Inhibition of the m6A Methyltransferase METTL3 Attenuates the Inflammatory Response in Fusarium solani-Induced Keratitis via the NF-κB Signaling Pathway

PurposeThe purpose of this study was to elucidate the effect of methyltransferase-like enzyme 3 (METTL3) on inflammation and the NF-κB signaling pathway in fungal keratitis (FK).MethodsWe established corneal stromal cell models and FK mouse models by incubation with Fusarium solani. The overall RNA N6-methyladenosine (m6A) level was determined using an m6A RNA methylation assay kit. The expression of METTL3 was quantified via real-time quantitative polymerase chain reaction (RT–PCR), Western blotting, and immunofluorescence. Subsequently, the level of tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) was identified by Western blotting and immunofluorescence. Moreover, we assessed the effect of METTL3 by transfecting cells with siRNA (in vitro) or adeno-associated virus (in vivo). Hematoxylin and eosin (H&E) staining and slit-lamp biomicroscopy were performed to evaluate corneal damage. Furthermore, the state of NF-κB signaling pathway activation was examined by Western blotting. In addition, RT–PCR and enzyme-linked immunosorbent assays (ELISAs) were performed to evaluate levels of the pro-inflammatory factors interleukin-1β (IL-1β), interleukin-6 (IL-6) and TNF-ɑ.ResultsOur data demonstrated that the levels of the RNA m6A methylation and METTL3 were dramatically increased and that the NF-κB signaling pathway was activated in Fusarium solani-induced keratitis. Inhibition of METTL3 decreased the level of TRAF6, downregulated the phospho-p65(p-p65)/p65 and phospho-IκB(p-IκB)/IκB protein ratios, simultaneously attenuating the inflammatory response and fungal burden in FK.ConclusionsOur research suggests that the m6A methyltransferase METTL3 regulates the inflammatory response in FK by modulating the NF-κB signaling pathway.     

Hyperkeratinization and Proinflammatory Cytokine Expression in Meibomian Glands Induced by Staphylococcus aureus

PurposeThis exploratory study aimed to investigate the morphological and pathological alterations of the meibomian gland (MG) with the Staphylococcus aureus crude extracts (SACEs) treatment.MethodsMouse MG explants were cultured and differentiated with or without SACEs for 48 hours. Explant''s viability and cell death were determined by thiazolyl blue tetrazolium bromide (MTT) assay and TUNEL assay. MG morphology was observed by Hematoxylin and Eosin staining. Lipid droplet production was detected by Nile Red staining and LipidTox immunostaining. The pro-inflammatory cytokines were detected by ELISA. The relative gene and protein expression in MG explants was determined via quantitative RT-PCR, immunostaining, and immunoblotting. The components of the SACEs were analyzed by immunoblotting and silver staining.ResultsOur findings demonstrated that the SACEs treatment induced overexpression of keratin 1 (Krt1) in the ducts and acini of MG explants, accompanied by a decrease in viability and an increase in cell death in explants. Furthermore, the SACEs treatment dose-dependently increased the levels of TNF-α, IL-1β, and IL-6 in MG explants. The SACEs treatment induced activation of the nuclear factor kappa B (NF-κB) and AIM2 (absent in melanoma 2)/ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) inflammasome signaling pathway in explants. Further investigation showed expression of the key adipogenesis-related molecule peroxisome proliferator-activated receptor γ was decreased after SACEs treatment. However, no change was found in the lipid synthesis of MG explants after treatment with the SACEs. Staphylococcal enterotoxins B (SEB) was detected in the SACEs. SEB induced the overexpression of Krt1 and IL-1β in ducts and acini of MG explants.ConclusionsOur findings confirm that Staphylococcus aureus induced hyperkeratinization and pro-inflammatory cytokines expression in MG explants ducts and acini. These effects might be mediated by SEB. Activation of the NF-κB and AIM2/ASC signaling pathway is involved in this process.     

High-Fat Diet Induces Inflammation of Meibomian Gland

PurposeTo determine if a high-fat diet (HFD) induces meibomian gland (MG) inflammation in mice.MethodsMale C57BL/6J mice were fed a standard diet (SD), HFD, or HFD supplemented with the peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist rosiglitazone for various durations. Body weight, blood lipid levels, and eyelid changes were monitored at regular intervals. MG sections were subjected to hematoxylin and eosin staining, LipidTox staining, TUNEL assay, and immunostaining. Quantitative RT-PCR and western blot analyses were performed to detect relative gene expression and signaling pathway activation in MGs.ResultsMG acinus accumulated more lipids in the mice fed the HFD. Periglandular CD45-positive and F4/80-positive cell infiltration were more evident in the HFD mice, and they were accompanied by upregulation of inflammation-related cytokines. PPAR-γ downregulation accompanied activation of the mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) signaling pathways in the HFD mice. There was increased acini cell apoptosis and mitochondria damage in mice fed the HFD. MG inflammation was ameliorated following a shift to the standard diet and rosiglitazone treatment in the mice fed the HFD.ConclusionsHFD-induced declines in PPAR-γ expression and MAPK and NF-κB signaling pathway activation resulted in MG inflammation and dysfunction in mice.     

Isotretinoin Impairs the Secretory Function of Meibomian Gland Via the PPARγ Signaling Pathway

PurposeTo investigate the effects of isotretinoin on the ocular surface and to explore the possible mechanisms.MethodsRats were treated with isotretinoin 20 mg/kg/d for five months and tested monthly for tear secretion, fluorescein staining, and infrared photography. After five months of treatment, tissues were harvested for routine staining to evaluate the morphological changes; and real-time polymerase chain reaction, Western blot, and immunohistochemistry to study the expression of associated genes and their products such as forkhead box protein O1 (FoxO1), forkhead box protein O3, peroxisome proliferator–activated receptor γ (PPARγ), adipose differentiation–related protein, elongation of very long chain fatty acids protein 4, fatty acid binding protein 4, matrix metalloproteinase-9, and interleukin-6.ResultsSystemically, isotretinoin-treated rats have a significantly lower body weight that controls and apparent skin damage. Locally, although there was no alteration in tear secretion, a significant corneal involvement indicated by increased fluorescein staining scores, and also the contrast of meibomian gland was significantly reduced but no significant atrophy of the acinus was found. In addition, isotretinoin causes a decrease in conjunctival goblet cells. Furthermore, isotretinoin treatment did not cause the upregulation of FoxO1 and inflammation related genes but significantly suppressed the expression of PPARγ pathway.ConclusionsIsotretinoin does not cause a significant atrophy of the acinus and a significant change of FoxO1 expression in the meibomian gland. Isotretinoin causes meibomian gland dysfunction, affecting meibocyte differentiation and qualitative and quantitative changes in the meibum, through PPARγ pathway.     

Lacrimal Gland Microenvironment Changes After Obstruction of Lacrimal Gland Ducts

PurposeTo investigate microenvironment changes of the lacrimal gland after obstruction of lacrimal gland ducts.MethodsThe ducts of rat exorbital lacrimal gland were ligated by sutures for different durations. After that, the sutures in some animals were released, and they were observed for 21 days to evaluate the recovery of the lacrimal gland. Slit lamp and tear secretion test was performed to evaluate ocular surface and lacrimal gland function. The lacrimal gland and cornea were harvested and processed for hematoxylin and eosin staining, oil red O staining, LipidTOX staining, Masson staining, quantitative real time polymerase chain reaction, and immunofluorescence staining.ResultsAfter the lacrimal gland ducts were blocked, tear secretion and the weight of the lacrimal gland were reduced. Incidence of corneal neovascularization increased after seven days. Intraglandular ducts dilated and acini destroyed. Long-term ligation induced fibrosis and lipid accumulation of the lacrimal glands. Inflammatory cell infiltrated and inflammatory factors upregulated. Proliferative and apoptotic cells increased. Structure of myoepithelial cells and basement membrane was destroyed. The p63 expression increased whereas Pax6 expression decreased. After suture release, tear secretion and structure of acini could recover in less than seven days after ligation, with a decrease in inflammatory cell infiltration and fibrosis relief. Apoptotic cells and proliferative cells increased at five days thereafter. The structure of the myoepithelial cells and basement membrane could not recover three days after ligation, and the number of mesenchymal cells increased in ligation after five to 14 days.ConclusionsBlockage of the lacrimal gland ducts results in dystrophy of lacrimal gland acini cells, inflammation, and lipid accumulation of the lacrimal gland microenvironment. Long-term duct blockage will cause irreversible lacrimal gland failure.     

Anti-inflammatory effects of hinokitiol on human corneal epithelial cells: an in vitro study

Purpose

This study assessed the anti-inflammatory effect and mechanism of action of hinokitiol in human corneal epithelial (HCE) cells.

Methods

HCE cells were incubated with different concentrations of hinokitiol or dimethylsulfoxide (DMSO), which served as a vehicle control. Cell viability was evaluated using Cell Counting Kit-8 (CCK-8) assay. After polyriboinosinic:polyribocytidylic acid (poly(I:C)) stimulus, cells with or without hinokitiol were evaluated for the mRNA and protein levels of interleukin-8 (IL-8), interleukin-6 (IL-6), and interleukin-1β (IL-1β) using real-time PCR analysis and an enzyme-linked immunosorbent assay (ELISA), respectively. Nuclear and cytoplasmic levels of nuclear factor kappa B (NF-κB) p65 protein and an inhibitor of NF-κB α (IκBα) were evaluated using western blotting.

Results

There were no significant differences among the treatment concentrations of hinokitiol compared with cells incubated in medium only. Incubating with 100 μM hinokitiol significantly decreased the mRNA levels of IL-8 to 58.77±10.41% (P<0.01), IL-6 to 64.64±12.71% (P<0.01), and IL-1β to 54.19±8.10% (P<0.01) compared with cells stimulated with poly(I:C) alone. The protein levels of IL-8, IL-6, and IL-1β had similar trend. Further analysis revealed that hinokitiol maintained the levels of IκBα and significantly reduced NF-κB p65 subunit translocation to the nucleus which significantly inhibiting the activation of the NF-κB signal pathway.

Conclusion

Hinokitiol showed a significant protective effect against ocular surface inflammation through inhibiting the NF-κB pathway, which may indicate the possibility to relieve the ocular surface inflammation of dry eye syndrome (DES).     

IL-36α Exerts Proinflammatory Effects in Aspergillus fumigatus Keratitis of Mice Through the Pathway of IL-36α/IL-36R/NF-κB

PurposeTo explore the role of IL-36α in corneas infected by Aspergillus fumigatus.MethodsThe experimental group was comprised of 15 corneas with fungal keratitis, and 15 healthy donor corneas were included in the control group. IL-36α was detected in normal and infected corneas of humans and C57BL/6 mice. Mice corneas were infected with A. fumigatus with or without pretreatment of recombinant mouse (rm) IL-36α and IL-36α neutralizing antibody (Ab). Primary macrophages were stimulated with 75% ethanol-killed A. fumigatus with or without pretreatment of rmIL-36α. The severity of the disease was documented by clinical score and photographs with a slit lamp. PCR, western blot, and immunostaining were used to determine the expression of IL-36α, IL-1β, IL-6, and TNF-α. Polymorphonuclear neutrophilic leukocyte infiltration was assessed by myeloperoxidase (MPO) assay and flow cytometry. Macrophage infiltration was tested by immunofluorescent staining and flow cytometry.ResultsIL-36α mRNA and protein were significantly elevated in human and mice corneas after infection. The rmIL-36α treatment of C57BL/6 mice increased clinical score, MPO levels, macrophage infiltration, and expression of the proinflammatory cytokines IL-1β, IL-6, and TNF-α compared with the infected controls, which showed a decrease due to IL-36α Ab treatment. In primary macrophages, IL-36α expression was also significantly increased by A. fumigatus. The rmIL-36α treatment upregulated IL-1β, IL-6, and phosphorylated nuclear factor (NF)-κB expression, which was significantly inhibited by rmIL-36Ra.ConclusionsIL-36α act as a proinflammatory cytokine in A. fumigatus keratitis by promoting the infiltration of neutrophils and macrophages and increasing the secretion of IL-1β, IL-6, and TNF-α, in addition to regulating expression of phosphorylated NF-κB.     

Melatonin Attenuates LPS-Induced Proinflammatory Cytokine Response and Lipogenesis in Human Meibomian Gland Epithelial Cells via MAPK/NF-κB Pathway

PurposeInflammation contributes to the development of meibomian gland dysfunction (MGD) under specific disease conditions, but the underlying mechanisms remain elusive. We examined whether lipopolysaccharide (LPS) induced a proinflammatory cytokine response and lipogenesis in human meibomian gland epithelial cells (HMGECs) and whether melatonin (MLT), a powerful anti-inflammatory regent in the eyes, could protect against LPS-induced disorders.MethodsHuman meibomian gland (MG) tissues and immortalized HMGECs were stained to identify Toll-like receptor (TLR) 4 and MLT receptors (MT₁ and MT₂). HMGECs were pretreated with or without MLT and then stimulated with LPS. Then, TLR4 activation, cytokine levels, lipid synthesis, apoptosis, autophagy, and MAPK/NF-κB factor phosphorylation in HMGECs were analyzed.ResultsTLR4, MT₁, and MT₂ were expressed in human MG acini and HMGECs. Pretreatment with MLT inhibited the TLR4/MyD88 signaling and attenuated proinflammatory cytokine response and lipogenesis in LPS-stimulated HMGECs, which manifested as decreased production of cytokines (IL-1β, IL-6, IL-8, and TNF-α), reduced lipid droplet formation, and downregulated expression of meibum lipogenic proteins (ADFP, ELOVL4, and SREBP-1). Phospho-histone H2A.X foci, lysosome accumulation, and cytoplasmic cleaved caspase 3/LC3B-II staining were increased in LPS-stimulated HMGECs, indicating enhanced cell death mediated by apoptosis and autophagy during LPS-induced lipogenesis. MLT downregulated cleaved caspase 3 levels and the Bax/Bcl-2 ratio to alleviate apoptosis and ameliorated the expression of Beclin 1 and LC3B-II to inhibit autophagy. The protective mechanisms of MLT include the inhibition of MAPK and NF-κB phosphorylation.ConclusionsMLT attenuated lipogenesis, apoptosis, and autophagy in HMGECs induced by proinflammatory stimuli, indicating the protective potential of MLT in MGD.     

Role of the Phospholipase C Pathway and Calcium Mobilization in Oxytocin-Induced Contraction of Lacrimal Gland Myoepithelial Cells

PurposeWe reported that oxytocin (OXT), added to freshly prepared lacrimal gland lobules, induced myoepithelial cell (MEC) contraction. In other systems, OXT activates phospholipase C (PLC) generating Inositol 1,4,5-trisphosphate (IP₃) which increases intracellular calcium concentration ([Ca²⁺]_i) causing contraction. The aim of the current study was to investigate the role of this pathway in OXT-induced contraction of MEC.MethodsTear volume was measured using the cotton thread method. Lacrimal gland MEC were isolated and propagated from α-smooth muscle actin (SMA)-green fluorescent protein (GFP) mice, in which MEC express GFP making them easily identifiable. RNA and protein samples were prepared for RT-PCR and Western blotting for G protein expression. Changes in [Ca²⁺]_i were measured in Fura-2 loaded MEC using a ratio imaging system. MEC contraction was monitored in real time and changes in cell size were quantified using ImageJ software.ResultsOXT applied either topically to surgically exposed lacrimal glands or delivered subcutaneously resulted in increased tear volume. OXT stimulated lacrimal gland MEC contraction in a dose-dependent manner, with a maximum response at 10⁻⁷ M. MEC express the PLC coupling G proteins, Gαq and Gα11, and their activation by OXT resulted in a concentration-dependent increase in [Ca²⁺]_i with a maximum response at 10⁻⁶ M. Furthermore, the activation of the IP₃ receptor to increase [Ca²⁺]_i is crucial for OXT-induced MEC contraction since blocking the IP₃ receptor with 2-APB completely abrogated this response.ConclusionsWe conclude that OXT uses the PLC/Ca²⁺ pathway to stimulate MEC contraction and increase lacrimal gland secretion.     

Molecular Hydrogen Attenuated N-methyl-N-Nitrosourea Induced Corneal Endothelial Injury by Upregulating Anti-Apoptotic Pathway

PurposePrevious work by our group has demonstrated the value of N-methyl-N-nitrosourea (MNU)-induced corneal endothelial decompensation in animal models. The aim of this study was to investigate the effect of molecular hydrogen (H₂) on MNU-induced corneal endothelial cell (CEC) injury and the underlying mechanism.MethodsMNU-induced animal models of CEC injury were washed with hydrogen-rich saline (HRS) for 14 days. Immunofluorescence staining, immunohistochemical staining, and corneal endothelial assessment were applied to determine architectural and cellular changes on the corneal endothelium following HRS treatment. MNU-induced cell models of CEC injury were co-cultured with H₂. The effect of H₂ was examined using morphological and functional assays.ResultsIt was shown that MNU could inhibit the proliferation and specific physiological functions of CECs by increasing apoptosis and decreasing the expression of ZO-1 and Na⁺/K⁺-ATPase, whereas H₂ improved the proliferation and physiological function of CECs by anti-apoptosis. Cell experiments further confirmed that H₂ could reverse MNU damage to CECs by decreasing oxidative stress injury, interfering with the NF-κB/NLRP3 pathway and the FOXO3a/p53/p21 pathway.ConclusionsThis study suggests that topical application of H₂ could protect CECs against corneal damage factors through anti-apoptotic effect, reduce the incidence and severity of corneal endothelial decompensation, and maintain corneal transparency.     

The Intraoperative Corneal Pachymetry Changes during Accelerated Corneal Cross-linking in Progressive Keratoconus Patients with Thin Corneas

PurposeTo report the intraoperative corneal pachymetry changes during accelerated corneal cross-linking (A-CXL) in progressive keratoconus patients with thin corneas.MethodsThirty-six eyes (mean age, 22.26 ± 4.02 years) with progressive keratoconic thin corneas (<400 μm without epithelium) who underwent A-CXL with ultraviolet (UV)-A (UVA) (9 mW/cm²) using isotonic riboflavin5-phosphate 0.1% with 1.1% hydroxypropyl methycellulose (RF-HPMC, MedioCROSS M) were included in this retrospective study. Intraoperative corneal pachymetric changes were noted before the procedure, after removal of epithelium, after RF-HPMC instillation, before and after UV irradiation. The mean of corneal pachymetric values were compared statistically.ResultsThe mean corneal pachymetry reduced from 415.72 ± 29.66 to 369.50 ± 23.45 μm after removal of the epithelium (p < 0.05). After the application of RF-HPMC solution the mean thinnest corneal pachymetry (TCP) increased to 412.89 ± 26.94 μm. Statistically significant increase was observed in TCP after saturation with RF-HPMC (p = 0.001). The mean corneal pachymetry before and after UVA irradiation was 419.86 ± 10.41 and 417.47 ± 8.25 μm, respectively (p > 0.05).ConclusionsIsotonic RF-HPMC lead to a significant increase in intraoperative mean TCP. RF-HPMC seems to be a favorable riboflavin option in keratoconus patients with thin corneas.     

Clinical Outcomes of Nanothin Descemet Stripping Automated Endothelial Keratoplasty in Korean Patients with Corneal Endothelial Dysfunction

PurposeTo evaluate the clinical outcomes of nanothin Descemet stripping automated endothelial keratoplasty (DSAEK) in Korean patients with corneal endothelial dysfunction.MethodsWe retrospectively reviewed medical records of the patients who underwent nanothin DSAEK (graft thickness ≤50 μm) due to corneal endothelial dysfunction and followed up more than 1 year. We evaluated best-corrected visual acuity (BCVA), central corneal thickness, and corneal endothelial cell density at preoperative and 1, 3, 6, and 12 months postoperatively.ResultsSixteen eyes of 16 patients with the mean follow-up period of 13.00 ± 0.96 months were included. The mean graft thickness after deswelling was 45.25 ± 4.59 μm (range, 38.0–50.0 μm). The mean logarithm of the minimum angle of resolution BCVA improved from 1.37 ± 0.53 preoperatively to 0.68 ± 0.46, 0.55 ± 0.35, 0.40 ± 0.25, and 0.39 ± 0.25 at 1, 3, 6, and 12 months postoperatively (p = 0.005, p < 0.001, p < 0.001, and p < 0.001), respectively. The mean central corneal thickness improved from 752.00 ± 129.11 to 555.75 ± 54.66 μm at 12 months postoperatively (p = 0.006). The mean graft endothelial cell density decreased from 2,859.62 ± 228.34 to 1,542.25 ± 627.34 cells/mm² at 12 months postoperatively (p = 0.012). The postoperative complications included increased intraocular pressure (n = 3, 18.75%) and graft dislocation (n = 1, 6.25%), all of which were successfully managed by anterior chamber paracentesis or rebubbling. No other serious complications were encountered.ConclusionsNanothin DSAEK produced significant and stable visual improvements without severe postoperative complications in Korean patients with corneal endothelial dysfunction.     

Effects of extract of Buddleja officinalis on partial inflammation of lacrimal gland in castrated rabbits with dry eye

AIM: To assess the effects of extract of Buddleja officinalis on tear secretion volume, tear film stability, expressions of TGF-β1, IL-1β, TNF-α in lacrimal gland of castrated rabbits with dry eye. METHODS: A total of 30 victory rabbits were divided averagely into normal group(A), model group(B), therapy group with low dose extract of Buddleja officinalis (C), therapy group with high dose extract of Buddleja officinalis (D) and therapy group with genistein (E). The dry eye model was established with orchiectomy on Group B, C, D, E. Group C, D, E were administered intragastrically with corresponding dose extract of Buddleja officinalis or genistein for 30 days. All rabbits were detected with SIT. TGF-β1, IL-1β, TNF-α were detected with immunohistochemistry and the ultrastructure of lacrimal gland was observed under transmission electron microscope. RESULTS: The SIT value of group C, D, E were respectively 13.167±4.957, 14.667±5.279, 8.667±0.516, obviously higher than that of group B 5.667±2.338 (P<0.01). The positive expression of IL-1β in acinar cell and glandular tube cell of group C, D were 0.470±0.048, 0.510±0.088, obviously lower than that of group B 0.770±0.118 (P<0.01). The positive expression of TNF-α of group C, D were 0.498±0.156, 0.435±0.069, obviously lower than that of group B 0.769±0.095 too (P<0.01). The positive expression of TGF-β1 of group C, D were 0.406±0.171, 0.497±0.147, obviously higher than that of group B 0.222±0.113(P<0.01). Any result of group C, D was positive compared with that of group E (P <0.05). Ultrastructure of the lacrimal gland of group C, D, E was well preserved, especially in D group it was remarkable. CONCLUSION: The extract of Buddleja officinalis can adjust lacrimal gland partial inflammation of dry eye.     

The relationship between corneal subbasal nerve density and corneal sensitivity in patients with Fuchs endothelial corneal dystrophy

Purpose:The aim of this study was to investigate the association between alterations in corneal subbasal nerve plexus and tactile corneal sensitivity in patients with Fuchs’ endothelial corneal dystrophy (FECD).Methods:This retrospective, cross-sectional study included 24 (10 M/14 F) patients with FECD and 25 age- and sex-matched (10 M/15 F) healthy subjects as controls. Subjects with FECD were classified as having early (grades 1 and 2) and late (grades 3 and 4) disease. All subjects underwent central corneal tactile sensitivity measurements with the Cochet–Bonnet esthesiometer (Luneau Ophthalmologie, Chartres, France) and subbasal nerve density evaluation using in vivo confocal microscopy (IVCM). Association between corneal nerve plexus density and corneal sensitivity alterations were evaluated using the Mann–Whitney U test and the Spearman correlation test.Results:Compared to healthy subjects (mean age = 60.4 ± 7.5 years), patients with FECD (mean age = 60.6 ± 8.0 years) had worse central corneal sensitivity scores (5.9 ± 0.1 cm vs. 4.2 ± 0.8 cm; P < 0.001), reduced corneal nerve fibers (3.4 ± 1.3 nerves/frame vs. 5.0 ± 0.9 nerves/frame; P < 0.001) and lower corneal subbasal nerve plexus densities (2229.4 ± 364.3 μm/mm² vs. 1901.6 ± 486.8 μm/mm²; P = 0.050). Patients with late stage FECD demonstrated lower subbasal nerve densities as compared to those with early disease (2204.3 ± 313.1 μm/mm² (range = 1523–2552 μm/mm²); 1397.1 ± 227.4 μm/mm² (range = 1120-1834 μm/mm²); P < 0.001). In the FECD group, subbasal nerve density was found to be directly correlated with corneal sensitivity scores (r = 0.457, P = 0.025).Conclusion:Progressive loss of the corneal subbasal nerve plexus appears to be a consistent feature of FECD. Reduction of the corneal nerve plexus parallels the decrease in corneal sensitivity in this patient population.     

The Therapeutic Roles of Recombinant Hsp90α on Cornea Epithelial Injury

PurposeThe purpose of this study was to explore the therapeutic role of heat shock protein 90 (Hsp90) in wound healing of injury cornea epithelium.MethodsThe right eye of C57BL/6N male mice were performed the debridement wounds in the center of the cornea using an algerbrush II blade. The injured area was determined by staining the cornea with fluorescein sodium and measured with image-J. Immunoblotting, ELISA and immunochemistry were used for determining protein expression. The quantitation PCR was performed to measure mRNA expression.ResultsHsp90α is upregulated at both the mRNA and protein levels, and is secreted extracellularly into the corneal stroma and tear film during the healing process after corneal injury in mice. This upregulation is associated with activation of HSF1. Administration of recombinant exogenous Hsp90α (eHsp90α) speeds up wound healing of injured corneal epithelium. The eHsp90α binds to low-density lipoprotein (LDL)-related protein-1 (LRP-1) on the corneal epithelial cells and increases phosphorylation of AKT at S473, which is associated with proliferation and migration corneal epithelial cells in vitro or vivo. Inhibition of AKT by its inhibitor LY294002 abolishes eHsp90α-induced migration and proliferation of corneal epithelial cells.ConclusionsHsp90α is upregulated and secreted after corneal injury and acts to promote the healing process. Recombinant Hsp90α may be a promising therapeutic drug candidate for corneal injury.     

Conjunctival Expression of Toll-Like Receptor 3 Plays a Pathogenic Role in the Formation of Ultraviolet Light–Induced Pterygium

PurposeToll-like receptor 3 (TLR3), as a damage-associated molecular pattern sensor, can detect self-RNA released from necrotic cells induced by ultraviolet B (UVB) radiation exposure. Pterygium formation is believed to be a tumorigenesis-like process induced by UVB exposure. In this study, we aimed to investigate the expression pattern of TLR3 in pterygium specimens and cultured pterygial epithelial cells (PECs).MethodsHuman pterygium and ipsilateral pterygium-free conjunctiva from the same patients were used in this study. The expression of TLR3 and nuclear factor-kappa B (NF-κB) was investigated in these specimens. PECs were exposed to UVB radiation to determine the effect of UVB on the expression of TLR3 and the activation of NF-κB.ResultsThe immunofluorescence study showed stronger TLR3 expression in superficial epithelial cells in the pterygial epithelium in comparison with the normal conjunctival epithelium. The expression of TLR3 decreased in intensity from the superficial epithelium toward the basal cell layer, implying a correlation between UVB exposure and TLR3 expression. Differential TLR3 expression patterns in pterygial and conjunctival tissues were also found in quantitative PCR analyses. PECs after UVB irradiation had higher protein levels of TLR3 and phospho-NF-κB than those of the PECs without irradiation. Immunofluorescence studies showed that UVB irradiation induced the nuclear translocation of NF-κB in the PECs. In PECs with the targeted TLR3 gene silencing, the expression of phospho-NF-κB was not induced by UVB irradiation.ConclusionsOur results indicate that UVB exposure, TLR3 expression, and NF-κB activation may be a critical sequence that leads to the formation of pterygium.