Density and distribution of dendritiform cells in the peripheral cornea of healthy subjects using in vivo confocal microscopy.

PURPOSE: To establish in a large healthy cohort, dendritiform cell (DC) density and morphological parameters in the central and peripheral cornea using in vivo confocal microscopy (IVCM). METHODS: A prospective, cross-sectional, observational study was conducted in 85 healthy volunteers (n = 85 eyes). IVCM images of corneal center and four peripheral zones were analyzed for DC density and morphology to compare means and assess correlations (p < 0.05 being statistically significant). RESULTS: Central corneas had lower DC density (40.83 ± 5.14 cells/mm2; mean ± SEM) as compared to peripheral corneas (75.42 ± 2.67 cells/mm2, p < 0.0001). Inferior and superior zones demonstrated higher DC density (105.01 ± 7.12 and 90.62 ± 4.62 cells/mm2) compared to the nasal and temporal zones (59.93 ± 3.42 and 51.77 ± 2.98 cells/mm2, p < 0.0001). Similarly, lower DC size, field and number of dendrites were observed in the central as compared to the average peripheral cornea (p < 0.0001), with highest values in the inferior zone (p < 0.001 for all, except p < 0.05 for number of dendrites in superior zone). DC parameters did not correlate with age or gender. Inter-observer reliability was 0.987 for DC density and 0.771-0.922 for morphology. CONCLUSION: In healthy individuals, the peripheral cornea demonstrates higher DC density and larger morphology compared to the center, with highest values in the inferior zone. We provide the largest normative cohort for sub-stratified DC density and morphology, which can be used in future clinical trials to compare differential changes in diseased states. Furthermore, as DC parameters in the peripheral zones are dissimilar, random sampling of peripheral cornea may be inaccurate.

Characterization of Resident Corneal Plasmacytoid Dendritic Cells and Their Pivotal Role in Herpes Simplex Keratitis.

The presence and potential functions of resident plasmacytoid dendritic cells (pDCs) in peripheral tissues is unclear. We report that pDCs constitutively populate naïve corneas and are increased during sterile injuries or acute herpes simplex virus 1 (HSV-1) keratitis. Their local depletion leads to severe clinical disease, nerve loss, viral dissemination to the trigeminal ganglion and draining lymph nodes, and mortality, while their local adoptive transfer limits disease. pDCs are the main source of HSV-1-induced IFN-α in the corneal stroma through TLR9, and they prevent re-programming of regulatory T cells (Tregs) to effector ex-Tregs. Clinical signs of infection are observed in pDC-depleted corneas, but not in pDC-sufficient corneas, following low-dose HSV-1 inoculation, suggesting their critical role in corneal antiviral immunity. Our findings demonstrate a vital role for corneal pDCs in the control of local viral infections.

Clinically Relevant Immune-Cellular Metrics of Inflammation in Meibomian Gland Dysfunction.

Purpose: To determine the reliability and clinical relevance of in vivo confocal microscopy (IVCM)-based immune-cellular metrics of palpebral conjunctival inflammation in meibomian gland dysfunction (MGD). Methods: Sixteen MGD patients and 13 reference controls included in this cross-sectional, retrospective study, had an ocular surface exam, symptom assessment (Ocular Surface Disease Index questionnaire [OSDI]), and palpebral conjunctival IVCM imaging. Bland-Altman analyses, intraclass correlation coefficient (ICCa), Lin's concordance correlation coefficient (ρc), receiver operating characteristic (ROC) analyses, and correlations were performed. Clinical outcome measures were symptom severity (OSDI scores), tear break-up time (TBUT), and corneal fluorescein staining (CFS grade). Results: Compared to controls, patients with MGD had variable symptom severity (average OSDI score: 48.3 ± 7.6, P = 0.0008, range: 8.3-85.42), shorter TBUT (6.8 ± 0.9 seconds, P = 0.002), comparable corneal staining (0.31 ± 0.19, P = 0.20), and greater conjunctival inflammation (epithelial immune cells [EIC]: 477.8 ± 54.2 vs. 123.3 ± 17.2 cells/mm2, P < 0.0001; intraglandular immune cells [IGIC]: 41.9 ± 3.3% vs. 20.33 ± 7.3%, P < 0.01). Immune-cellular metrics had high inter- and intraobserver agreement (ρc: 0.86-0.94; ICCa and Cronbach's α: 0.85-0.97, P < 0.0001). EIC correlated positively with OSDI (rs: 0.49, P = 0.03), while both EIC and IGIC correlated inversely with TBUT (rs: -0.47, -0.45, P < 0.05), and had high accuracy in detecting inflammation (ROC area under the curve [AUC]: 0.97 and 0.89, P ≤ 0.001). Conclusions: EIC and IGIC are increased in highly symptomatic patients with MGD that have minimal corneal staining, and correlate with symptoms and clinical signs. EIC and IGIC may provide reliable and clinically relevant metrics of inflammation.

Corneal Nerve and Epithelial Cell Alterations in Corneal Allodynia: An In Vivo Confocal Microscopy Case Series.

PURPOSE: To investigate morphological changes of the corneal epithelium and subbasal nerves in patients with corneal allodynia using in vivo confocal microscopy (IVCM). DESIGN: Case-control study of patients with corneal allodynia and healthy controls. METHODS: Ten eyes of six patients were diagnosed with corneal allodynia at a single center and compared to fifteen healthy eyes. IVCM of the central cornea was performed on all subjects and controls. Images were retrospectively analyzed numbers of total corneal subbasal nerves, main trunks and branches, total nerve length and density, nerve branching, and tortuosity, superficial and basal epithelial cell densities, and superficial epithelial cell size. RESULTS: Corneal allodynia was seen in patients with dry eye disease, recurrent corneal erosion syndrome, exposure to ultraviolet radiation, and Accutane use. Compared to controls, patients with corneal allodynia had a significant decrease in the total numbers of subbasal nerves (P=.014), nerve branches (P=.006), total nerve length (P=.0029), total nerve density (P=.0029) and superficial and basal epithelial cell densities (P=.0004, P=.0036) with an increase in superficial epithelial cell size (P=.016). There were no statistically significant differences in the number of subbasal nerve main trunks (P=.09), nerve branching (P=.21), and nerve tortuosity (P=.05). CONCLUSIONS: Corneal IVCM enables near-histological visualization and quantification of the cellular and neural changes in corneal allodynia. Regardless of etiology, corneal allodynia is associated with decreased corneal epithelial cell densities, increased epithelial cell size, and decreased numbers and lengths of subbasal nerves despite an unremarkable slit-lamp examination. Therefore, IVCM may be useful in the management of patients with corneal allodynia.

In Vivo Confocal Microscopy of Corneal Nerves in Health and Disease.

In vivo confocal microscopy (IVCM) is becoming an indispensable tool for studying corneal physiology and disease. Enabling the dissection of corneal architecture at a cellular level, this technique offers fast and noninvasive in vivo imaging of the cornea with images comparable to those of ex vivo histochemical techniques. Corneal nerves bear substantial relevance to clinicians and scientists alike, given their pivotal roles in regulation of corneal sensation, maintenance of epithelial integrity, as well as proliferation and promotion of wound healing. Thus, IVCM offers a unique method to study corneal nerve alterations in a myriad of conditions, such as ocular and systemic diseases and following corneal surgery, without altering the tissue microenvironment. Of particular interest has been the correlation of corneal subbasal nerves to their function, which has been studied in normal eyes, contact lens wearers, and patients with keratoconus, infectious keratitis, corneal dystrophies, and neurotrophic keratopathy. Longitudinal studies have applied IVCM to investigate the effects of corneal surgery on nerves, demonstrating their regenerative capacity. IVCM is increasingly important in the diagnosis and management of systemic conditions such as peripheral diabetic neuropathy and, more recently, in ocular diseases. In this review, we outline the principles and applications of IVCM in the study of corneal nerves in various ocular and systemic diseases.

In Vivo Confocal Microscopy in Dry Eye Disease Associated With Chronic Graft-Versus-Host Disease.

PURPOSE: To compare densities of corneal epithelial dendritic cells (DCs), corneal subbasal nerves, and conjunctival epithelial immune cells (EICs) between patients with dry eye disease (DED) with and without graft-versus-host disease (GVHD) using in vivo confocal microscopy (IVCM). METHODS: This study included 54 patients who had moderate to severe DED either associated with (n = 33) or without (n = 21) chronic GVHD. In addition to evaluating clinical parameters of DED, images obtained by laser-scanning IVCM of the central cornea and superior tarsal conjunctiva were analyzed to measure densities of corneal epithelial DCs, corneal subbasal nerves, and conjunctival EICs. RESULTS: Although there were no significant differences between GVHD and non-GVHD groups in symptom scores, the GVHD group had significantly worse corneal fluorescein staining, tear break-up time, and Schirmer's scores than the non-GVHD group. Corneal epithelial DC density, corneal subbasal nerve density, and conjunctival EIC density were 148 ± 135 cells/mm2, 16.3 ± 6.1 mm/mm2, and 670 ± 267 cells/mm2, respectively, in the GVHD group; and 122 ± 99 cells/mm2, 18.3 ± 5.1 mm/mm2, and 572 ± 271 cells/mm2, respectively, in the non-GVHD group. After adjusting for clinical parameters, including the DED severity, none of the IVCM parameters was significantly different between the GVHD versus non-GVHD groups (P = 0.82, P = 0.21, and P = 0.60, respectively). CONCLUSIONS: In GVHD-associated DED, cellular changes in the cornea and conjunctiva observed by IVCM were similar to those seen in patients who have non-GVHD dry eye with the same level of disease severity. Therefore, corneal and conjunctival IVCM findings in GVHD-associated DED are possibly reflective of the local disease (DED) severity rather than the underlying systemic disease process.

Validity and Reliability of a Novel Ocular Pain Assessment Survey (OPAS) in Quantifying and Monitoring Corneal and Ocular Surface Pain.

PURPOSE: To validate the Ocular Pain Assessment Survey (OPAS), specifically designed to measure ocular pain and quality of life for use by eye care practitioners and researchers. DESIGN: A single-center cohort study was conducted among patients with and without corneal and ocular surface pain at initial and follow-up visits over a 6-month period. The content of the OPAS was guided by literature review, a body of experts, and incorporating conceptual frameworks from existing pain questionnaires. The Wong-Baker FACES Pain Rating Scale served as the gold standard for measuring the intensity of ocular pain. PARTICIPANTS: A total of 102 patients aged 18 to 80 years completed the OPAS at the initial visit. A total of 21 patients were followed up after treatment. METHODS: Indices of validity and internal consistency (Spearman's rank-order, rs, or Pearson's correlation coefficients, rp), and coefficient of reliability (Cronbach's α) were determined in addition to equivalence testing, exploratory factor analysis (EFA), and diagnostic analysis. MAIN OUTCOME MEASURES: Eye pain intensity was the primary outcome measure, and interference with quality of life (QoL), aggravating factors, associated factors, associated non-eye pain intensity, and self-reported symptomatic relief were the secondary outcome measures. RESULTS: The OPAS had criterion validity at both initial (rs = 0.71; n = 102; P < 0.01) and follow-up visits (rs = 0.97; n = 21; P < 0.01). Equivalence tests yielded OPAS and gold standard equivalence for both the initial and follow-up visits. The EFA supported 6 subscales (eye pain intensity at 24 hours and 2 weeks, non-eye pain intensity, QoL, aggravating factors, and associated factors) confirming multidimensionality. Cronbach's α >0.83 for all subscales established strong internal consistency, which correlated with the gold standard, including 24-hour eye pain intensity and QoL interference scores (rp = 0.81, 0.64, respectively P < 0.001). At follow-up, reduction in pain scores was accompanied by improvement in all dimensions of the OPAS. Percentage change in QoL correlated to percentage change in the gold standard (rp = 0.53; P < 0.05). The OPAS was sensitive (94%), specific (81%), and accurate (91%), with a diagnostic odds ratio >50. CONCLUSIONS: The OPAS is a valid, reliable, and responsive tool with strong psychometric and diagnostic properties in the multidimensional quantification of corneal and ocular surface pain intensity, and QoL.

Correlation between human tear cytokine levels and cellular corneal changes in patients with bacterial keratitis by in vivo confocal microscopy.

PURPOSE: We investigated bilateral tear cytokine levels in patients with unilateral bacterial keratitis (BK) as associated with in vivo confocal microscopic (IVCM) alterations in corneal nerves and dendritiform immune cells (DCs). METHODS: A total of 54 (13 BK, 13 contralateral, 28 healthy controls) tear samples was collected prospectively and analyzed by multiplex microbeads assay. The IVCM of the central cornea was performed on the same day, and assessed for corneal nerve and DC alterations. RESULTS: Interleukin-1ß, IL-6, and IL-8 were significantly elevated only in affected eyes (66.6 ± 26.8, 7174 ± 2430, and 810 ± 315 ρg/mL, respectively; P = 0.04, P < 0.001, and P < 0.001, respectively), compared to healthy controls (13.0 ± 4.0, 171.8 ± 32.1, and 56.5 ± 33.8 ρg/mL). Levels of chemokine ligand 2 (CCL-2), IL-10, and IL-17a were elevated only in contralateral eyes (813 ± 478, 86.7 ± 38.3, and 3350 ± 881 ρg/mL, respectively; P = 0.02, P = 0.01, and P = 0.04, respectively), compared to controls (73.7 ± 25.3, 17.5 ± 4.9, and 1350 ± 337 ρg/mL). Triggering receptor expressed on myeloid cells (TREM)-1 was significantly elevated in affected (551 ± 231 ρg/mL, P = 0.02) and contralateral unaffected (545 ± 298 ρg/mL, P = 0.03) eyes compared to controls (31.3 ± 12.4 ρg/mL). The density of DCs was significantly increased in affected (226.9 ± 37.3 cells/mm(2), P < 0.001) and unaffected (122.3 ± 23.7 cells/mm(2), P < 0.001) eyes compared to controls (22.7 ± 5.9 cells/mm(2)). Sub-basal nerve density significantly decreased in affected (3337 ± 1615 µm/mm(2), P < 0.001) and contralateral (13,230 ± 1635 µm/mm(2), P < 0.001) eyes compared to controls (21,200 ± 545 µm/mm(2)). Levels of IL-1ß, IL-6, and IL-8 were significantly correlated with DC density (R = 0.40, R = 0.55, and R = 0.31, all P < 0.02) and nerve density (R = -0.30, R = -0.53, and R = -0.39, all P < 0.01). CONCLUSIONS: Proinflammatory tear cytokines are elevated bilaterally in patients with unilateral BK, and are correlated strongly with alterations in DCs and nerve density as detected by IVCM.

Image-guided evaluation and monitoring of treatment response in patients with dry eye disease.

BACKGROUND: Dry eye disease (DED) is one of the most common ocular disorders worldwide. The pathophysiological mechanisms involved in the development of DED are not well-understood, and thus treating DED has been a significant challenge for ophthalmologists. Most of the currently available diagnostic tests demonstrate low correlation to patient symptoms and have low reproducibility. METHODS: Recently, sophisticated in vivo imaging modalities have become available for patient care, namely, in vivo confocal microscopy (IVCM) and optical coherence tomography (OCT). These emerging modalities are powerful and non-invasive, allowing real-time visualization of cellular and anatomical structures of the cornea and ocular surface. Here we discuss how, by providing both qualitative and quantitative assessment, these techniques can be used to demonstrate early subclinical disease, grade layer-by-layer severity, and allow monitoring of disease severity by cellular alterations. Imaging-guided stratification of patients may also be possible in conjunction with clinical examination methods. CONCLUSIONS: Visualization of subclinical changes and stratification of patients in vivo allows objective image-guided evaluation of tailored treatment response based on cellular morphological alterations specific to each patient. This image-guided approach to DED may ultimately improve patient outcomes and make it possible to study the efficacy of novel therapies in clinical trials.

Gene therapy in corneal transplantation.

Corneal transplantation is the most commonly performed organ transplantation. Immune privilege of the cornea is widely recognized, partly because of the relatively favorable outcome of corneal grafts. The first-time recipient of corneal allografts in an avascular, low-risk setting can expect a 90% success rate without systemic immunosuppressive agents and histocompatibility matching. However, immunologic rejection remains the major cause of graft failure, particularly in patients with a high risk for rejection. Corticosteroids remain the first-line therapy for the prevention and treatment of immune rejection. However, current pharmacological measures are limited in their side-effect profiles, repeated application, lack of targeted response, and short duration of action. Experimental ocular gene therapy may thus present new horizons in immunomodulation. From efficient viral vectors to sustainable alternative splicing, we discuss the progress of gene therapy in promoting graft survival and postulate further avenues for gene-mediated prevention of allogeneic graft rejection.

Targeted intraceptor nanoparticle therapy reduces angiogenesis and fibrosis in primate and murine macular degeneration.

Monthly intraocular injections are widely used to deliver protein-based drugs that cannot cross the blood-retina barrier for the treatment of leading blinding diseases such as age-related macular degeneration (AMD). This invasive treatment carries significant risks, including bleeding, pain, infection, and retinal detachment. Further, current therapies are associated with a rate of retinal fibrosis and geographic atrophy significantly higher than that which occurs in the described natural history of AMD. A novel therapeutic strategy which improves outcomes in a less invasive manner, reduces risk, and provides long-term inhibition of angiogenesis and fibrosis is a felt medical need. Here we show that a single intravenous injection of targeted, biodegradable nanoparticles delivering a recombinant Flt23k intraceptor plasmid homes to neovascular lesions in the retina and regresses CNV in primate and murine AMD models. Moreover, this treatment suppressed subretinal fibrosis, which is currently not addressed by clinical therapies. Murine vision, as tested by OptoMotry, significantly improved with nearly 40% restoration of visual loss induced by CNV. We found no evidence of ocular or systemic toxicity from nanoparticle treatment. These findings offer a nanoparticle-based platform for targeted, vitreous-sparing, extended-release, nonviral gene therapy.

Corneal Allograft Rejection: Immunopathogenesis to Therapeutics.

Corneal transplantation is among the most successful solid organ transplants. However, despite low rejection rates of grafts in the 'low-risk' setting, rejection can be as high as 70% when grafted into 'high-risk' recipient beds. Under normal homeostatic conditions, the avascular cornea provides a unique environment that facilitates immune and angiogenic privilege. An imbalance in pro-inflammatory, angiogenic and lymphangiogenic mediators leads to a breakdown in corneal immune privilege with a consequent host response against the donor graft. Recent developments in lamellar and endothelial keratoplasties have reduced the rates of graft rejection even more, while providing improved visual outcomes. The corneal layer against which an immune response is initiated, largely determines reversibility of the acute episode. While epithelial and stromal graft rejection may be treated with topical corticosteroids with higher success, acute endothelial rejection mandates a more aggressive approach to therapy due to the lack of regenerative capacity of this layer. However, current immunosuppressive regimens come with the caveat of ocular and systemic side effects, making prolonged aggressive treatment undesirable. With the advent of biologics, efficacious therapies with a superior side effect profile are on the horizon. In our review we discuss the mediators of ocular immune privilege, the roles of cellular and molecular immune players in graft rejection, with a focus on human leukocyte antigen and antigen presenting cells. Furthermore, we discuss the clinical risk factors for graft rejection and compare rates of rejection in lamellar and endothelial keratoplasties to traditional penetrating keratoplasty. Lastly, we present the current and upcoming measures of therapeutic strategies to manage and treat graft rejection, including an overview of biologics and small molecule therapy.

Nanoparticle-mediated delivery of shRNA.VEGF-a plasmids regresses corneal neovascularization.

PURPOSE: To determine the efficacy of a plasmid containing a small hairpin RNA expression cassette (pSEC.shRNA) against VEGF-A-loaded poly(lactic co-glycolic acid) nanoparticles (PLGA NPs) in the sustained regression of murine corneal neovascularization. METHODS: PLGA nanoparticles were loaded with pSEC.shRNA.VEGF-A plasmids using the double emulsion-solvent evaporation method. KNV was induced in BALB/c mice by mechanical-alkali injury. Four weeks after induction of KNV, the mice were randomly divided to receive one of four treatments intrastromally: pSEC.shRNA.VEGF-A PLGA NPs (2 µg plasmid); naked pSEC.shRNA.VEGF-A plasmid only (2 µg plasmid); control blank PLGA NPs (equivalent dry weight of NPs); and vehicle. Two and five days after intervention, corneas were harvested to determine VEGF-A gene and protein expression using reverse transcriptase polymerase chain reaction and ELISA, respectively. Four weeks after intervention, corneas were photographed, mice sacrificed, and the corneal whole mounts were immunostained for CD31 (panendothelial cell marker). Immunofluorescence microscopy was performed and the neovascular area was quantitated. RESULTS: VEGF-A mRNA (49.6 ± 12.4 vs. 82.9 ± 6.0%, P < 0.01) and protein (4.0 ± 5.2 vs. 20.0 ± 7.5 ρg VEGF-A/mg total protein, P < 0.05) expression were significantly reduced in pSEC.shRNA.VEGF-A PLGA NP-treated corneas as compared with control blank NP. The pSEC.shRNA.VEGF-A PLGA NP-treated corneas showed significant regression in the mean fractional areas of KNV (0.125 ± 0.042; 12.5%, P <0.01) compared with both naked plasmid only (0.283 ± 0.004; 28.3%) and control (blank NPs = 0.555 ± 0.072, 55.5%) at 4 weeks post-treatment. CONCLUSIONS: The pSEC.shRNA.VEGF-A-loaded PLGA NPs are an effective, nonviral, nontoxic, and sustainable form of gene therapy for the regression of murine KNV.

Assessment of axial length measurements in mouse eyes.

PURPOSE: To compare measurements of murine ocular axial lengths (ALs) made with 780 nm partial coherence interferometry (PCI) and 1310 nm spectral domain-optical coherence tomography (SD-OCT). METHODS: AL was measured at postnatal day (P) 58 in C57BL/6J mice. Repeated AL measurements were taken using a custom-made 780 nm PCI and a commercial 1310 nm SD-OCT. Intra- and interuser variability was assessed along the central optical axis and 2-degree off-axes angles with the SD-OCT. Data were collected and analyzed using Cronbach alpha (α), Bland-Altman coefficient of repeatability, agreement plots, and intraclass correlation coefficients (ICC). RESULTS: AL measurements agreed well between the two instruments (3.262 ± 0.042 mm for PCI; 3.264 ± 0.047 mm for SD-OCT; n = 20 eyes). The ICC for PCI compared with SD-OCT was 0.92, confirming high agreement between the two instruments. Intrauser ICC for the PCI and SD-OCT were 0.814 and 0.995, respectively. Similarly, interuser ICC for PCI and SD-OCT were 0.970 and 0.943, respectively. Using SD-OCT, a 2-degree misalignment of the eye along the horizontal meridian produced mean differences in AL of -0.002 ± 0.017 mm relative to the centrally aligned images, whereas similar misalignment along the vertical meridian created 0.005 ± 0.018 mm differences in AL measurements. CONCLUSIONS: AL measurements from the 780 nm PCI and 1310 nm SD-OCT correlate well. Multiple statistical indices indicate that both instruments have good precision and agreement for measuring murine ocular AL in vivo. Although the vertical meridian had the greater variability in AL in the small mouse eye; 2-degree off-axes differences were within the SD of centrally aligned AL.

Corneal transparency: genesis, maintenance and dysfunction.

Optimal vision is contingent upon transparency of the cornea. Corneal neovascularization, trauma and, surgical procedures such as photorefractive keratectomy and graft rejection after penetrating keratoplasty can lead to corneal opacification. In this article we identify the underlying basis of corneal transparency and factors that compromise the integrity of the cornea. With evidence from work on animal models and clinical studies, we explore the molecular mechanisms of both corneal avascularity and its dysfunction. We also seek to review therapeutic regimens that can safely salvage and restore corneal transparency.

Mediators of ocular angiogenesis.

Angiogenesis is the formation of new blood vessels from pre-existing vasculature. Pathologic angiogenesis in the eye can lead to severe visual impairment. In our review, we discuss the roles of both pro-angiogenic and anti-angiogenic molecular players in corneal angiogenesis, proliferative diabetic retinopathy, exudative macular degeneration and retinopathy of prematurity, highlighting novel targets that have emerged over the past decade.