Optometry and Vision Sciences - Research Publications
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ItemNo Preview AvailableReversibility of retinal ganglion cell dysfunction due to chronic IOP elevation.(Association for Research in Vision and Ophthalmology, 2018-07-01)Purpose : To determine the duration of chronic IOP elevation beyond which ganglion cell function can no longer recover using the mouse circumlimbal suture model. Methods : IOP elevation was induced in anaesthetized (isoflurane) adult male C57BL6/J mice by attaching a circumlimbal suture (nylon, 10/0) around the equator of one eye, with the contralateral eye serving as a control. The suture was left in place for 8, 12 and 16 weeks (n=27, 23 and 27), respectively, and animals underwent electroretinography and optical coherence tomography at these time points. In two other groups, the suture was removed after 8 and 12 weeks (n=26 and 28), and the capacity for recovery assessed 4 weeks later. IOP was measured weekly (Tonolab). Retinal ganglion cell (RGC) function (or integrity) was assessed with the positive scotopic threshold response (pSTR) and retinal nerve fibre layer (RNFL) thickness. Data (mean ± SEM) were compared using t-test (control vs. treatment) and one-way ANOVA (within groups). Results : IOP in sutured eyes was higher than control eyes (8wk: 17.1 ± 0.3 vs. 26.8 ± 0.6 mmHg, 12wk: 13.8 ± 0.3 vs. 19.5 ± 0.5 mmHg, 16wk: 17.1 ± 0.2 vs. 27.4 ± 0.6 mmHg; all P<0.001). After suture removal, IOP returned to levels comparable to control eyes (8+4wk: 16.9 ± 0.3 vs. 16.1 ± 0.3 mmHg; P=0.08, 12+4wk: 17.3 ± 0.2 vs. 17.1 ± 0.3 mmHg; P=0.5). With IOP elevation, RGC function declined to 75% ± 8% (8wk), 78% ± 7% (12wk) and 59% ± 4% (16wk, all P<0.001) of control eyes. RNFL thinning was also evident (8wk: 84% ± 4%, 12wk: 83% ± 5%; 16wk: 83% ± 3%; P<0.001) but no change in total retinal thickness was noted (P=0.33). Suture removal at week 8 facilitated full recovery of RGC function (97% ± 7%, P=0.9 vs. baseline) 4 weeks later. However, there was no recovery in RNFL thickness (87% ± 3%, P<0.001 vs. baseline). When the suture was removed at week 12, neither function (79% ± 9%, P<0.05) nor RNFL thickness recovered (89% ± 3%, P<0.01) 4 weeks later. Conclusions : RGC dysfunction can be recovered 4 weeks after an 8-week period of mild IOP elevation, but not after a 12-week period. Beyond 12 weeks, IOP reversal only served to prevent further functional decline. This identifies a critical chronic IOP duration that results in irreversible ganglion cell dysfunction. This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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ItemOmega-3 supplementation is neuroprotective to corneal nerves in dry eye disease: a pilot study(WILEY, 2017-07)PURPOSE: To investigate whether oral, long-chain omega-3 (ω-3) essential fatty acid (EFA) supplementation, for 3 months, induces changes to the central corneal sub-basal nerve plexus in dry eye disease and whether nerve alterations correlate with clinical findings. METHODS: This prospective, comparative study involved the final 12 participants enrolled in a randomised, double-masked, placebo-controlled clinical trial of 60 participants with moderate dry eye disease. Participants received either placebo (olive oil 1500 mg/day; n = 4) or ω-3 EFA supplements (~1000 mg/day eicosapentaenoic acid + ~500 mg/day docosahexaenoic acid; n = 8) for 90 days. The main outcome measure was the mean change in central corneal sub-basal plexus nerve parameters between days one and 90, quantified using in vivo confocal microscopy. Secondary outcomes included mean change in tear osmolarity, corneal dendritic cell density and basal epithelial cell density. RESULTS: Compared with baseline, the reduction in OSDI score and tear osmolarity at day 90 were greater in the ω-3 EFA group than the placebo group (OSDI: ω-3 EFA, mean ± SEM: -15.6 ± 2.8 vs placebo: -2.8 ± 4.1 units, t5 = 2.6, p = 0.04; tearosmolarity: ω-3 EFA: -22.63 ± 5.7 vs placebo: -8 ± 2.7 mOsmol/L, t9 = 2.3, p = 0.04). At day 90, corneal total nerve branch density (CTBD: 91.1 ± 8.6 vs 45.1 ± 13.4 branches/mm2 , F1,10 = 14, p = 0.004) and corneal nerve branch density on the main fibre (CNBD: 63.4 ± 6.5 vs 27.9 ± 11.5 branches/mm2 , F1,10 = 6, p = 0.03) were higher in the ω-3 EFA group compared with placebo. Relative to day 1, CNBD (branches/mm2 ) increased at day 90 in the ω-3 EFA group (+20.0 ± 9.2, t8 = 3.2 p = 0.01) compared with placebo (-10.8 ± 3.2). Similar changes were evident for corneal nerve fibre length (CNFL, mm/mm2 ), which increased from baseline at day 90 in the omega-3 EFA group (+2.9 ± 1.6, t8 = 3.4 p = 0.01) compared with placebo (-2.7 ± 0.5). There was a negative correlation between CTBD and tear osmolarity (r10 = -0.70, p = 0.01). No significant changes were observed for basal epithelial cell or corneal dendritic cell density. CONCLUSION: These pilot study findings suggest that ω-3 EFA supplementation imparts neuroprotective effects in the corneal sub-basal plexus that correlate with the extent of tear osmolarity normalisation.
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ItemA Case of Mistaken Identity: CD11c-eYFP+ Cells in the Normal Mouse Brain Parenchyma and Neural Retina Display the Phenotype of Microglia, Not Dendritic Cells(WILEY-BLACKWELL, 2016-08)Under steady-state conditions the central nervous system (CNS) is traditionally thought to be devoid of antigen presenting cells; however, putative dendritic cells (DCs) expressing enhanced yellow fluorescent protein (eYFP) are present in the retina and brain parenchyma of CD11c-eYFP mice. We previously showed that these mice carry the Crb1(rd8) mutation, which causes retinal dystrophic lesions; therefore we hypothesized that the presence of CD11c-eYFP(+) cells within the CNS may be due to pathology associated with the Crb1(rd8) mutation. We generated CD11c-eYFP Crb1(wt/wt) mice and compared the distribution and immunophenotype of CD11c-eYFP(+) cells in CD11c-eYFP mice with and without the Crb1(rd8) mutation. The number and distribution of CD11c-eYFP(+) cells in the CNS was similar between CD11c-eYFP Crb1(wt/wt) and CD11c-eYFP Crb1(rd8/rd8) mice. CD11c-eYFP(+) cells were distributed throughout the inner retina, and clustered in brain regions that receive input from the external environment or lack a blood-brain barrier. CD11c-eYFP(+) cells within the retina and cerebral cortex of CD11c-eYFP Crb1(wt/wt) mice expressed CD11b, F4/80, CD115 and Iba-1, but not DC or antigen presentation markers, whereas CD11c-eYFP(+) cells within the choroid plexus and pia mater expressed CD11c, I-A/I-E, CD80, CD86, CD103, DEC205, CD8α and CD135. The immunophenotype of CD11c-eYFP(+) cells and microglia within the CNS was similar between CD11c-eYFP Crb1(wt/wt) and CD11c-eYFP Crb1(rd8/rd8) mice; however, CD11c and I-A/I-E expression was significantly increased in CD11c-eYFP Crb1(rd8/rd8) mice. This study demonstrates that the overwhelming majority of CNS CD11c-eYFP(+) cells do not display the phenotype of DCs or their precursors and are most likely a subpopulation of microglia. GLIA 2016. GLIA 2016;64:1331-1349.
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ItemRegional and functional heterogeneity of antigen presenting cells in the mouse brain and meninges(WILEY, 2019-05)The central nervous system (CNS) is considered to be immune privileged, owing in part to the absence of major histocompatibility (MHC) class II+ cells in the healthy brain parenchyma. However, systemic inflammation can activate microglia to express MHC class II, suggesting that systemic inflammation may be sufficient to mature microglia into functional antigen presenting cells (APCs). We examined the effects of systemic lipopolysaccharide (LPS)-induced inflammation on the phenotype and function of putative APCs within the mouse brain parenchyma, as well as its supporting tissues-the choroid plexus and meninges. Microglia isolated from different regions of the brain demonstrated significant heterogeneity in their ability to present antigen to naïve OT-II CD4+ T cells following exposure to systemic LPS. Olfactory bulb microglia (but not cortical microglia) intimately interacted with T cells in vivo and stimulated T cell proliferation in vitro, albeit in the absence of co-stimulation. In contrast, myeloid cells within the choroid plexus and meninges were immunogenic and upregulated the co-stimulatory molecule CD80 following systemic inflammation. Dural APCs, which clustered around LYVE-1+ lymphatics, were more efficient at stimulating naïve T cell proliferation than choroid plexus APCs, suggesting that the dura may be an under-appreciated site for immune interactions. This study has highlighted the functional diversity of myeloid cells within the sub-compartments of the CNS and its supporting tissues. Furthermore, these findings demonstrate that systemic inflammation can mature selected microglia populations and choroid plexus/meningeal myeloid cells into functional APCs, which may contribute to the pathogenesis of neuroinflammation and neurodegenerative diseases.
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ItemMacrophage physiology in the eye(SPRINGER, 2017-04)The eye is a complex sensory organ composed of a range of tissue types including epithelia, connective tissue, smooth muscle, vascular and neural tissue. While some components of the eye require a high level of transparency to allow light to pass through unobstructed, other tissues are characterized by their dense pigmentation, which functions to absorb light and thus control its passage through the ocular structures. Macrophages are present in all ocular tissues, from the cornea at the anterior surface through to the choroid/sclera at the posterior pole. This review will describe the current understanding of the distribution, phenotype, and physiological role of ocular macrophages, and provide a summary of evidence pertaining to their proposed role during pathological conditions.
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ItemOptical Coherence Tomography Reveals Changes to Corneal Reflectivity and Thickness in Individuals with Tear Hyperosmolarity(ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2017-05)PURPOSE: To investigate whether tear hyperosmolarity, a feature of dry eye disease (DED), affects central corneal thickness (CCT), corneal light reflectivity, and/or tear film reflectivity. METHODS: This prospective, cross-sectional study involved 48 participants (38 with hyperosmolar tears and 10 controls with normo-osmolar tears). Symptoms and signs of DED (tear osmolarity, sodium fluorescein tear break-up time, ocular surface staining, Schirmer test) were assessed. CCT, and the reflectivity of the cornea and the tear-epithelial interface were quantified relative to background noise using Fourier-domain optical coherence tomography (FD-OCT). RESULTS: CCT of eyes with severe tear hyperosmolarity, defined as eyes in the upper quartile of the hyperosmolar group, was less than control eyes (539.1 ± 7.4 vs. 583.1 ± 15.0 μm, P = 0.02) and eyes with less severe tear hyperosmolarity, defined as hyperosmolar eyes in the lower quartile (622.7 ± 5.8 μm, P < 0.0001). CCT showed a negative linear relationship with tear osmolarity for values above 316 mOsmol/L (R2 = 0.17, P = 0.01). Central corneal reflectivity was lower in hyperosmolar eyes than normo-osmolar eyes (45.1 ± 0.3 vs. 48.1 ± 0.6 pixels, P = 0.02); the greatest relative difference was in the anterior stroma, where corneal reflectivity was 4.7 ± 1.9% less in hyperosmolar eyes (P < 0.01). Peak reflectivity of the tear-epithelial interface was 4.8% ± 3.5% higher in the hyperosmolar group than the normo-osmolar tear group (P = 0.04). CONCLUSION: Individuals with significant tear hyperosmolarity and clinical signs of symptoms of DED show reduced CCT and altered corneal reflectivity. TRANSLATIONAL RELEVANCE: Anterior segment FD-OCT provides novel insight into corneal microstructural differences in individuals with DED.
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ItemCharacterization of the Circumlimbal Suture Model of Chronic IOP Elevation in Mice and Assessment of Changes in Gene Expression of Stretch Sensitive Channels.(Frontiers Media SA, 2017)To consider whether a circumlimbal suture can be used to chronically elevate intraocular pressure (IOP) in mice and to assess its effect on retinal structure, function and gene expression of stretch sensitive channels. Anesthetized adult C57BL6/J mice had a circumlimbal suture (10/0) applied around the equator of one eye. In treated eyes (n = 23) the suture was left in place for 12 weeks whilst in sham control eyes the suture was removed at day two (n = 17). Contralateral eyes served as untreated controls. IOP was measured after surgery and once a week thereafter. After 12 weeks, electroretinography (ERG) was performed to assess photoreceptor, bipolar cell and retinal ganglion cell (RGC) function. Retinal structure was evaluated using optical coherence tomography. Retinae were processed for counts of ganglion cell density or for quantitative RT-PCR to quantify purinergic (P2x7, Adora3, Entpd1) or stretch sensitive channel (Panx1, Trpv4) gene expression. Immediately after suture application, IOP spiked to 33 ± 3 mmHg. After 1 day, IOP had recovered to 27 ± 3 mmHg. Between weeks 2 and 12, IOP remained elevated above baseline (control 14 ± 1 mmHg, ocular hypertensive 19 ± 1 mmHg). Suture removal at day 2 (Sham) restored IOP to baseline levels, where it remained through to week 12. ERG analysis showed that 12 weeks of IOP elevation reduced photoreceptor (-15 ± 4%), bipolar cell (-15 ± 4%) and ganglion cell responses (-19 ± 6%) compared to sham controls and respective contralateral eyes (untreated). The retinal nerve fiber layer was thinned in the presence of normal total retinal thickness. Ganglion cell density was reduced across all quadrants (superior -12 ± 5%; temporal, -7% ± 2%; inferior -9 ± 4%; nasal -8 ± 5%). Quantitative RT-PCR revealed a significant increase in Entpd1 gene expression (+11 ± 4%), whilst other genes were not significantly altered (P2x7, Adora3, Trpv4, Panx1). Our results show that circumlimbal ligation produces mild chronic ocular hypertension and retinal dysfunction in mice. Consistent with a sustained change to purinergic signaling we found an up-regulation of Entpd1.
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ItemLaser scanning in vivo confocal microscopy (IVCM) for evaluating human corneal sub-basal nerve plexus parameters: protocol for a systematic review(BMJ PUBLISHING GROUP, 2017-11)INTRODUCTION: Laser scanning in vivo confocal microscopy (IVCM) enables non-invasive, high-resolution imaging of the cornea. In recent years, there has been a vast increase in researchers using laser scanning IVCM to image and quantify corneal nerve parameters. However, a range of methodological approaches have been adopted. The primary aim of this systematic review is to critically appraise the reported method(s) of primary research studies that have used laser scanning IVCM to quantify corneal sub-basal nerve plexus (SBNP) parameters in humans, and to examine corneal nerve parameters in healthy individuals. METHODS AND ANALYSIS: A systematic review of primary studies that have used laser scanning IVCM to quantify SBNP parameters in humans will be conducted. Comprehensive electronic searches will be performed in Ovid MedLine, Embase and the Cochrane Library. Two reviewers will independently assess titles and abstracts, and exclude studies not meeting the inclusion criteria. For studies judged eligible or potentially eligible, full texts will be independently assessed by two reviewers to determine eligibility. A third reviewer will resolve any discrepancies in judgement. Risk of bias will be assessed using a custom tool, covering five methodological domains: participant selection, method of image capture, method of image analysis, data reporting and other sources of bias. A systematic narrative synthesis of findings will be provided. A multilevel random-effects meta-analysis will be performed for corneal nerve parameters derived from healthy participants. This review will be reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses statement. ETHICS AND DISSEMINATION: As this review considers published data, ethical approval is not required. We foresee that this synthesis will serve as a reference for future studies, and can be used to inform best practice standards for using IVCM in clinical research. A manuscript reporting the results of the review will be published and may also be presented at scientific conferences.
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ItemNo Preview AvailableTLR9 and TLR7/8 activation induces formation of keratic precipitates and giant macrophages in the mouse cornea(FEDERATION AMER SOC EXP BIOL, 2015-01)Macrophage adherence to the inner corneal surface and formation of MGCs in the stroma are common signs of chronic inflammation following corneal infection. To determine whether macrophage adherence (known clinically as KPs) and giant cell formation were specific to innate immune activation via particular TLR ligands, macrophage activation was examined in a murine model of TLR-mediated corneal inflammation. The corneal epithelium was debrided and highly purified TLR ligands were topically applied once to the cornea of TLR7(-/-), TLR9(-/-), Cx3cr1(gfp/+), CD11c(eYFP), and IL-4(-/-) mice. At 1 week post-treatment macrophage activation and phenotype was evaluated in the cornea. Treatment with TLR2, TLR3, TLR4, and TLR5 ligands caused an increase in the number of activated stromal macrophages in the central cornea at 1 week post-treatment. However, treatment with TLR9 ligand CpG-ODN and the TLR7/8 ligand R848/Resiquimod led to an accumulation of macrophages on the corneal endothelium and formation of multinucleated giant macrophages in the corneal stroma. We suggest that giant cell formation, which is a characteristic feature of granuloma formation in many tissues, may be a unique feature of TLR9- and TLR7/8-mediated macrophage activation.
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ItemNo Preview AvailableRetinal Microglial Activation Following Topical Application of Intracellular Toll-Like Receptor Ligands(ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2015-11)PURPOSE: We previously have reported that application of the intracellular toll-like receptor (TLR)-9 ligand CpG-ODN onto the injured corneal surface induces widespread inflammation within the eye, including the retina. We tested the hypothesis that topical application of two other intracellular TLR agonists, Poly I:C and R848, would cause retinal microglial activation and migration into the subretinal space. METHODS: C57BL/6J wild-type and Cx3cr1gfp/+ mice were anesthetized and received central corneal abrasions followed by topical application of Poly I:C (TLR3 agonist), R848 (TLR7/8 agonist), or CpG-ODN (TLR9 agonist). Eyes were imaged in vivo by using spectral-domain optical coherence tomography to assess and quantify vitreous cells and retinal edema. Tissues were processed for whole-mount immunofluorescence staining or gene expression studies. Microglial activation was determined by morphologic changes, major histocompatibility complex (MHC) class II reactivity, and migration to the subretinal space. Expression of proinflammatory cytokine gene IL-6, IL-1β, IFN-γ, and MCP-1 in retinal tissues were analyzed. RESULTS: At 24 hours, topical treatment with CpG-ODN and R848, but not Poly I:C, led to altered microglial morphology. One week after CpG-ODN and R848-treatment, eyes exhibited vitritis and mild inner retinal edema, increased number of subretinal Iba-1+ cells, and an increase in MHC II+ cells in the neural retina. Proinflammatory cytokine genes were upregulated after R848 treatment, whereas in the CpG-ODN group, only IL-1β and MCP-1 were significantly upregulated. Retinal microglial activation was not observed in the Poly I:C-treated group. CONCLUSIONS: Topical application of CpG-ODN and R848, but not Poly I:C, to the damaged corneal surface can cause activation and migration of retinal microglia.