Ocular Surface最新文献

Purpose

Human donor corneas are an essential control tissue for corneal research. We utilized whole mount immunofluorescence (WM-IF) to evaluate how the storage affects the tissue integrity and putative limbal stem cells in human and porcine corneas. Moreover, we compare this information with the marker expression patterns observed in human pluripotent stem cell (hPSC)-derived LSCs.

Methods

The expression of putative LSC markers was analyzed with WM-IF and the fluorescence intensity was quantified in human donor corneas stored for 1–30 days, and in porcine corneas processed 0–6 h after euthanasia. The results were compared with the staining of human and porcine corneal cryosections and with both primary and hPSC-derived LSC cultures.

Results

WM-IF analyses emerged as a more effective method when compared to tissue sections for visualizing the expression of LSC markers within human and porcine corneas. Storage duration was a significant factor influencing the expression of LSC markers, as human tissues stored longer exhibited notable epithelial degeneration and lack of LSC markers. Porcine corneas replicated the expression patterns observed in fresh human tissue. We validated the diverse expression patterns of PAX6 in the limbal-corneal region, which aligned with findings from hPSC-LSC differentiation experiments.

Conclusions

WM-IF coupled with quantification of fluorescence intensities proved to be a valuable tool for investigating LSC marker expression in both human and porcine tissues ex vivo. Prolonged storage significantly influences the expression of LSC markers, underscoring the importance of fresh human or substitute control tissue when studying limbal stem cell biology.

Corneal neovascularization (CoNV) is the second leading common cause of vision impairment worldwide and is a blinding pathological alteration brought on by ocular trauma, infection, and other factors. There are some limitations in the treatment of CoNV, hence it's critical to look into novel therapeutic targets. The corneal epithelial barrier, which is the initial barrier of the ocular surface, is an important structure that shields the eye from changes in the internal environment or invasion by the external environment. This study sought to collate evidence on the regulation of corneal epithelial barrier injury on the activation of vascular endothelial cells (VECs), basement membrane (BM) degradation, differentiation, migration, and proliferation of VECs, vascular maturation and stability, and other key processes in CoNV, so as to provide a novel concept for CoNV therapy targeting corneal epithelial barrier repair.

Purpose

Ocular surface hydration is critical for eye health and its impairment can lead to dry eye disease. Extracellular calcium-sensing receptor (CaSR) is regulator of ion transport in epithelial cells expressing cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel. CFTR is also a major ion channel in ocular surface epithelia, however the roles of CaSR in ocular surface are not well studied. This study aims to investigate expression and functional roles of CaSR in ocular surface.

Methods

CaSR immunostaining was performed in mouse and human cornea and conjunctiva. Ocular surface potential difference (OSPD) and tear fluid volume measurements were performed in mice with topically applied cinacalcet (CaSR activator) and NPS-2143 (CaSR inhibitor).

Results

CaSR is expressed in corneal and conjunctival epithelia of mice and humans. Topically administered CaSR activator cinacalcet inhibits cAMP agonist forskolin-induced Cl⁻ secretion and CFTR activity up to 90 %. CaSR inhibitor NPS-2143 stimulates CFTR-mediated Cl⁻ secretion in mouse ocular surface, after which cAMP agonist forskolin had minimal additional secretory effects. Single dose NPS-2143 treatment (as an eye drop) increases tear fluid volume in mice by ∼60 % compared to vehicle treatment. NPS-2143 effect on tear volume lasts at least 8 h after single dose.

Conclusions

CaSR is a key regulator of ocular surface ion transport and CaSR inhibition promotes Cl⁻ and tear secretion in the ocular surface. If they are found to be effective in in dry eye models, CaSR inhibitors (currently in clinical development) can potentially be repurposed as novel prosecretory treatments for dry eye disease.

Filamentary keratitis (FK) is a clinical sign of underlying ocular and systemic conditions. FK can cause significant irritation, tearing, and photophobia in the eye. It is a refractory debilitating condition caused by dry eye that affects the day-to-day activities of patients. The etiopathogenesis of FK is not well known; there are numerous predisposing causes. The condition starts as a sub-epithelial or Bowman's membrane dysfunction and leads to the shedding of epithelial cells that take a strand-like form and attach to the cornea. These strands are surrounded by mucin and continue to elongate to become filaments. The filament formation is further aided by the shearing action caused by eyelid movements. Several management approaches, such as addressing the underlying causes of filamentary keratitis, administering copious lubricants, topical corticosteroids, mucolytic agents, bandage contact lenses, punctal plugs, and mechanical removal of filaments are available. The prognosis is fair, and most cases resolve with occasional recurrences. Traditionally FK has been treated with lubricants, mechanical removal, and bandage contact lenses. The newer treatments are topical immunomodulators especially that treat filamentary keratitis associated with aqueous deficient dry eye. The review describes the treatment as well as pathogenesis.

Purpose

To investigate the delayed diagnosis of chronic ocular graft-versus-host disease (coGVHD) after allogeneic hematopoietic stem cell transplantation (alloHCT), and further analyze potential confounding factors.

Methods

This cross-sectional study included 118 patients newly diagnosed as coGVHD after alloHCT at Zhongshan Ophthalmic Center, Sun Yat-sen University. All participants finished the flow path of medical history taking, detailed ophthalmological examination and questionnaire-based survey. coGVHD was diagnosed and graded by International Chronic Ocular GVHD Consensus Group (ICOGCG) criteria. Lag time of diagnosis was defined as interval between noting of ocular symptoms and confirmed diagnosis of coGVHD (T_N-D). We further compared the clinical parameters between groups categorized by the median T_N-D as medium and long delay groups.

Results

The median T_N-D was 6.3 [IQR 2.8–14.5] months. Most coGVHD patients underwent delayed diagnosis of coGVHD longer than 3 months (70 %, 83 of 118), with 90 of 118 diagnosed as severe coGVHD (76 %). The long delay group exhibited higher ICOGCG scores (10 [IQR 9–10.5] vs. 9 [IQR 8–10], P = 0.039) and more pronounced ocular signs, including conjunctival injection, meibomian gland loss, fibrotic tarsal conjunctiva, symblepharon, and corneal complications (all P < 0.05). Delayed diagnosis was strikingly correlated with seeking ophthalmic medical care twice or more prior to diagnosis (adjusted OR = 5.42, 95%CI: 1.40–21.06, P = 0.015) and accurate knowledge of ocular discomfort symptoms in coGVHD (adjusted OR = 0.29, 95%CI: 0.08–1.00, P = 0.050).

Conclusions

Delayed diagnosis of coGVHD, associated with disease severity, was common among alloHCT recipients in southern China. Improving patient education and the awareness of ophthalmologists may facilitate early diagnosis of coGVHD.

Purpose

Polyunsaturated fatty acids (PUFA) are a source of bioactive lipids regulating inflammation and its resolution.

Methods

Changes in PUFA metabolism were compared between lacrimal glands (LGs) from young and aged C57BL/6 J mice using a targeted lipidomics assay, as was the gene expression of enzymes involved in the metabolism of these lipids.

Results

Global reduction in PUFAs and their metabolites was observed in aged LGs compared to young controls, averaging between 25 and 66 % across all analytes. ꞷ-6 arachidonic acid (AA) metabolites were all reduced in aged LGs, where the changes in prostaglandin E2 (PGE2) and lipoxin A4 (LXA4) were statistically significant. Several other 5-lipoxygenase (5-LOX) mediated metabolites were significantly reduced in the aged LGs, including D-series resolvins (e.g., RvD4, RvD5, and RvD6). Along with the RvDs, several ꞷ-3 docosahexaenoic acid (DHA) metabolites such as 14-HDHA, neuroprotectin D1 (NPD1), Maresin 2 (MaR2), and MaR 1 metabolite (22-COOH-MaR1) were significantly reduced in aged LGs. Similarly, ꞷ-3 eicosapentaenoic acid (EPA) and its metabolites were significantly reduced in aged LGs, where the most significantly reduced was 18-HEPE. Using metabolite ratios (product:precursor) for specific metabolic conversions as surrogate enzymatic measures, reduced 12-LOX activity was identified in aged LGs.

Conclusion

In this study, global reduction of PUFAs and their metabolites was found in the LGs of aged female C57BL/6 J compared to young controls. A consistent reduction was observed across all detected lipid analytes except for ꞷ-3 docosapentaenoic acid (DPA) and its special pro-resolving mediator (SPM) metabolites in aged mice, suggesting an increased risk for LG inflammation.

Purpose

Stevens-Johnson syndrome (SJS) is characterised as an immuno-inflammatory condition with potentially blinding ocular sequelae. Therefore, we have investigated the ocular surface immune cell profile and correlated it with secreted tear molecular factors and clinical ocular sequelae in SJS patients.

Methods

21 patients (42 eyes) with chronic ocular SJS and 16 healthy controls (20 eyes) were included in the study. Severity, types of keratopathies and ocular surface (OS) manifestations were determined. OS wash samples from study subjects were used to determine the status of 13 immune cell subsets using flow cytometry. Levels of 42 secreted immuno-inflammatory factors were measured by flow cytometry-based multiplex ELISA in tear samples.

Results

Neutrophils (Total, activated), neutrophils/NK cells ratio, neutrophils/T cells ratio were significantly (p < 0.05) elevated in SJS, while, proportions of T cells and NKT cells were significantly lower in SJS patients. Positive association between neutrophils and chronic ocular surface complication score (COCS) was observed, whereas, a negative association was noted between NK cells and COCS. Tear fluid levels of IL-6, IL-8, IL-18, IFNα/β/γ, TNFα, LIF, IL-8, HGF, sTNFR-I, NGAL, Granzyme, Perforins, MMP9/TIMP1 ratio were significantly higher in SJS. Loss of Limbal niche correlated significantly with immune profile and clinical sequelae. Increased neutrophils, decreased NK cells and specific set of altered secreted immuno-inflammatory mediators including bFGF, and IL-8 were observed in SJS patients with different types of keratopathies compared to those without keratopathy.

Conclusion

Distinct ocular surface immune profile variations were observed to correlate with clinical stages of chronic ocular SJS. Our findings uncover novel mechanisms and potential for targeted therapy in chronic ocular SJS patients.

Purpose

To investigate the global transcriptional landscape of lacrimal gland cell populations in the GVHD mouse model.

Methods

Single-cell RNA sequencing and further bioinformatic analysis of dissociated lacrimal gland (LG) cells from the mouse model were performed. Parts of transcriptional results were confirmed by immunofluorescence staining.

Results

We identified 23 cell populations belonging to 11 cell types. In GVHD LG, the proportion of acinar cells, myoepithelial cells, and endothelial cells was remarkably decreased, while T cells and macrophages were significantly expanded. Gene expression analysis indicated decreased secretion function, extracellular matrix (ECM) synthesis, and increased chemokines of myoepithelial cells. A newly described epithelial population named Lrg1^high epithelial cells, expressing distinct gene signatures, was exclusively identified in GVHD LG. The fibroblasts exhibited an inflammation gene pattern. The gene pattern of endothelial cells suggested an increased ability to recruit immune cells and damaged cell-cell junctions. T cells were mainly comprised of Th2 cells and effective memory CD8⁺ T cells. GVHD macrophages exhibited a Th2 cell-linked pattern.

Conclusions

This single-cell atlas uncovered alterations of proportion and gene expression patterns of cell populations and constructed cell-cell communication networks of GVHD LG. These data may provide some new insight into understanding the development of ocular GVHD.