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.

N-acetylcysteine amide and di- N-acetylcysteine amide protect retinal cells in culture via an antioxidant action.

Reactive oxygen species (ROS) play a significant role in toxicity to the retina in a variety of diseases. N-acetylcysteine (NAC), N-acetylcysteine amide (NACA) and the dimeric di-N-acetylcysteine amide (diNACA) were evaluated in terms of protecting retinal cells, in vitro, in a variety of stress models. Three types of rat retinal cell cultures were utilized in the study: macroglial-only cell cultures, neuron-only retinal ganglion cell (RGC) cultures, and mixed cultures containing retinal glia and neurons. Ability of test agents to attenuate oxidative stress in all cultures was ascertained. In addition, capability of agents to protect against a variety of alternate clinically-relevant stressors, including excitotoxins and mitochondrial electron transport chain inhibitors, was also evaluated. Capacity of test agents to elevate cellular levels of reduced glutathione under normal and compromised conditions was also determined. NAC, NACA and diNACA demonstrated concentration-dependent cytoprotection against oxidative stress in all cultures. These three compounds, however, had differing effects against a variety of alternate insults to retinal cells. The most protective agent was NACA, which was most potent against the most stressors (including oxidative stress, mitochondrial impairment by antimycin A and azide, and glutamate-induced excitotoxicity). Similar to NAC, NACA increased glutathione levels in non-injured cells, although diNACA did not, suggesting a different, unknown mechanism of antioxidant activity for the latter. In support of this, diNACA was the only agent to attenuate rotenone-induced toxicity in mitochondria. NAC, NACA and diNACA exhibited varying degrees of antioxidant activity, i.e., protected cultured rat retinal cells from a variety of stressors which were designed to mimic aspects of the pathology of different retinal diseases. A general rank order of activity was observed: NACA ≥ diNACA > NAC. These results warrant further exploration of NACA and diNACA as antioxidant therapeutics for the treatment of retinal diseases, particularly those involving oxidative stress. Furthermore, we have defined the battery of tests carried out as the "Wood, Chidlow, Wall and Casson (WCWC) Retinal Antioxidant Indices"; we believe that these are of great value for screening molecules for potential to reduce retinal oxidative stress in a range of retinal diseases.

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.

Characterization of the novel spontaneously immortalized rat Müller cell line SIRMu-1.

Müller cells (MCs) play a crucial role in the retina, and cultured MC lines are an important tool with which to study MC function. Transformed MC lines have been widely used; however, the transformation process can also lead to unwanted changes compared to the primary cells from which they were derived. To provide an alternative experimental tool, a novel monoclonal spontaneously immortalized rat Müller cell line, SIRMu-1, was derived from primary rat MCs and characterized. Immunofluorescence, western blotting and RNA sequencing demonstrate that the SIRMu-1 cell line retains similar characteristics to cultured primary MCs in terms of expression of the MC markers cellular retinaldehyde-binding protein, glutamine synthetase, S100, vimentin and glial fibrillary acidic protein at both the mRNA and protein levels. Both the cellular morphology and overall transcriptome of the SIRMu-1 cells are more similar to primary rat MCs than the commonly used rMC-1 cells, a well-described, transformed rat MC line. Furthermore, SIRMu-1 cells proliferate rapidly, have an effectively indefinite life span and a high transfection efficiency. The expression of Y chromosome specific genes confirmed that the SIRMu-1 cells are derived from male MCs. Thus, the SIRMu-1 cell line represents a valuable experimental tool to study roles of MCs in both physiological and pathological states.

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.

Macrophage Elastase Induces TRAIL-mediated Tumor Cell Death through Its Carboxy-Terminal Domain.

RATIONALE: Macrophage elastase (matrix metalloproteinase [MMP]-12) is a potent protease that contributes to the lung destruction that accompanies cigarette smoking; it simultaneously inhibits lung tumor angiogenesis and metastasis by catalyzing the formation of antiangiogenic peptides. Recent studies have revealed novel nonproteolytic functions of MMP12, including antimicrobial activity through a peptide within its C-terminal domain (CTD). OBJECTIVES: To determine whether the MMP12 CTD contributes to its antitumor activity in lung cancer. METHODS: We used recombinant MMP12 peptide fragments, including its catalytic domain, CTD, and a 20 amino acid peptide within the CTD (SR20), in an in vitro system to delineate their effects on non-small cell lung cancer cell proliferation and apoptosis. We translated our findings to two murine models of lung cancer, including orthotopic human xenograft and KrasLSL/G12D mouse models of lung cancer. MEASUREMENTS AND MAIN RESULTS: We show that SR20 triggers tumor apoptosis by up-regulation of gene expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor, death receptor 4, sensitizing cells to an autocrine loop of TRAIL-mediated cell death. We then demonstrate the therapeutic efficacy of SR20 against two murine models of lung cancer. CONCLUSIONS: The MMP12 CTD initiates TRAIL-mediated tumor cell death through its conserved SR20 peptide.

Mouse Genome-Wide Association Study of Preclinical Group II Pulmonary Hypertension Identifies Epidermal Growth Factor Receptor.

Pulmonary hypertension (PH) is associated with features of obesity and metabolic syndrome that translate to the induction of PH by chronic high-fat diet (HFD) in some inbred mouse strains. We conducted a genome-wide association study (GWAS) to identify candidate genes associated with susceptibility to HFD-induced PH. Mice from 36 inbred and wild-derived strains were fed with regular diet or HFD for 20 weeks beginning at 6-12 weeks of age, after which right ventricular (RV) and left ventricular (LV) end-systolic pressure (ESP) and maximum pressure (MaxP) were measured by cardiac catheterization. We tested for association of RV MaxP and RV ESP and identified genomic regions enriched with nominal associations to both of these phenotypes. We excluded genomic regions if they were also associated with LV MaxP, LV ESP, or body weight. Genes within significant regions were scored based on the shortest-path betweenness centrality, a measure of network connectivity, of their human orthologs in a gene interaction network of human PH-related genes. WSB/EiJ, NON/ShiLtJ, and AKR/J mice had the largest increases in RV MaxP after high-fat feeding. Network-based scoring of GWAS candidates identified epidermal growth factor receptor (Egfr) as having the highest shortest-path betweenness centrality of GWAS candidates. Expression studies of lung homogenate showed that EGFR expression is increased in the AKR/J strain, which developed a significant increase in RV MaxP after high-fat feeding as compared with C57BL/6J, which did not. Our combined GWAS and network-based approach adds evidence for a role for Egfr in murine PH.

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.

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.

Establishing human lacrimal gland cultures from biopsy-sized tissue specimens.

OBJECTIVES: To establish cultures of human lacrimal gland from patient-derived, biopsy-sized, tissue specimens. METHODS: Tissue was obtained after surgical removal from patients without dry eye disease undergoing routine procedures. Samples were subjected to mechanical and enzymatic digestion and resulting cell suspensions were plated onto collagen-coated glass coverslips and grown for up to 21 days. Cultures were analysed by immunocytochemistry and light microscopy, and resultant cellular distributions were compared to those in sections of fixed human lacrimal gland tissue. RESULTS: Dissociation of biopsy-sized pieces of human lacrimal gland and seeding onto coated surfaces allowed development of a mixed population of cells in vitro. Within 7-14 days, cellular aggregation was observed and by 21 days many cells had organised themselves into distinct three-dimensional complexes. Immunohistochemistry revealed a heterogeneous population of cells, including epithelial, myoepithelial, mesenchymal and progenitor cells. Some of the epithelia labelled positively for lysozyme and lactoferrin. CONCLUSIONS: Collection and dissociation of biopsy-sized pieces of human lacrimal gland leads to a cellular preparation that can proliferate in vitro and organise into three-dimensional structures. This is the first report detailing that biopsy-collected specimens of human lacrimal gland can be used to establish cell cultures.

Interleukin-6 is an efficacious marker of axonal transport disruption during experimental glaucoma and stimulates neuritogenesis in cultured retinal ganglion cells.

It is increasingly recognised that chronically activated glia contribute to the pathology of various neurodegenerative diseases, including glaucoma. One means by which this can occur is through the release of neurotoxic, proinflammatory factors. In the current study, we therefore investigated the spatio-temporal patterns of expression of three such cytokines, IL-1ß, TNFα and IL-6, in a validated rat model of experimental glaucoma. First, only weak evidence was found for increased expression of IL-1ß and TNFα following induction of ocular hypertension. Second, and much more striking, was that robust evidence was uncovered showing IL-6 to be synthesised by injured retinal ganglion cells following elevation of intraocular pressure and transported in an orthograde fashion along the nerve, accumulating at sites of axonal disruption in the optic nerve head. Verification that IL-6 represents a novel marker of disrupted axonal transport in this model was obtained by performing double labelling immunofluorescence with recognised markers of fast axonal transport. The stimulus for IL-6 synthesis and axonal transport during experimental glaucoma arose from axonal injury rather than ocular hypertension, as the response was identical after optic nerve crush and bilateral occlusion of the carotid arteries, each of which is independent of elevated intraocular pressure. Moreover, the response of IL-6 was not a generalised feature of the gp130 family of cytokines, as it was not mimicked by another family member, ciliary neurotrophic factor. Finally, further study suggested that IL-6 may be an early part of the endogenous regenerative response as the cytokine colocalised with growth-associated membrane phosphoprotein-43 in some putative regenerating axons, and potently stimulated neuritogenesis in retinal ganglion cells in culture, an effect that was additive to that of ciliary neurotrophic factor. These data comprise clear evidence that IL-6 is actively involved in the attempt of injured retinal ganglion cells to regenerate their axons.

Definition of glaucoma: clinical and experimental concepts.

Glaucoma is a term describing a group of ocular disorders with multi-factorial etiology united by a clinically characteristic intraocular pressure-associated optic neuropathy. It is not a single entity and is sometimes referred to in the plural as the glaucomas. All forms are potentially progressive and can lead to blindness. The diverse conditions that comprise glaucoma are united by a clinically characteristic optic neuropathy: glaucomatous optic neuropathy (GON). Evidence suggests that the primary site of neurological injury is at the optic nerve head. This fact enables the conditions to be grouped, irrespective of the causal mechanism(s). The term experimental glaucoma implies model resemblance to the human condition. We propose that 'experimental glaucoma' be restricted to animal models with demonstrable features of GON and/or evidence of a primary axonopathy at the optic nerve head. A fundamental inadequacy in this framework is any reference to the pathogenesis of GON, which remains unclear.