Intravital imaging of the human cornea reveals the differential effects of season on innate and adaptive immune cell morphodynamics.

PURPOSE: While the external environment has been shown to shape the systemic human immune landscape, defining the in vivo immune status of peripheral tissues has remained a technical challenge. We recently developed functional in vivo confocal microscopy (Fun-IVCM) for dynamic, longitudinal imaging of corneal immune cells in living humans. This study investigated the effect of seasonal-driven environmental factors on the density, morphology and dynamic behavior of human corneal immune cell subsets. DESIGN: Longitudinal, observational clinical study. PARTICIPANTS: Sixteen healthy participants (18-40 years) attended two visits in distinct seasons in Melbourne, Australia (Visit 1: Spring/Summer: November-December 2021; Visit 2: Autumn/Winter: April-June 2022). METHODS: Environmental data were collected over each period. Participants underwent ocular surface examinations and corneal Fun-IVCM (Heidelberg HRT-3, Rostock Corneal Module). Volume scans (80µm) were acquired at 5.5±1.5 minute intervals, for up to five timepoints. Time-lapse videos were created to analyze corneal immune cells, comprising epithelial T cells and dendritic cells (DCs), and stromal macrophages. Tear cytokines were analyzed using multiplex bead-based immunoassay. MAIN OUTCOME MEASURES: Difference in the density, morphological and dynamic parameters of corneal immune cell subsets over the study periods. RESULTS: Visit 1 was characterized by higher temperature, lower humidity, and higher air particulate and pollen levels than Visit 2. Clinical ocular surface parameters, and the density of immune cell subsets were similar across visits. At Visit 1 (Spring/Summer), corneal epithelial DCs were larger and more elongated, with a lower dendrite probing speed (0.38±0.21 vs 0.68±0.33µm/min, p<0.001) relative to Visit 2; stromal macrophages were more circular and had less dynamic activity (Visit 1: 7.2±1.9 vs Visit 2: 10.3±3.7 'dancing index', p<0.001). T cell morphology and dynamics were unchanged across periods. Basal tear levels of IL-2 and CXCL10 were lower during Spring/Summer. CONCLUSION: This novel study shows that the in vivo morphodynamics of innate corneal immune cells (DCs, macrophages) are modified by environmental factors, but such effects are not evident for adaptive immune cells (T cells). The cornea is a potential non-invasive, in vivo 'window' to season-dependent changes to the human immune system, with capacity to yield new insight into environmental influences on immune regulation.

Evaluating the efficacy and safety of therapeutic interventions for corneal neuropathy: A systematic review.

Corneal neuropathy involves corneal nerve damage that disrupts ocular surface integrity, negatively impacting quality-of-life from pain and impaired vision. Any ocular or systemic condition that damages the trigeminal nerve can lead to corneal neuropathy. However, the condition currently does not have standardized diagnostic criteria or treatment protocols. The primary aim of this systematic review was to evaluate the efficacy and safety of interventions for treating corneal neuropathy. Randomized controlled trials (RCTs) that investigated corneal neuropathy treatments were eligible if the intervention(s) was compared to a placebo or active comparator. Comprehensive searches were conducted in Ovid MEDLINE, Ovid Embase and clinical trial registries from inception to July 2022. The Cochrane Risk-of-Bias 2 tool was used to assess study methodological quality. Certainty of the body of evidence was assessed using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach. Overall, 20 RCTs were included. Evaluated interventions comprised regenerative therapies (n=6 studies), dietary supplements (n=4), anti-glycemic agents (n=3), combination therapy (n=3), supportive therapies (n=2) and systemic pain pharmacotherapies (n=2). Nine RCTs were judged at high risk of bias for most outcomes. Definitions for corneal neuropathy in the populations varied substantially across studies, consistent with lack of consensus on diagnostic criteria. A diverse range of outcomes were quantified, likely reflecting absence of an agreed core outcome. There was insufficient evidence to draw definitive conclusions on the efficacy or safety of any intervention. There was low or very low certainty evidence for several neuroregenerative agents and dietary supplements for improving corneal nerve fiber length in corneal neuropathy due to dry eye disease and diabetes. Low or very low certainty evidence was found for neuroregenerative therapies and dietary supplements not altering corneal immune cell density. This review identifies a need to standardize the clinical definition of corneal neuropathy and define a minimum set of core outcome measures. Together, this will provide a foundation for improved phenotyping of clinical populations in studies, and improve capacity to synthesize data to inform evidence-based based care. Protocol registration: PROSPERO ID: CRD42022348475.

Shaking Up Our Approach: The Need for Characterization and Optimization of Pre-clinical Models of Infant Abusive Head Trauma.

Traumatic brain injuries (TBIs) are a large societal and individual burden. In the first year of life, the vast majority of these injuries are the result of inflicted abusive events by a trusted caregiver. Abusive head trauma (AHT) in infants, formerly known as shaken baby syndrome, is the leading cause of inflicted mortality and morbidity in this population. In this review we address clinical diagnosis, symptoms, prognosis, and neuropathology of AHT, emphasizing the burden of repetitive AHT. Next, we consider existing animal models of AHT, and we evaluate key features of an ideal model, highlighting important developmental milestones in children most vulnerable to AHT. We draw on insights from other injury models, such as repetitive, mild TBIs (RmTBIs), post-traumatic epilepsy (PTE), hypoxic-ischemic injuries, and maternal neglect, to speculate on key knowledge gaps and underline important new opportunities in pre-clinical AHT research. Finally, potential treatment options to facilitate healthy development in children following an AHT are considered. Together, this review aims to drive the field toward optimized, well-characterized animal models of AHT, which will allow for greater insight into the underlying neuropathological and neurobehavioral consequences of AHT.

Ocular development after highly effective modulator treatment early in life.

Highly effective cystic fibrosis (CF) transmembrane conductance regulator (CFTR) modulator therapies (HEMT), including elexacaftor-tezacaftor-ivacaftor, correct the underlying molecular defect causing CF. HEMT decreases general symptom burden by improving clinical metrics and quality of life for most people with CF (PwCF) with eligible CFTR variants. This has resulted in more pregnancies in women living with CF. All HEMT are known to be able pass through the placenta and into breast milk in mothers who continue on this therapy while pregnant and breast feeding. Toxicity studies of HEMT in young rats demonstrated infant cataracts, and case reports have reported the presence of congenital cataracts in early life exposure to HEMT. This article reviews the evidence for how HEMT influences the dynamic and interdependent processes of healthy and abnormal lens development in the context of HEMT exposure during pregnancy and breastfeeding, and raises questions that remain unanswered.

Redefining the human corneal immune compartment using dynamic intravital imaging.

The healthy human cornea is a uniquely transparent sensory tissue where immune responses are tightly controlled to preserve vision. The cornea contains immune cells that are widely presumed to be intraepithelial dendritic cells (DCs). Corneal immune cells have diverse cellular morphologies and morphological alterations are used as a marker of inflammation and injury. Based on our imaging of corneal T cells in mice, we hypothesized that many human corneal immune cells commonly defined as DCs are intraepithelial lymphocytes (IELs). To investigate this, we developed functional in vivo confocal microscopy (Fun-IVCM) to investigate cell dynamics in the human corneal epithelium and stroma. We show that many immune cells resident in the healthy human cornea are T cells. These corneal IELs are characterized by rapid, persistent motility and interact with corneal DCs and sensory nerves. Imaging deeper into the corneal stroma, we show that crawling macrophages and rare motile T cells patrol the tissue. Furthermore, we identify altered immune cell behaviors in response to short-term contact lens wear (acute inflammatory stimulus), as well as in individuals with allergy (chronic inflammatory stimulus) that was modulated by therapeutic intervention. These findings redefine current understanding of immune cell subsets in the human cornea and reveal how resident corneal immune cells respond and adapt to chronic and acute stimuli.

Topical Decorin Reduces Corneal Inflammation and Imparts Neuroprotection in a Mouse Model of Benzalkonium Chloride-induced Corneal Neuropathy.

Purpose: We evaluated the neuroprotective and immunomodulatory effects of topical decorin in a murine model of benzalkonium chloride (BAK)-induced corneal neuropathy. Methods: Topical BAK (0.1%) was administered daily to both eyes of female C57BL/6J mice (n = 14) for 7 days. One group of mice received topical decorin (1.07 mg/mL) eye drops to one eye and saline (0.9%) to the contralateral eye; the other group received saline eye drops to both eyes. All eye drops were given three times daily over the experimental period. A control group (n = 8) received daily topical saline only, instead of BAK. Optical coherence tomography imaging was performed before (at day 0) and after (day 7) treatment to evaluate the central corneal thickness. Whole-mount immunofluorescence staining was performed to evaluate the density of corneal intraepithelial nerves and immune cells. Results: BAK-exposed eyes showed corneal epithelial thinning, infiltration of inflammatory macrophages and neutrophils, and a lower density of intraepithelial nerves. No change to the corneal stromal thickness or dendritic cell density was observed. After BAK exposure, decorin-treated eyes had a lower density of macrophages and less neutrophil infiltration and a higher nerve density than the saline-treated group. Contralateral eyes from the decorin-treated animals showed fewer macrophages and neutrophils relative to saline-treated animals. A negative correlation was found between corneal nerve density and macrophage or neutrophil density. Conclusions: Topical decorin provides neuroprotective and anti-inflammatory effects in a chemical model of BAK-induced corneal neuropathy. The attenuation of corneal inflammation by decorin may contribute to decreasing corneal nerve degeneration induced by BAK.

Developing culturally safe education practices in optometry schools across Australia and Aotearoa New Zealand.

Access to culturally safe health services is a basic human right, however through the lasting effects of colonisation, oppression, and systemic racism, the individual and community health of Indigenous peoples in Australia and Aotearoa New Zealand have been severely impacted. The Aboriginal and Torres Strait Islander Health and Cultural Safety Strategy of the Australian Health Practitioners Regulation Agency, and the Standards of Cultural Competence and Cultural Safety of the Optometrists and Dispensing Opticians Board of New Zealand, recognise the importance of access to safe health care for Aboriginal, Torres Strait Islander and Maori patients, which encompasses both clinical competency and cultural safety. Universities have an ongoing responsibility to ensure their learning and teaching activities result in graduates being able to provide culturally safe practice. This article highlights the emergence of culturally safe practices in the Australian and Aotearoa New Zealand optometry curricula over the last five years incorporating Indigenous ways of knowing, being and doing into the curricula, understanding the local Indigenous histories and contexts, the adoption of online cultural education modules, and clinical placement partnerships with local Indigenous communities. Whilst there is still much work to do to achieve the goal of graduating culturally safe optometrists, this paper focuses on features that enable or impede progress in the development of culturally safe practices within the optometry programmes to improve eye health equity for Indigenous recognise the diversity of Indigenous cultures across Australia and NZ.

Topographical Distribution and Phenotype of Resident Meibomian Gland Orifice Immune Cells (MOICs) in Mice and the Effects of Topical Benzalkonium Chloride (BAK).

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.

Neuroimmune crosstalk in the cornea: The role of immune cells in corneal nerve maintenance during homeostasis and inflammation.

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.

Defining an Optimal Sample Size for Corneal Epithelial Immune Cell Analysis Using in vivo Confocal Microscopy Images.

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.

Corneal tissue-resident memory T cells form a unique immune compartment at the ocular surface.

The eye is considered immune privileged such that immune responses are dampened to protect vision. As the most anterior compartment of the eye, the cornea is exposed to pathogens and can mount immune responses that recruit effector T cells. However, presence of immune memory in the cornea is not defined. Here, we use intravital 2-photon microscopy to examine T cell responses in the cornea in mice. We show that recruitment of CD8+ T cells in response to ocular virus infection results in the formation of tissue-resident memory T (TRM) cells. Motile corneal TRM cells patrol the cornea and rapidly respond in situ to antigen rechallenge. CD103+ TRM cell generation requires antigen and transforming growth factor ß. In vivo imaging in humans also reveals highly motile cells that patrol the healthy cornea. Our study finds that TRM cells form in the cornea where they can provide local protective immunity.

The effect of topical decorin on temporal changes to corneal immune cells after epithelial abrasion.

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 (ß-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-ß) 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-ß and CSPG4 proteoglycan likely regulate decorin-mediated innate immune cell responses and nerve regeneration after injury.

Corneal immune cell morphometry as an indicator of local and systemic pathology: A review.

The corneal epithelium contains a population of resident immune cells commonly referred to as dendritic cells (DCs), or Langerhans cells. A unique advantage of the transparent cornea being situated at the surface of the eye is that these cells can be readily visualised using in vivo confocal microscopy. Over the past decade, interest in the involvement of corneal DCs in a range of ocular and systemic diseases has surged. For most studies, the number of corneal DCs has been the main outcome of interest. However, more recently attention has shifted towards understanding how DC morphology may provide insights into the inflammatory status of the cornea, and in some cases, the health of the peripheral nervous system. In this review, we provide examples of recent methodologies that have been used to classify and measure corneal DC morphology and discuss how this relates to local and systemic inflammatory conditions in humans and rodents.

Distribution of Corneal TRPV1 and Its Association With Immune Cells During Homeostasis and Injury.

Purpose: Given the role of corneal sensory nerves during epithelial wound repair, we sought to examine the relationship between immune cells and polymodal nociceptors following corneal injury. Methods: Young C57BL/6J mice received a 2 mm corneal epithelial injury. One week later, corneal wholemounts were immunostained using ß-tubulin-488, TRPV1 (transient receptor potential ion channel subfamily V member-1, a nonselective cation channel) and immune cell (MHC-II, CD45 and CD68) antibodies. The sum length of TRPV1+ and TRPV1- nerve fibers, and their spatial association with immune cells, was quantified in intact and injured corneas. Results: TRPV1+ nerves account for ∼40% of the nerve fiber length in the intact corneal epithelium and ∼80% in the stroma. In the superficial epithelial layers, TRPV1+ nerve terminal length was similar in injured and intact corneas. In intact corneas, the density (sum length) of basal epithelial TRPV1+ and TRPV1- nerve fibers was similar, however, in injured corneas, TRPV1+ nerve density was higher compared to TRPV1- nerves. The degree of physical association between TRPV1+ nerves and intraepithelial CD45+ MHC-II+ CD11c+ cells was similar in intact and injured corneas. Stromal leukocytes co-expressed TRPV1, which was partially localized to CD68+ lysosomes, and this expression pattern was lower in injured corneas. Conclusions: TRPV1+ nerves accounted for a higher proportion of corneal nerves after injury, which may provide insights into the pathophysiology of neuropathic pain following corneal trauma. The close interactions of TRPV1+ nerves with intraepithelial immune cells and expression of TRPV1 by stromal macrophages provide evidence of neuroimmune interactions in the cornea.

Altered Corneal Epithelial Dendritic Cell Morphology and Phenotype Following Acute Exposure to Hyperosmolar Saline.

Purpose: The purpose of this study was to assess the morphological and phenotypic responses of corneal epithelial dendritic cells (DCs) to acute topical hyperosmolar stress, given a pathogenic role for tear hyperosmolarity in dry eye disease (DED). Methods: C57BL/6J mice were anesthetized and received 350 mOsm/L (physiological; n = 5 mice), 450 mOsm/L (n = 6), or 600 mOsm/L (n = 6) saline on a randomly assigned eye. Corneas were harvested 2 hours later. Immunofluorescent staining was performed using CD45, CD86, and CD68 antibodies to investigate DC morphology (density, viability, field area, circularity, and dendritic complexity) and immunological phenotype. Flow cytometry was used to confirm CD86 and CD68 expression in CD11c+ DCs, using C57BL/6J mice that received topical applications of 350 mOsm/L, 450 mOsm/L, or 600 mOsm/L (n = 5 per group) bilaterally for 2 hours. Results: Following exposure to 450 mOsm/L topical saline for 2 hours, DCs in the central and peripheral cornea were larger (field area: Pcentral = 0.005, Pperipheral = 0.037; circularity: Pcentral = 0.026, and Pperipheral = 0.013) and had higher expression of CD86 compared with 350 mOsm/L controls (immunofluorescence: P < 0.0001; flow cytometry: P = 0.0058). After application of 600 mOsm/L saline, DC morphology was unchanged, although the percentage of fragmented DCs, and phenotypic expression of CD86 (immunofluorescence: P < 0.0001; and flow cytometry: P = 0.003) and CD68 (immunofluorescence: P = 0.024) were higher compared to 350 mOsm/L controls. Conclusions: Short-term exposure to mild hyperosmolar saline (450 mOsm/L) induced morphological and phenotypic maturation in corneal epithelial DCs. More severe hyperosmolar insult (600 mOsm/L) for 2 hours appeared toxic to these cells. These data suggest that hyperosmolar conditions activate corneal DCs, which may have implications for understanding DC activation in DED.

Morphometric Changes to Corneal Dendritic Cells in Individuals With Mild Cognitive Impairment.

PURPOSE: There has been increasing interest in identifying non-invasive, imaging biomarkers for neurodegenerative disorders of the central nervous system (CNS). The aim of this proof-of-concept study was to investigate whether corneal sensory nerve and dendritic cell (DC) parameters, captured using in vivo confocal microscopy (IVCM), are altered in individuals with mild cognitive impairment (MCI) and Alzheimer's disease (AD). METHODS: Fifteen participants were recruited from the Australian Imaging Biomarkers and Lifestyle (AIBL) study in Melbourne, VIC, Australia. The cohort consisted of cognitively normal (CN) individuals (n = 5), and those with MCI (n = 5) and AD (n = 5). Participants underwent a slit lamp examination of the anterior segment, followed by corneal imaging using laser-scanning in vivo confocal microscopy (IVCM) of the central and inferior whorl regions. Corneal DC density, field area, perimeter, circularity index, aspect ratio, and roundness were quantified using Image J. Quantitative data were derived for corneal nerve parameters, including nerve fiber length (CNFL), fiber density (CNFD), branch density (CNBD), and diameter. RESULTS: Corneal DC field area and perimeter were greater in individuals with MCI, relative to CN controls, in both the central and inferior whorl regions (p < 0.05 for all comparisons). In addition, corneal DCs in the whorl region of MCI eyes had lower circularity and roundness indices and a higher aspect ratio relative to CNs (p < 0.05 for all comparisons). DC density was similar across participant groups in both corneal regions. There was a trend toward lower quantitative parameters for corneal nerve architecture in the AD and MCI groups compared with CN participants, however, the inter-group differences did not reach statistical significance. Central corneal nerve diameters were similar between groups. CONCLUSION: This study is the first to report morphological differences in corneal DCs in humans with MCI. These differences were evident in both the central and mid-peripheral cornea, and in the absence of significant nerve abnormalities or a difference in DC density. These findings justify future large-scale studies to assess the utility of corneal IVCM and DC analysis for identifying early stage pathology in neurodegenerative disorders of the CNS.

The effect of high-fat diet-induced metabolic disturbance on corneal neuroimmune features.

PURPOSE: The highly innervated cornea is susceptible to nerve loss secondary to systemic diseases such as diabetes and metabolic disturbances caused by high-fat diet. In this study, we characterize the effect of high-fat diet on the mouse corneal neuroimmune phenotype, including changes to corneal nerve density and resident immune cells, alongside the clinical assessment of corneal thickness and endothelial cell density. METHODS: Male C57Bl6/J mice, aged 10 weeks, were fed a high-fat diet (60 kcal% fat, 5.2 kcal/g) or control diet (10 kcal%, 3.8 kcal/g) for 16 weeks. At the study endpoint, metabolic parameters (HbA1c, weight, fasting glucose, body fat) were measured to confirm metabolic disturbance. Clinical imaging of the anterior segment was performed using optical coherence tomography to measure the corneal epithelial and stromal thickness. Corneal sensory nerves were visualized using flatmount immunostaining and confocal microscopy. The topographical distribution and density of sensory nerves (BIII-tubulin+), intraepithelial CD45+ and MHC- II+ cells, stromal macrophages (IBA1+CD206+) and endothelial cells (ZO-1+) were analysed using FIJI. RESULTS: High-fat diet mice had significantly higher blood HbA1c, higher body weight, a higher percentage of body fat and elevated fasting glucose compared to the control diet mice. Corneal epithelial and stromal thickness was similar in both groups. The sum length of the basal nerve plexus was lower in the central and peripheral cornea of mice fed a high-fat diet. In contrast, the sum length of superficial nerve terminals was similar between groups. Epithelial immune cell density was two-fold higher in the central corneas of high-fat diet mice compared to control diet mice. IBA1+CD206+ macrophage density was similar in the anterior stroma of both groups but was significantly higher in the posterior stroma of the peripheral cornea in the high-fat diet mice compared to controls. The percentage of nerve-associated MHC-II+ cells in the epithelium and stroma was higher in HFD mice compared to controls. Endothelial cell density was similar in the corneas of high-fat diet mice compared to controls. CONCLUSION: Together with corneal neuropathy, corneal immune cells in mice fed a high-fat diet were differentially affected depending on their topographical distribution and location within cornea, and appeared in closer proximity to epithelial and stromal nerves, suggesting a local neuroimmune disruption induced by systemic metabolic disturbance.

Retinal Functional and Structural Changes in the 5xFAD Mouse Model of Alzheimer's Disease.

Alzheimer's disease is characterized by the aberrant deposition of protein in the brain and is the leading cause of dementia worldwide. Increasingly, there have been reports of the presence of these protein hallmarks in the retina. In this study, we assayed the retina of 5xFAD mice, a transgenic model of amyloid deposition known to exhibit dementia-like symptoms with age. Using OCT, we found that the retinal nerve fiber layer was thinner in 5xFAD at 6, 12, and 17 months of age compared with wild-type littermates, but the inner plexiform layer was thicker at 6 months old. Retinal function showed reduced ganglion cell responses to light in 5xFAD at 6, 12, and 17 months of age. This functional loss was observed in the outer retina at 17 months of age but not in younger mice. We showed using immunohistochemistry and ELISA that soluble and insoluble amyloid was present in the retina and brain at all ages. In conclusion, we report that amyloid is present in brain and retina of 5xFAD mice and that the pattern of neuronal dysfunction occurs in the inner retina at the early ages and progresses to encompass the outer retina with age. This implies that the inner retina is more sensitive to amyloid changes in early disease and that the outer retina is also affected with disease progression.

In vivo immune cell dynamics in the human cornea.

In vivo confocal microscopy (IVCM) allows the evaluation of the living human cornea at the cellular level. The non-invasive nature of this technique longitudinal, repeated examinations of the same tissue over time. Image analysis of two-dimensional time-lapse sequences of presumed immune cells with and without visible dendrites at the corneal sub-basal nerve plexus in the eyes of healthy individuals was performed. We demonstrated evidence that cells without visible dendrites are highly dynamic and move rapidly in the axial directions. A number of dynamic cells were observed and measured from three eyes of different individuals. The total average displacement and trajectory speeds of three cells without visible dendrites (N = 9) was calculated to be 1.12 ± 0.21 and 1.35 ± 0.17 µm per minute, respectively. One cell with visible dendrites per cornea was also analysed. Tracking dendritic cell dynamics in vivo has the potential to significantly advance the understanding of the human immune adaptive and innate systems. The ability to observe and quantify migration rates of immune cells in vivo is likely to reveal previously unknown insights into corneal and general pathophysiology and may serve as an effective indicator of cellular responses to intervention therapies.