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Purpose: The purpose of this study is to analyze bilateral changes of matrix metalloproteinase-9 (MMP-9) expression in the tear film in patients with unilateral neurotrophic keratitis., Methods: We included 24 eyes from 24 subjects with normal corneal sensitivity according to Cochet-Bonnet corneal esthesiometry in the control group and 25 subjects with the unilateral neurotrophic keratitis in the unilateral decrease of corneal sensitivity (UDCS) group. The expression of MMP-9 in tears was assessed using point-of-care immunoassay. The clinical parameters including meibomian gland plugging, quality of the secreted meibum, tear secretion using Schirmer I without anesthesia, and corneal erosions were evaluated among the control, affected, and contralateral unaffected eyes in the UDCS group., Results: Tear MMP-9 grades in affected (2.5 ± 1.0) and contralateral eyes (2.6 ± 0.9) in the UDCS group were higher than those in control eyes (1.5 ± 0.7, P = 0.0003 and P = 0.0006, respectively). The lacrimal secretion decreased in the affected eyes but preserved in the contralateral eyes. There was no difference in corneal erosion scores between control eyes and contralateral eyes in the UDCS group. Tear MMP-9 grades in the contralateral eyes were significantly correlated with the meibum quality grades in the contralateral eyes (r = 0.525 and P = 0.025)., Conclusions: Patients with the unilateral neurotrophic keratitis demonstrated a significant bilateral increase of the MMP-9 expression in the tear film as compared with controls. Despite the tearing reflex and corneal surface barrier being preserved in the unaffected contralateral eyes, it is necessary to pay attention to the possible attenuation of meibomian gland function in the opposite eyes as well., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

The kinetics of antigen-presenting cells (APCs) vary depending on their resident tissues and the manner of immunization. We investigated the long-term changes in mature APC and T-cell subsets over 4 weeks in the ocular surface in murine models of corneal quiescent or potent sterile inflammation, and allosensitization using partial (PT), syngeneic (Syn), and allogeneic (Allo) corneal transplantation. In PT, CD11b int CD11c hi MHCII hi CD86 hi cells increased until 4 weeks with an increase in IFNγ hi T cells. In Syn, both CD11b int CD11c hi MHCII hi CD86 hi and CD11b hi CD11c hi MHCII hi CD86 hi APC subsets increased until 4 weeks with a brief increase in CD69 hi T cells at 2 weeks. In Allo, CD11b int CD11c hi MHCII hi CD86 hi and CD11b hi CD11c hi MHCII hi CD86 hi APC subsets increased until 4 weeks, and an early increase in CD69 hi T cells was observed at 2 weeks followed by a late increase in IFNγ hi T cells at 4 weeks. The frequency of the IFNγ hi T cell subset was positively correlated with the frequency of the CD11b int CD11c hi MHCII hi CD86 hi subset, indicating the existence of APC-T cell interaction in the ocular surface. Together, the results indicate that allosensitization in mature APCs leads to T-cell activation in the ocular surface, whereas sterile inflammation merely induces a brief and non-specific T-cell activation in the ocular surface.

Purpose: This study investigated the effect of a unilateral cut of the corneal nerve on the bilateral ocular surface and tear secretory function., Methods: Seven-week-old female BALB/c mice were divided into control and nerve-cutting (NC) groups (n = 60). The left cornea was partially incised with a 2-mm circular trephine through the upper half of the stromal layer. Lissamine green corneal staining and tear volume measurements were conducted, and corneal whole-mount staining using class III β-tubulin antibody was performed to assess corneal nerves. Flow cytometric analyses for dendritic cells (DCs), CD4+/CD8+ and regulatory T cells and ELISA for neuropeptides were performed., Results: The grading of corneal staining increased in the NC group, while the tear volume decreased over the 4 weeks. The nerve density decreased in bilateral corneas over 2 weeks. At day 14, CD11b+ or CD11c+ DCs and the mature DCs expressing CD86 or MHCII increased in bilateral cornea/conjunctiva. At day 28, CD11c+CD86hi, CD11c+MHCIIhi, Th17 and IFN-γ-secreting CD8+ T cells highly increased in bilateral draining lymph nodes. CD4+CD25hiFoxp3hi and CD8+CD25hiFoxp3hi regulatory T cells notably increased in the spleen. In ELISA, neuropeptide Y, calcitonin gene-related peptide, and vasoactive intestinal peptide were generally suppressed in the extraorbital lacrimal glands at day14., Conclusions: The unilateral corneal nerve severing resulted in activation of the immune cells on the ocular surface and dysregulated lacrimal secretion bilaterally through the bidirectional neuronal signals. It suggests that the unilateral corneal nerve damage may alter immune homeostasis and mechanistically participate in the development of bilateral inflammatory disorders such as dry eye.

Background: Angiogenin (ANG), a component of tears, is involved in the innate immune system and is related with inflammatory disease. We investigated whether ANG has an immune modulatory function in human corneal fibroblasts (HCFs)., Methods: HCFs were cultured from excised corneal tissues. The gene or protein expression levels of interleukin (IL)-1beta (β), IL-4, IL-6, IL-8, IL-10, complements, toll-like receptor (TLR)4, myeloid differentiation primary response gene (MYD)88, TANK-binding kinase (TBK)1, IkappaB kinase-epsilon (IKK-ε) and nuclear factor-kappaB (NF-κB) were analyzed with or without ANG treatment in tumor necrosis factor-alpha (TNF-α)- or lipopolysaccharide (LPS)-induced inflammatory HCFs by real-time polymerase chain reaction (PCR), Western blotting and immunocytochemistry. Inflammatory cytokine profiles with or without ANG were evaluated through immunodot blot analysis in inflammatory HCFs. Corneal neovascularization and opacity in a rat model of corneal alkali burn were evaluated after application of ANG eye drops., Results: ANG decreased the mRNA levels of IL-1β, IL-6, IL-8, TNF-α receptor (TNFR)1, 2, TLR4, MYD88, and complement components except for C1r and C1s and elevated the mRNA expression of IL-4 and IL-10. Increased signal intensity of IL-6, IL-8 and monocyte chemotactic protein (MCP)-1 and MCP-2 induced by TNF-α or LPS was weakened by ANG treatment. ANG reduced the protein levels of IKK-ε by either TNF-α and LPS, and decreased TBK1 production induced by TNF-α, but not induced by LPS. The expression of NF-κB in the nuclei was decreased after ANG treatment. ANG application lowered corneal neovascularization and opacity in rats compared to controls., Conclusion: These results demonstrate that ANG reduces the inflammatory response induced by TNF-α or LPS in HCFs through common suppression of IKK-ε-mediated activation of NF-κB. This may support the targeting of immune-mediated corneal inflammation by using ANG.

Purpose: Previously, we reported that keratocyte-conditioned medium (KCM) facilitates the differentiation of human mesenchymal stem cells (hMSCs) into corneal keratocyte-like cells. This study is designed to investigate the roles of insulin-like growth factor binding protein 2 (IGFBP2) for the regulation of corneal fibroblast differentiation as a newly unveiled component of KCM., Methods: Immunodot blot analysis was performed to identify the factors that are highly secreted, especially in KCM. Then, we investigated whether IGFBP2 differentiates hMSCs into keratocyte-like cells and whether maintains the phenotypes of keratocyte in human corneal fibroblasts (HCFs) by analyzing expression patterns of alpha-smooth muscle actin (α-SMA) and keratocyte markers including keratocan, lumican and aldehyde dehydrogenase 1 family member A1 (ALDH1A1). Furthermore, to specify the role of IGFBP2, the expression of α-SMA and keratocyte markers was determined in transforming growth factor β 1 (TGFβ1)-induced corneal myofibroblast and in HCFs after knockdown of IGFBP2., Results: The most prominent factor in both KCM and amniotic membrane extract was IGFBP2. Insulin-like growth factor binding protein 2 increased the expression of IGFBP2, keratocan, and ALDH1A1, and decreased α-SMA expression in hMSCs and HCFs. Insulin-like growth factor binding protein 2 inhibited TGFβ1-induced upregulation of α-SMA and increased expressions of keratocan and ALDH1A1 in HCFs. Furthermore, the knockdown of IGFBP2 increased α-SMA expression and decreased ALDH1A1 level in HCFs., Conclusions: Insulin-like growth factor binding protein 2 is strongly associated with restoration of keratocyte phenotype in HCFs. Our results show an important novel role of IGFBP2 in regulation of corneal fibroblast differentiation and suggest that IGFBP2 can be a therapeutic candidate for corneal antifibrotic strategy.

The purpose of this study was to elucidate the origin and cellular composition of retrocorneal membranes (RCMs) associated with chemical burns using immunohistochemical staining for primitive cell markers. Six cases of RCMs were collected during penetrating keratoplasty. We examined RCMs with hematoxylin and eosin (H&E), periodic acid-Schiff (PAS) staining and immunohistochemical analysis using monoclonal antibodies against hematopoietic stem cells (CD34, CD133, c-kit), mesenchymal stem cells (beta-1-integrin, TGF-β, vimentin, hSTRO-1), fibroblasts (FGF-β, α-smooth muscle actin), and corneal endothelial cells (type IV collagen, CD133, VEGF, VEGFR1). Histologic analysis of RCMs revealed an organized assembly of spindle-shaped cells, pigment-laden cells, and thin collagenous matrix structures. RCMs were positive for markers of mesenchymal stem cells including beta-1-integrin, TGF-β, vimentin, and hSTRO-1. Fibroblast markers were also positive, including FGF-β and α-smooth muscle actin (SMA). In contrast, immunohistochemical staining was negative for hematopoietic stem cell markers including CD34, CD133 and c-kit as well as corneal endothelial cell markers such as type IV collagen, CD133 except VEGF and VEGFR1. Pigment-laden cells did not stain with any antibodies. The results of this study suggest that RCMs consist of a thin collagen matrix and fibroblast-like cells and may be a possible neogenetic structure produced from a lineage of bone marrow-derived mesenchymal stem cells.

Angiogenin (ANG) is reportedly multifunctional, with roles in angiogenesis and autoimmune diseases. This protein is involved in the innate immune system and has been implicated in several inflammatory diseases. Although ANG may be involved in the anti-inflammatory response, there is no evidence that it has direct anti-inflammatory effects. In this study we sought to determine whether ANG has an anti-inflammatory effect in human corneal fibroblasts (HCFs) exposed to media containing tumor necrosis factor-alpha (TNF-α). We found that ANG reduced the mRNA expression of interleukin-1 beta (IL-1β), -6, -8 and TNF-α receptors (TNFR) 1 and 2. In contrast, ANG increased the mRNA expression of IL-4 and -10. Protein levels of TANK-binding kinase 1 (TBK1) were reduced by ANG in HCFs treated with TNF-α. Moreover, ANG diminished the expression of IL-6 and -8 and monocyte chemotactic protein- (MCP-) 1. The protein expression of nuclear factor-κB (NF-κB) was downregulated by ANG treatment. These findings suggest that ANG suppressed the TNF-α-induced inflammatory response in HCFs through inhibition of TBK1-mediated NF-κB nuclear translocation. These novel results are likely to play a significant role in the selection of immune-mediated inflammatory therapeutic targets and may shed light on the pathogenesis of immune-mediated inflammatory diseases.

Aims: This study aimed to report corneal steep island (CSI) formation following primary pterygium surgery and to identify preoperative pterygium morphological features that predict the likelihood of CSI. Methods: A total of 93 eyes from 84 subjects with primary nasal pterygium, who underwent pterygium excision combined with conjunctival-limbal autograft, were included in this retrospective longitudinal cohort study. CSI was defined using anterior segment swept-source optical coherence tomography (AS SS-OCT). Eyes were divided into two groups: those with postoperative CSI formation (Group 1) and those without postoperative CSI (Group 2). We compared postoperative anterior corneal astigmatism (ACA, in diopters [D]) and root mean square (RMS) values of anterior corneal lower-order (LoA) and higher-order aberrations (HoA) between the groups. Baseline clinical severity grades of pterygium based on the pre-established pterygium body morphology and vascularity, ACA, and AS SS-OCT-guided pterygium morphological profiles (horizontal invasion length [HIL, mm], height [μm], thickness (μm), and residual corneal thickness [RCT]/central corneal thickness [CCT] ratio [RCT/CCT]) were also compared. Results: Postoperative CSI occurred in 26 eyes (28.0%) with a maximum follow-up duration of 22.9±27.4 months. Group 1 exhibited significantly higher postoperative anterior corneal RMS LoA and HoA, as well as the RMS values of the 4th to 6th orders. Although clinical severity grades of pterygium did not differ between groups, baseline ACA was higher in Group 1 (4.56±5.49 D vs. 2.70±3.80 D, P = 0.009). HIL (4.49±0.84 mm vs. 3.77±1.29 mm, P = 0.010) was higher in Group 1, while pterygium height (930.8±84.4 μm vs. 999.3±128.0 μm, P = 0.015) and RCT/CCT ratio (1.07±0.13 vs. 1.14±0.16, P = 0.049) were lower in Group 1. Conclusions: CSI may develop after primary pterygium surgery, particularly in patients with relatively higher preoperative ACA, longer HIL, and shorter height. Given that CSI can significantly increase both lower and higher-order aberrations, it is crucial to anticipate CSI probability and inform patients before surgery. [ABSTRACT FROM AUTHOR]

Purpose: To propose the optimal value of baseline corneal astigmatism and pterygial morphological profiles for primary pterygium surgery to restore the corneal optical properties. Methods: We analysed 93 eyes from 84 subjects with nasal‐only primary pterygium who underwent pterygium excision with conjunctival‐limbal autograft and were assessed perioperatively using anterior segment swept‐source optical coherence tomography (AS SS‐OCT). We collected data on anterior corneal astigmatism (ACA) and root mean square (RMS) values for anterior corneal lower‐ (LoA) and higher‐order aberrations (HoA) as corneal optical properties using AS SS‐OCT. Using preoperative ACA and four pterygial morphological profiles (horizontal invasion length [HIL], height, thickness and the ratio of residual corneal thickness [RCT] to central corneal thickness [CCT]) measured in AS SS‐OCT, we plotted receiver operating characteristic (ROC) curves. These curves aimed to determine cut‐off values predicting a perioperative decrease exceeding 50% in ACA, RMS LoA and RMS HoA, as well as postoperative residual ACA higher than 1.25D. Results: Preoperative ACA > 1.42D (AUC = 0.934) and >3.60D (AUC = 0.946) proved most effective in identifying subjects with perioperative decrease exceeding in ACA and RMS LoA, respectively. HIL > 3.34 mm (AUC = 0.941) was most effective in distinguishing subjects with perioperative reduction exceeding 50% in RMS HoA. Preoperative ACA > 5.78D (AUC = 0.776) and HIL > 5.03 mm (AUC = 0.700) significantly distinguished subjects with postoperative residual ACA higher than 1.25D. Conclusion: Optimizing the restoration of corneal astigmatism and aberrations after pterygium surgery may be facilitated by determining the optimal surgical timing based on preoperative ACA and HIL values. [ABSTRACT FROM AUTHOR]

Abstract: Culturing corneal keratocytes is difficult because keratocytes growing in a monolayer rapidly lose their stellate morphology and cease to express keratocyte markers such as keratocan, lumican and aldehyde dehydrogenase 1 family, member A1 (ALDH1A1). Conversely, mesenchymal stem cells (MSCs) can be easily expanded in cell culture, and they have a variety of differentiation pathways. We studied the feasibility of using MSCs as a source for corneal tissue engineering. Based on the observation that keratocytes have MSC-like properties, similar to bone marrow-derived MSCs (BM-MSCs), we hypothesized that MSCs would differentiate into corneal keratocyte-like cells in keratocyte-conditioned medium (KCM). We measured changes in the expression of keratocyte markers through quantitative real-time polymerase chain reaction (qRT-PCR) and found that human MSC''s cultured in KCM expressed both keratocan and ALDH1A1. Western blot analysis demonstrated that human MSCs cultured in KCM steadily increased their expression of lumican and ALDH1A1, while they lost expression of α-smooth muscle actin (α-SMA). Immunocytochemistry indicated that human MSCs grown in KCM acquired characteristics similar to those of keratocytes. These results suggest that KCM can direct human MSCs to differentiate into keratocyte-like cells. [Copyright &y& Elsevier]