with an emphasis on the retina is discussed.

Effect of subconjunctival glucose on retinal ganglion cell survival in experimental retinal ischaemia and contrast sensitivity in human glaucoma.

PURPOSE: This study aims to evaluate the effect of subconjunctival glucose on the retinal ganglion cells (RGCs) in experimental retinal ischaemia and contrast sensitivity in humans with primary open-angle glaucoma (POAG). METHODS: First, we measured the intravitreal concentration of glucose at various time points after a subconjunctival injection of 100 µl of 50% glucose to Sprague-Dawley rats. Next, treatment and control groups received 50% subconjunctival glucose and iso-osmotic (8%) saline, respectively, 1 h prior to a unilateral ischaemic retinal injury; 7 days later, the damage profiles were compared using RGC and axon counts. Subsequently, we conducted a double-blind, crossover, pilot clinical study in seven eyes of five pseudophakic subjects with severe POAG. Subjects received either 0.3 mL of 50% glucose subconjunctivally or iso-osmotic (8%) saline, then vice versa after a 2-3 week 'wash-out' period; change in contrast sensitivity from baseline was the primary outcome. RESULTS: Subconjunctival glucose preserved approximately 60% of Brn3a-positive RGCs in all retinal zones compared with an 80% loss in control retinas, and rescued approximately 40% of the axonal loss. In the human trial, the contrast sensitivity at 12 cycles/degree was 0.24 log units greater than baseline (95% confidence interval 0.12-0.36; P < 0.001). CONCLUSIONS: Subconjunctival glucose partially protects RGC somata and axons against an ischaemic insult and temporarily recovers contrast sensitivity in patients with severe POAG. Although an unlikely therapeutic strategy for POAG, the findings motivate further bioenergetic-based research in glaucoma and other optic nerve and retinal diseases, where energy failure may be part of the pathogenesis.

Cancer-like metabolism of the mammalian retina.

The retina, like many cancers, produces energy from glycolysis even in the presence of oxygen. This phenomenon is known as aerobic glycolysis and eponymously as the Warburg effect. In recent years, the Warburg effect has become an explosive area of study within the cancer research community. The expanding knowledge about the molecular mechanisms underpinning the Warburg effect in cancer promises to provide a greater understanding of mammalian retinal metabolism and has motivated cancer researchers to target the Warburg effect as a novel treatment strategy for cancer. However, if the molecular mechanisms underlying the Warburg effect are shared by the retina and cancer, treatments targeting the Warburg effect may have serious adverse effects on retinal metabolism. Herein, we provide an updated understanding of the Warburg effect in mammalian retina.

Glucose-induced temporary visual recovery in primary open-angle glaucoma: a double-blind, randomized study.

PURPOSE: To investigate the effect of topical glucose on visual parameters in individuals with primary open-angle glaucoma (POAG). DESIGN: Double-blind, randomized, crossover study. PARTICIPANTS: Nondiabetic pseudophakic patients with definite POAG were recruited; 29 eyes of 16 individuals participated in study 1. A follow-up study (study 2) included 14 eyes of 7 individuals. INTERVENTION: Eyes were randomly allocated to receive 50% glucose or saline eye drops every 5 minutes for 60 minutes. MAIN OUTCOME MEASURES: The contrast sensitivity and best-corrected logarithm of the minimum angle of resolution (logMAR). RESULTS: The 50% glucose reached the vitreous in pseudophakic but not phakic individuals. Glucose significantly improved the mean contrast sensitivity at 12 cycles/degree compared with 0.9% saline by 0.26 log units (95% confidence interval [CI], 0.13-0.38; P < 0.001) and 0.40 log units (95% CI, 0.17-0.60; P < 0.001) in the follow-up study. The intraocular pressure, refraction, and central corneal thickness were not affected by glucose; age was not a significant predictor of the response. CONCLUSIONS: Topical glucose temporarily improves psychophysical visual parameters in some individuals with POAG, suggesting that neuronal energy substrate delivery to the vitreous reservoir may recover function of "sick" retinal neurons.

Effects of a conventional photocoagulator and a 3-ns pulse laser on preconditioning responses and retinal ganglion cell survival after optic nerve crush.

Previous research has demonstrated that laser photocoagulation treatment of the monkey retina affords protection against experimental glaucoma-induced retinal ganglion cell (RGC) loss in areas overlying laser spots. The underlying mechanism is unknown, but it is conceivable that the laser acted as a preconditioning stimulus, inducing localised, endogenous production of survival factors. The related purposes of the current study were firstly to examine whether preconditioning pathways are activated by either a conventional photocoagulator (CW) laser or a photoreceptor-sparing, short-pulse duration (2RT) laser in the rat retina, and secondly, to examine whether such preconditioning with either laser improves RGC survival after optic nerve (ON) crush. Pigmented rats were randomly assigned to one of three groups: sham, CW, 2RT. For the preconditioning study, laser spots were applied randomly to each retina in the posterior hemisphere of the eye taking care to avoid major blood vessels. Animals were killed at 6 h, 1d, and 7d after laser treatment, then analysed by qPCR, immunohistochemistry or Western immunoblotting. For the neuroprotection study, laser spots were administered to the mid-central retina of the right eye. The left eye served as a control. In two experiments, rats were lasered either 24 h or 7 days before ON crush, then killed a further 7 days later. Wholemount retinas were prepared and double labelling immunofluorescence performed. Nestin labelling allowed visualization of laser spots. Brn3a labelling identified viable RGCs. Photomicrographs of Brn3a labelling were taken in areas overlying nestin-positive laser spots. Quantification of Brn3a RGCs was then performed. Both the CW and 2RT lasers induced local glial cell activation. Moreover, both lasers induced localized upregulations of a number of well-documented (CNTF, FGF-2 Hsp27, pAKT) or putative (cFOS, ATF-3, IL-6) RGC survival factors. However, neither laser caused sustained increases in other factors associated with neuronal preconditioning, such as BDNF, Hsp70, IGF-1, bcl-2, and nitric oxide synthase. As regards neuroprotection, analysis of the data revealed that ON crush resulted in the loss of approximately 70% of Brn3a-labelled RGCs after 1 week. Neither the CW nor the 2RT laser augmented Brn3a-positive RGC survival in areas overlying and neighbouring laser spots. This was the case irrespective of whether lasering occurred 1 or 7 days before the ON crush. Our results showed that the CW and 2RT lasers both stimulated de novo synthesis of certain genes that are well-known RGC survival factors and/or that have been implicated in preconditioning-induced neuroprotection studies. Despite these findings, neither laser augmented survival of RGCs when delivered prior to ON crush.

Immune priming and experimental glaucoma: the effect of prior systemic lipopolysaccharide challenge on tissue outcomes after optic nerve injury.

BACKGROUND: Microglial activation is a prominent feature throughout the optic pathway in experimental glaucoma. Pro-inflammatory microglial activation may contribute to neurodegeneration through the release of pro-inflammatory cytokines and other inflammatory mediators. Systemic administration of lipopolysaccharide stimulates microglia to produce pro-inflammatory cytokines and chemoattractants. A preliminary investigation demonstrated pro-inflammatory microglial activation throughout the optic pathway following systemic lipopolysaccharide challenge. The aim of the current work was to investigate whether microglial priming with lipopolysaccharide would exacerbate optic nerve injury in rats following experimental glaucoma. METHODS: Adult female Sprague-Dawley rats were divided into lipopolysaccharide treatment (n = 15) and saline treatment groups (n = 15). Microglial priming was induced with a 2.5-mg/kg intraperitoneal injection of lipopolysaccharide; control animals received saline. Experimental glaucoma was induced 48 h later in the right eyes of animals by laser photocoagulation of the trabecular meshwork. Animals were sacrificed 9 days after laser treatment. RESULTS: The estimated number of axons per optic nerve was 51 327 ± 3868 (mean ± standard error of the mean) in the lipopolysaccharide group and 54 569 ± 6687 (mean ± standard error of the mean) in the saline group. Optic nerve axon counts were not significantly different between lipopolysaccharide and saline groups (P = 0.67). CONCLUSIONS: Systemic lipopolysaccharide challenge had no discernible effect on optic nerve injury in laser-induced experimental glaucoma. These findings do not support the hypothesis that this model of experimental glaucoma involves inflammation and instead suggest that microglial activation may occur secondary to chronic neurodegeneration.