Optometry and Vision Sciences - Research Publications
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ItemNeuroimmune crosstalk in the cornea: The role of immune cells in corneal nerve maintenance during homeostasis and inflammation(PERGAMON-ELSEVIER SCIENCE LTD, 2022-11)In the cornea, resident immune cells are in close proximity to sensory nerves, consistent with their important roles in the maintenance of nerves in both homeostasis and inflammation. Using in vivo confocal microscopy in humans, and ex vivo immunostaining and fluorescent reporter mice to visualize corneal sensory nerves and immune cells, remarkable progress has been made to advance our understanding of the physical and functional interactions between corneal nerves and immune cells. In this review, we summarize and discuss recent studies relating to corneal immune cells and sensory nerves, and their interactions in health and disease. In particular, we consider how disrupted corneal nerve axons can induce immune cell activity, including in dendritic cells, macrophages and other infiltrating cells, directly and/or indirectly by releasing neuropeptides such as substance P and calcitonin gene-related peptide. We summarize growing evidence that the role of corneal intraepithelial immune cells is likely different in corneal wound healing versus other inflammatory-dominated conditions. The role of different types of macrophages is also discussed, including how stromal macrophages with anti-inflammatory phenotypes communicate with corneal nerves to provide neuroprotection, while macrophages with pro-inflammatory phenotypes, along with other infiltrating cells including neutrophils and CD4+ T cells, can be inhibitory to corneal re-innervation. Finally, this review considers the bidirectional interactions between corneal immune cells and corneal nerves, and how leveraging this interaction could represent a potential therapeutic approach for corneal neuropathy.
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ItemTopographical Distribution and Phenotype of Resident Meibomian Gland Orifice Immune Cells (MOICs) in Mice and the Effects of Topical Benzalkonium Chloride (BAK)(MDPI, 2022-09)Meibomian gland orifices (MGOs) are located along the eyelid margin and secrete meibum into the tear film. The profile of resident innate immune cells (ICs) at this site is not well understood. The distribution and phenotype of resident ICs around MGOs in mice was investigated and herein defined as MGO-associated immune cells (MOICs). The effect of topical 0.1% benzalkonium chloride (BAK) on MOICs was also assessed. Eyelids from healthy CD11ceYFP and Cx3cr1gfp/gfp mice aged three or seven months were compared. ICs were identified as CD11c+, Cx3cr1+, and MHC-II+ using four-colour immunostaining and confocal microscopy. MOIC density was variable but clustered around MGOs. There were more CD11c+ MOICs in three-month-old compared with seven-month-old mice (three-month-old: 893 ± 449 cells/mm2 vs. seven-month-old: 593 ± 493 cells/mm2, p = 0.004). Along the eyelid margin, there was a decreasing gradient of CD11c+ MOIC density in three-month-old mice (nasal: 1003 ± 369 cells/mm2, vs. central: 946 ± 574 cells/mm2, vs. temporal: 731 ± 353 cells/mm2, p = 0.044). Cx3cr1-deficient mice had two-fold fewer MHC-II+ MOICs, suggesting a role for Cx3cr1 receptor signaling in meibomian gland surveillance. CD11c+ MOIC density was lower in BAK-exposed eyes compared to saline-treated controls, suggesting a change in homeostasis. This study provides novel insight into resident ICs located at MGOs, and their contribution to MG homeostasis.
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ItemDefining an Optimal Sample Size for Corneal Epithelial Immune Cell Analysis Using in vivo Confocal Microscopy Images(FRONTIERS MEDIA SA, 2022-06-01)PURPOSE: In vivo confocal microscopy (IVCM) images are frequently used to quantify corneal epithelial immune cell (IC) density in clinical studies. There is currently limited evidence to inform the selection of a representative image sample size to yield a reliable IC density estimate, and arbitrary numbers of images are often used. The primary aim of this study was to determine the number of randomly selected, unique IVCM images required to achieve an acceptable level of accuracy when quantifying epithelial IC density, in both the central and peripheral cornea. The secondary aim was to evaluate the consistency and precision of an image selection approach where corneal epithelial IC density was quantified from "three representative images" selected independently by three experienced observers. METHODS: All combinations of two to 15 non-overlapping IVCM images were used for deriving IC density estimates, for both the central and peripheral cornea, in 20 healthy participants; the density value from averaging quantifications in the 16 images was defined as the "true mean". IC density estimates were compared with the true mean in each corneal region using a mean ratio. Intraclass correlation coefficients (ICCs) were used to evaluate the consistency of the mean ratios of IC density estimates derived from the method involving the manual selection of "three representative images" by the observers. The precision of the IC density estimates was compared to a scenario involving three randomly selected images. RESULTS: A total of 12 randomly selected, non-overlapping IVCM images were found to be required to produce a corneal epithelial IC density estimate that was within 30% of the true mean, 95% of the time, for the central cornea; seven such images produced an equivalent level of precision in the peripheral cornea. Mean ratios of corneal IC density estimates derived from "three representative images" methods had poor consistency between observers (ICC estimates <0.5) and similar levels of precision when compared with using three randomly selected images (p > 0.05 for all comparisons), in both the central and peripheral cornea. CONCLUSIONS: Data presented in this study can inform image selection methods, and the sample size required for a preferred level of accuracy, when quantifying IC densities in the central and peripheral corneal epithelium using IVCM images.
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ItemThe effect of topical decorin on temporal changes to corneal immune cells after epithelial abrasion(BMC, 2022-04-12)BACKGROUND: Corneal immune cells interact with corneal sensory nerves during both homeostasis and inflammation. This study sought to evaluate temporal changes to corneal immune cell density in a mouse model of epithelial abrasion and nerve injury, and to investigate the immunomodulatory effects of topical decorin, which we have shown previously to promote corneal nerve regeneration. METHODS: Bilateral corneal epithelial abrasions (2 mm) were performed on C57BL/6J mice. Topical decorin or saline eye drops were applied three times daily for 12 h, 24 h, 3 days or 5 days. Optical coherence tomography imaging was performed to measure the abrasion area. The densities of corneal sensory nerves (β-tubulin III) and immune cells, including dendritic cells (DCs; CD11c+), macrophages (Iba-1+) and neutrophils (NIMP-R14+) were measured. Cx3cr1gfp/gfp mice that spontaneously lack resident corneal intraepithelial DCs were used to investigate the specific contribution of epithelial DCs. Neuropeptide and cytokine gene expression was evaluated using qRT-PCR at 12 h post-injury. RESULTS: In decorin-treated corneas, higher intraepithelial DC densities and lower neutrophil densities were observed at 24 h after injury, compared to saline controls. At 12 h post-injury, topical decorin application was associated with greater re-epithelialisation. At 5 days post-injury, corneal stromal macrophage density in the decorin-treated and contralateral eyes was lower, and nerve density was higher, compared to eyes treated with saline only. Lower expression of transforming growth factor beta (TGF-β) and higher expression of CSPG4 mRNA was detected in corneas treated with topical decorin. There was no difference in corneal neutrophil density in Cx3cr1gfp/gfp mice treated with or without decorin at 12 h. CONCLUSIONS: Topical decorin regulates immune cell dynamics after corneal injury, by inhibiting neutrophils and recruiting intraepithelial DCs during the acute phase (< 24 h), and inhibiting macrophage density at the study endpoint (5 days). These immunomodulatory effects were associated with faster re-epithelialisation and likely contribute to promoting sensory nerve regeneration. The findings suggest a potential interaction between DCs and neutrophils with topical decorin treatment, as the decorin-induced neutrophil inhibition was absent in Cx3cr1gfp/gfp mice that lack corneal epithelial DCs. TGF-β and CSPG4 proteoglycan likely regulate decorin-mediated innate immune cell responses and nerve regeneration after injury.
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ItemThe neuroregenerative effects of topical decorin on the injured mouse cornea(BMC, 2020-05-04)BACKGROUND: The cornea is innervated with a rich supply of sensory nerves that play important roles in ocular surface health. Any injury or pathology of the corneal nerves increases the risk of dry eye disease and infection. This study aims to evaluate the therapeutic potential of topical decorin to improve corneal nerve regeneration in a mouse model of sterile epithelial abrasion injury. METHODS: Bilateral central corneal epithelial abrasions (2-mm, Alger Brush) were performed on young C57BL/6 J mice to remove the corneal sensory nerves. Decorin, or vehicle, was applied topically, three times per day for 1 week or every 2 h for 6 h. Spectral-domain optical coherence tomography was performed to measure the abrasion area and corneal thickness. Wholemount immunofluorescence staining was used to assess sensory nerve regeneration (β-tubulin III) and immune cell density (CD45, Iba1, CD11c). To investigate the specific role of dendritic cells (DCs), Cx3cr1gfp/gfp mice, which spontaneously lack resident corneal epithelial DCs, were also investigated. The effect of prophylactic topical administration of recombinant human decorin (applied prior to the abrasion) was also investigated. Nerve tracing (NeuronJ software) was performed to compare recovery of basal nerve axons and superficial nerve terminals in the central and peripheral cornea. RESULTS: At 6 h after injury, topical decorin application was associated with greater intraepithelial DC recruitment but no change in re-epithelialisation or corneal thickness, compared to the vehicle control. One week after injury, sub-basal nerve plexus and superficial nerve terminal density were significantly higher in the central cornea in the decorin-treated eyes. The density of corneal stromal macrophages in the decorin-treated eyes and their contralateral eyes was significantly lower compared to saline-treated corneas. No significant improvement in corneal nerve regeneration was observed in Cx3cr1gfp/gfp mice treated with decorin. CONCLUSIONS: Decorin promotes corneal epithelial nerve regeneration after injury. The neuroregenerative effect of topical decorin was associated with a higher corneal DC density during the acute phase, and fewer macrophages at the study endpoint. The corneal neuroregenerative effects of decorin were absent in mice lacking intraepithelial DCs. Together, these findings support a role for decorin in DC-mediated neuroregeneration following corneal abrasion injury.
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ItemNovel alterations in corneal neuroimmune phenotypes in mice with central nervous system tauopathy(BMC, 2020-04-28)BACKGROUND: Tauopathy in the central nervous system (CNS) is a histopathological hallmark of frontotemporal dementia (FTD) and Alzheimer's disease (AD). Although AD is accompanied by various ocular changes, the effects of tauopathy on the integrity of the cornea, which is densely innervated by the peripheral nervous system and is populated by resident dendritic cells, is still unknown. The aim of this study was to investigate if neuroimmune interactions in the cornea are affected by CNS tauopathy. METHODS: Corneas from wild type (WT) and transgenic rTg4510 mice that express the P301L tau mutation were examined at 2, 6, 8, and 11 months. Clinical assessment of the anterior segment of the eye was performed using spectral domain optical coherence tomography. The density of the corneal epithelial sensory nerves and the number and field area of resident epithelial dendritic cells were assessed using immunofluorescence. The immunological activation state of corneal and splenic dendritic cells was examined using flow cytometry and compared between the two genotypes at 9 months of age. RESULTS: Compared to age-matched WT mice, rTg4510 mice had a significantly lower density of corneal nerve axons at both 8 and 11 months of age. Corneal nerves in rTg4510 mice also displayed a higher percentage of beaded nerve axons and a lower density of epithelial dendritic cells compared to WT mice. From 6 months of age, the size of the corneal dendritic cells was significantly smaller in rTg4510 compared to WT mice. Phenotypic characterization by flow cytometry demonstrated an activated state of dendritic cells (CD86+ and CD45+ CD11b+CD11c+) in the corneas of rTg4510 compared to WT mice, with no distinct changes in the spleen monocytes/dendritic cells. At 2 months of age, there were no significant differences in the neural or immune structures between the two genotypes. CONCLUSIONS: Corneal sensory nerves and epithelial dendritic cells were altered in the rTg4510 mouse model of tauopathy, with temporal changes observed with aging. The activation of corneal dendritic cells prior to the gradual loss of neighboring sensory nerves suggests an early involvement of corneal immune cells in tau-associated pathology originating in the CNS.