Mitophagy and mitochondrion-related expression profiles in response to physiological and pathological hypoxia in the corneal epithelium.

To study the mitochondrial and cellular responses to physiological and pathological hypoxia, corneal epithelial cells were preconditioned under 21% O2, 8% O2 or 1% O2. The cell survival rate, mitochondrial fluorescence and mitophagy flux were quantified using flow cytometry. After RNA sequencing, gene set enrichment analysis (GSEA) was performed. When the oxygen level decreased from 21% to 8%, mitochondrial fluorescence decreased by 45% (p < 0.001), accompanied by an 80% increase in mitophagy flux (p < 0.001). When the oxygen level dropped to 1%, the cell survival rate and mitochondrial fluorescence decreased, while mitophagy flux further increased (each p < 0.001). Comparison of 1% O2 vs. 21% O2 revealed enrichment of the HYPOXIA hallmark. Most of the significantly enriched mitochondrion-related gene sets were involved in apoptosis. The corresponding foremost leading edge genes belonged to the BCL-2 family. Corneal epithelial cell fate decisions under hypoxia may involve noncanonical pathways of mitophagy.

Ductal Hyperkeratinization and Acinar Renewal Abnormality: New Concepts on Pathogenesis of Meibomian Gland Dysfunction.

Meibomian gland dysfunction (MGD) is a functional and morphological disorder of the meibomian glands which results in qualitative or quantitative alteration in meibum secretion and is the major cause of evaporative dry eye (EDE). EDE is often characterized by tear film instability, increased evaporation, hyperosmolarity, inflammation, and ocular surface disorder. The precise pathogenesis of MGD remains elusive. It has been widely considered that MGD develops as a result of ductal epithelial hyperkeratinization, which obstructs the meibomian orifice, halts meibum secretion, and causes secondary acinar atrophy and gland dropout. Abnormal self-renewal and differentiation of the acinar cells also play a significant role in MGD. This review summarizes the latest research findings regarding the possible pathogenesis of MGD and provides further treatment strategies for MGD-EDE patients.

TAP1, a potential immune-related prognosis biomarker with functional significance in uveal melanoma.

BACKGROUND: TAP1 is an immunomodulation-related protein that plays different roles in various malignancies. This study investigated the transcriptional expression profile of TAP1 in uveal melanoma (UVM), revealed its potential biological interaction network, and determined its prognostic value. METHODS: CIBERSORT and ESTIMATE bioinformatic methods were used on data sourced from The Cancer Genome Atlas database (TCGA) to determine the correlation between TAP1 expression, UVM prognosis, biological characteristics, and immune infiltration. Gene set enrichment analysis (GSEA) was used to discover the signaling pathways associated with TAP1, while STRING database and CytoHubba were used to construct protein-protein interaction (PPI) and competing endogenous RNA (ceRNA) networks, respectively. An overall survival (OS) prognostic model was constructed to test the predictive efficacy of TAP1, and its effect on the in vitro proliferation activity and metastatic potential of UVM cell line C918 cells was verified by RNA interference. RESULTS: There was a clear association between TAP1 expression and UVM patient prognosis. Upregulated TAP1 was strongly associated with a shorter survival time, higher likelihood of metastasis, and higher mortality outcomes. According to GSEA analysis, various immunity-related signaling pathways such as primary immunodeficiency were enriched in the presence of elevated TAP1 expression. A PPI network and a ceRNA network were constructed to show the interactions among mRNAs, miRNAs, and lncRNAs. Furthermore, TAP1 expression showed a significant positive correlation with immunoscore, stromal score, CD8+ T cells, and dendritic cells, whereas the correlation with B cells and neutrophils was negative. The Cox regression model and calibration plots confirmed a strong agreement between the estimated OS and actual observed patient values. In vitro silencing of TAP1 expression in C918 cells significantly inhibited cell proliferation and metastasis. CONCLUSIONS: This study is the first to demonstrate that TAP1 expression is positively correlated with clinicopathological factors and poor prognosis in UVM. In vitro experiments also verified that TAP1 is associated with C918 cell proliferation, apoptosis, and metastasis. These results suggest that TAP1 may function as an oncogene, prognostic marker, and importantly, as a novel therapeutic target in patients with UVM.

The inhibition of p38 MAPK blocked inflammation to restore the functions of rat meibomian gland epithelial cells.

Meibomian glands (MGs) are vital for ocular surface health. However, the roles of inflammation in the progression of meibomian gland dysfunction (MGD) are largely unknown. In this study, the roles of the inflammation factor interleukin-1ß (IL-1ß) via the p38 mitogen-activated protein kinases (MAPK) signaling pathway on rat meibomian gland epithelial cells (RMGECs) were explored. Eyelids from adult rat mice at 2 months and 2 years of age were stained with specific antibodies against IL-1ß to identify inflammation levels. RMGECs were exposed to IL-1ß and/or SB203580, a specific inhibitor of p38 MAPK signaling pathway, for 3 days. Cell proliferation, keratinization, lipid accumulation, and matrix metalloproteinases 9 (MMP9) expression were evaluated by MTT assay, polymerase chain reaction (PCR), immunofluorescence staining, apoptosis assay, lipid staining, and Western blot analyses. We found that IL-1ß was significantly higher in the terminal ducts of MGs in rats with age-related MGD than in young rats. IL-1ß inhibited cell proliferation, suppressed lipid accumulation and peroxisome proliferator activator receptor γ (PPARγ) expression, and promoted apoptosis while activating the p38 MAPK signaling pathway. Cytokeratin 1 (CK1), a marker for complete keratinization, and MMP9 in RMGECs were also up-regulated by IL-1ß. SB203580 effectively diminished the effects of IL-1ß on differentiation, keratinization, and MMP9 expression by blocking IL-1ß-induced p38 MAPK activation, although it also inhibited cell proliferation. The inhibition of the p38 MAPK signaling pathway blocked IL-1ß-induced differentiation reduction, hyperkeratinization, and MMP9 overexpression of RMGECs, which provides a potential therapy for MGD.

Prostaglandin F2α Regulates Adipogenesis by Modulating Extracellular Signal-Regulated Kinase Signaling in Graves' Ophthalmopathy.

Prostaglandin F2α (PGF2α), the first-line anti-glaucoma medication, can cause the deepening of the upper eyelid sulcus due to orbital lipoatrophy. However, the pathogenesis of Graves' ophthalmopathy (GO) involves the excessive adipogenesis of the orbital tissues. The present study aimed to determine the therapeutic effects and underlying mechanisms of PGF2α on adipocyte differentiation. In this study primary cultures of orbital fibroblasts (OFs) from six patients with GO were established. Immunohistochemistry, immunofluorescence, and Western blotting (WB) were used to evaluated the expression of the F-prostanoid receptor (FPR) in the orbital adipose tissues and the OFs of GO patients. The OFs were induced to differentiate into adipocytes and treated with different incubation times and concentrations of PGF2α. The results of Oil red O staining showed that the number and size of the lipid droplets decreased with increasing concentrations of PGF2α and the reverse transcription-polymerase chain reaction (RT-PCR) and WB of the peroxisome proliferator-activated receptor γ (PPARγ) and fatty-acid-binding protein 4 (FABP4), both adipogenic markers, were significantly downregulated via PGF2α treatment. Additionally, we found the adipogenesis induction of OFs promoted ERK phosphorylation, whereas PGF2α further induced ERK phosphorylation. We used Ebopiprant (FPR antagonist) to interfere with PGF2α binding to the FPR and U0126, an Extracellular Signal-Regulated Kinase (ERK) inhibitor, to inhibit ERK phosphorylation. The results of Oil red O staining and expression of adipogenic markers showed that blocking the receptor binding or decreasing the phosphorylation state of the ERK both alleviate the inhibitory effect of PGF2a on the OFs adipogenesis. Overall, PGF2α mediated the inhibitory effect of the OFs adipogenesis through the hyperactivation of ERK phosphorylation via coupling with the FPR. Our study provides a further theoretical reference for the potential application of PGF2α in patients with GO.

Limbal niche cells can reduce the angiogenic potential of cultivated oral mucosal epithelial cells.

BACKGROUND: Autologous cultivated oral mucosal epithelial transplantation (COMET) is an important treatment for limbal stem cell deficiency. However, peripheral corneal neovascularization after surgery hinders its application. This study aims to employ a culture system using allogenic limbal niche cells (LNCs) instead of mouse-derived 3T3 cells as a feeder layer that could relieve postoperative neovascularization. METHODS: Rat oral mucosal epithelial cells (OMECs) were co-cultured with rat LNCs or 3T3 cells. Cultivated oral mucosal epithelial cells (COMECs) of different culture systems were identified by hematoxylin and eosin staining and immunocytochemistry. The expression levels of the angiogenesis-related factors were analyzed by RT-qPCR and western blotting/ELISA. Angiogenic potential was reconfirmed by cell viability and tube formation assays with human umbilical vein endothelial cells (HUVECs). RESULTS: COMECs were obtained from both culture systems successfully. Immunocytochemistry showed approximately equal percentages of positive staining cells for p63α (p = 0.9177), ABCG2 (p = 0.526), Ki67 (p = 0.0987), and CK3 (p = 0.4000) in COMECs of different groups. RT-qPCR and western blotting/ELISA showed that COMECs of the LNC group expressed a significantly lower amount of basic fibroblast growth factor (bFGF) (p = 0.0038 for RT-qPCR, p = 0.0026 for western blotting) but more pigment epithelium-derived factor (PEDF) (p = 0.0172 for RT-qPCR, p = 0.0253 for western blotting) and soluble fms-like tyrosine kinase-1 (sFlt-1) (p < 0.0001 for RT-qPCR, p = 0.0064 for ELISA) than the COMECs of the 3T3 group. Furthermore, compared with COMECs of the 3T3 group, COMECs of the LNC group could reduce the viability (p = 0.0002) and tube formation (p = 0.0002) of HUVECs. CONCLUSIONS: LNCs could substitute 3T3 cells for expanding OMECs in vitro, and the COMECs obtained in this system are less likely to induce postsurgical neovascularization, which provides an alternative option for an ex vivo culture system and promotes the application of COMET.

p70S6K activation promotes the transdifferentiation of fibroblasts to myofibroblasts in pterygium tissue growth on the cornea.

OBJECTIVE: To investigate the role of p70S6K in the transdifferentiation of fibroblasts to myofibroblasts in pterygium tissue growth on the cornea. RESULTS: Quantitative real-time PCR and immunohistochemistry showed that p70S6K expression was higher in pterygium tissues than in normal conjunctival tissues. Higher p70S6K RNA expression levels were correlated with higher pterygium grades. Additionally, western blot analysis revealed that phosphorylated (activated) p70S6K (p-p70S6K) expression was significantly correlated with α-smooth muscle actin (α-SMA, a hallmark of transdifferentiation) expression in cultured human pterygium fibroblasts (HPFs). Furthermore, p70S6K knockdown and the specific mTOR inhibitor rapamycin decreased the expression levels of p-p70S6K and α-SMA in cultured fibroblasts from grade T3 pterygium. CONCLUSIONS: p70S6K activation promotes the transdifferentiation of pterygium fibroblasts to myofibroblasts. Thus, targeting p70S6K may be a useful strategy in the management of pterygium.

Pharmacokinetics and retinal toxicity of various doses of intravitreal triamcinolone acetonide in rabbits.

PURPOSE: To compare the pharmacokinetics and retinal toxicity of various doses of intravitreal triamcinolone acetonide (TA) in rabbits. METHODS: The rabbits received intravitreal injections of 4 mg and 8 mg TA. The drug concentrations were determined with high-performance liquid chromatography after extraction from the vitreous at various time points. The main pharmacokinetics parameters were calculated with 3p97 pharmacokinetics software. The intraocular pressure, electroretinography, and pathological examinations were evaluated before and after intravitreal injection of different doses of TA. RESULTS: The half-life of intravitreal injection of 4 mg and 8 mg TA was 24 days and 34 days, respectively. No significant differences were found in intraocular pressure (p>0.05) and the electroretinography b-wave amplitudes (p>0.05) among the rabbits before and after intravitreal injection of 4 mg and 8 mg TA. Light and electron microscopy did not show any retinal damage in any group. CONCLUSIONS: Intravitreal injection of 4 mg and 8 mg TA are safe for the rabbit retina. The injection of 8 mg TA produced a longer vitreous half-life and had a prolonged effect on the retina. This conclusion may be referenced in the clinical application of TA in retinal diseases.

Anterior Segment Biometry During Accommodation After Posterior Chamber Phakic Implantable Collamer Lens Implantation.

PURPOSE: To evaluate the dynamic changes in anterior segment parameters during accommodation following Implantable Collamer Lens (ICL) implantation with swept-source optical coherence tomography (SS-OCT). METHODS: Under the accommodation of 0.00 diopters (D), 3.00 D, and maximum amplitude, SS-OCT was used to examine the anterior segment parameters, including ICL vault, ICL depth (the distance between the corneal endothelium and the posterior surface of ICL), crystalline lens thickness, anterior chamber depth, and various parameters of the anterior chamber angle, comprising angle opening distance, angle recess area, trabecular iris space area, and trabecular iris angle. RESULTS: During accommodation, the ICL vault showed a significant decrease from baseline (536 ± 278 µm) to 3.00 D (522 ± 281 µm), followed by an increase from 3.00 D to maximum amplitude (548 ± 306 µm) (analysis of variance [ANOVA], P < .001). Four eyes (2.61%) exhibited a decrease in ICL vault to less than 100 µm (47 ± 32 µm) at maximum accommodation. The ICL depth decreased significantly as accommodation increased (ANOVA, P < .001). Crystalline lens thickness increased, whereas anterior chamber depth decreased during accommodation (ANOVA, P < .001). The anterior chamber angle widened during 3.00 D of accommodation but narrowed at maximum accommodation, leading to significant changes in the angle opening distance, angle recess area, trabecular iris space area, and trabecular iris angle during accommodation (ANOVA, P < .001 for all). CONCLUSIONS: The anterior segment, including ICL vault and anterior chamber angle, undergo significant dynamic changes during accommodation. These accommodative changes may require careful monitoring for the surgery design of ICL implantation. [J Refract Surg. 2024;40(3):e164-e172.].

Differential expression of antimicrobial peptides in human fungal keratitis.

AIM: To analyze a series of antimicrobial peptides (AMPs) in corneal tissue from individuals with fungal keratitis (FK) during the active phase of the fungus infection and after healing. METHODS: Patients undergone lamellar keratoplasty for the treatment of severe FK or corneal scar had their corneal buttons sampled. Quantitative real-time polymerase chain reaction (PCR) was used to ascertain the gene expression of human beta-defensin (HBD)-1, -2, -3, -9, S100A7, 8, 9, and LL-37. RESULTS: All AMPs' messenger ribonucleic acid (mRNA) expression was considerably elevated in all samples (n=12). In contrast to controls, where HBD-2, -3, and S100A7 mRNAs were expressed at very low levels, it was discovered that HBD-1, -9, S100A8, S100A9, and LL-37 were constitutively expressed in all healed samples (n=4). HBD-1, -2 -3, S100A7, and LL-37 mRNAs were significantly increased in all active FK samples (n=8). The levels of HBD-9, S100A8, and S100A9 mRNAs were moderately upregulated in all active FK samples. Subgroup comparison showed that HBD-2 was significantly increased in Fusarium keratitis samples (n=5), and LL-37 mRNAs were significantly enhanced in Aspergillus keratitis samples (n=3). Whereas there was not significantly increased of HBD-1, -3, -9, S100A7, 8, 9 mRNA in Aspergillus keratitis samples compared with Fusarium keratitis samples. CONCLUSION: AMPs expression is increased in active FK, but not all AMPs are equally expressed. HBD-2 and LL-37 expression levels are the highest, showing some specificity of AMP expression related to FK. Human AMPs, particularly HBD-2 may play a significant role in Fusarium keratitis and LL-37 might be the key player in Aspergillus keratitis.

Conjunctival Limbal Autograft Combined with Amnion-Assisted Conjunctival Epithelial Redirection for Unilateral Total Limbal Stem Cell Deficiency after Severe Chemical Burn.

(1) Background: To evaluate the efficacy of conjunctival limbal autograft (CLAU) combined with the amnion-assisted conjunctival epithelial redirection (ACER) procedure for patients with unilateral total limbal stem cell deficiency (LSCD) caused by severe chemical burn. (2) Methods: A retrospective interventional case series of unilateral total LSCD after chemical burn who underwent CLAU combined with ACER surgery between September 2021 and July 2023 was collected. Outcome measures included epithelialization of the cornea with donor limbus-derived epithelium, best corrected visual acuity (BCVA), and complications. (3) Results: Nine males and one female were included in this study. The mean age was 40.9 ± 9.63 (range, 26 to 55) years. The average duration between injury and CLAU combined with the ACER procedure was 7.67 ± 3.97 (range, 4 to 18) months. All patients achieved corneal epithelialization and improved BCVA. Postoperative complications occurred in four cases, including delayed corneal epithelial healing in one case, delayed amniotic membrane dissolution and detachment in two cases, and recurrence of symblepharon in one case. No complications were noted in the healthy donor eyes. (4) Conclusions: CLAU combined with ACER is a safe and effective treatment for unilateral total LSCD caused by severe chemical burn. This combined surgery restores visual function for patients with corneal blindness caused by chemical burn, reducing the burden on the families and society.

Two-step strategy-conjunctival flap covering surgery combined with secondary deep anterior lamellar keratoplasty for the treatment of high-risk fungal keratitis.

AIM: To investigate whether the two-step strategy [conjunctival flap covering surgery (CFCS) combined with secondary deep anterior lamellar keratoplasty (DALK)] is effective for patients with high-risk fungal keratitis (FK). METHODS: In this noncomparative, retrospective case series, 10 subjects (6 males, 4 females) with a mean age of 56.5±7.1 (range 47-72)y with high-risk FK undergone the two-step strategy were included. Reported outcome measures were healing of the corneal ulcer, recurrence of FK, reject reaction, improvement in best corrected visual acuity (BCVA) and relevant complications. RESULTS: The average diameter of corneal infiltrates was 7.50±0.39 mm, ranging from 6.94 to 8.13 mm. The mean depth of corneal infiltrates was 422.4±77.1 µm, ranging from 350 to 535 µm. The mean corneal thickness was 597.4±117.3 µm, ranging from 458 to 851 µm. Hypopyon and endothelial plaques were presented in all patients. The period between the two steps was 3.65±0.9 (ranging from 3 to 5)mo. The graft diameter was 7.75±0.39 mm. At the last follow-up (average 9.25±3.39, ranging from 5.5 to 17mo), no fungal recurrence or graft rejection appeared, and all patients showed improvement of BCVA. One patient suffered from liver function impairment due to oral voriconazole for 4wk and recovered spontaneously after 1wk of drug withdrawal. CONCLUSION: The two-step strategy is safe and effective in the treatment of high-risk FK by transforming intentional therapeutic penetrating keratoplasty during acute infection to later optical DALK. It is a practical strategy, especially in areas lacking fresh donor corneas and eye bank services.

Characteristics of In Vitro Culture and In Vivo Confocal Microscopy in Patients with Fungal Keratitis in a Tertiary Referral Hospital in Central China.

PURPOSE: To investigate the characteristics of in vitro culture and in vivo confocal microscopy (IVCM) in patients with fungal keratitis (FK) presented in a tertiary referral hospital in central China. METHODS: In this noncomparative retrospective study, patients with the diagnosis of FK between October 2021 and November 2022 were reviewed. An IVCM and fungal culture (corneal scraping specimens) were performed, and the characteristics were analyzed. RESULTS: During October 2021 and November 2022, 85 patients were diagnosed with FK. From 63 culture-positive cases, 8 species of fungus were identified. The proportions of isolated fungal species were Fusarium and Aspergillus equally accounting for 33.3% (21 of 63), Alternaria 9.5% (6 of 63), Curvularia 6.3% (4 of 63), Scedosporium apiospermum 6.3% (4 of 63), Paecilomyces lilacinus 3.2% (2 of 63), Exserohilum 3.2% (2 of 63), and Candida 4.8% (3 of 63), respectively. In positive culture cases, IVCM was found to be positive for hyphae or spores in 61 of 63 patients (96.8%). Different fungal species had a variety of cultural characteristics and IVCM manifestations. CONCLUSIONS: In a tertiary referral hospital in central China, Fusarium species, Aspergillus species, and Alternaria species were the 3 most common isolated fungal pathogens, and the proportion of Aspergillus species was significantly higher than that in other regions of China. Careful lesion depth examination by IVCM and OCT should be taken before lamellar keratoplasty to avoid postoperative recurrence. Identifying the IVCM image and culture characteristics will facilitate rapid diagnosis and proper treatment, but IVCM cannot yet replace fungal cultures to distinguish between different fungal species.

Botulinum Neurotoxin Type a Injection Combined with Absorbable Punctal Plug Insertion: An Effective Therapy for Blepharospasm Patients with Dry Eye.

Blepharospasm patients often have dry eye manifestations. Botulinum neurotoxin type A (BoNT-A) injection has been the main management for blepharospasm and absorbable punctal plug (APP) insertion is shown to be effective in the treatment of dry eye. However, there have been no studies investigating the combined treatment of BoNT-A and APP in blepharospasm patients with dry eye. In this retrospective study, 17 blepharospasm patients with dry eye treated by BoNT-A injection and 12 receiving BoNT-A plus APP treatment were enrolled. The efficacy was evaluated according to the Jankovic rating scale, Ocular Surface Disease Index (OSDI), fluorescein staining (FL), fluorescein tear break-up time (FBUT) and Schirmer I test (SIT). Both BoNT-A and BoNT-A+APP treatment effectively reduced the functional impairment of blepharospasm. At baseline, all the patients had high OSDI scores (BoNT-A group: 82.48 ± 7.37, BoNT-A+APP group: 78.82 ± 4.60, p = 0.112), but relatively low degrees of FL (BoNT-A group: 3.18 ± 1.01, BoNT-A+APP group: 3.50 ± 1.24, p = 0.466), FBUT (BoNT-A group: 1.71 ± 0.77, BoNT-A+APP group: 2.17 ± 0.58, p = 0.077) and SIT (BoNT-A group: 2.53 ± 0.99, BoNT-A+APP group: 3.17 ± 1.23, p = 0.153). After treatment, OSDI, FL, FBUT and SIT were all obviously restored in the two groups. When comparing the changing rates, only OSDI (BoNT-A group: -52.23% ± 15.57%, BoNT-A+APP group: -61.84% ± 9.10%, p = 0.047) and FL (BoNT-A group: -22.55% ± 25.98%, BoNT-A+APP group: -41.94% ± 14.46%, p = 0.016) showed significant differences between the two groups. This study suggests that OSDI is not applicable in the diagnosis of dry eye among blepharospasm patients. For blepharospasm patients with severe dry eye symptoms, especially those with fluorescein staining in the cornea, the combined treatment of BoNT-A and APP is more effective than using BoNT-A alone.

Amniotic membrane transplantation combined with conjunctival flap covering surgery for the treatment of corneal perforations in fungal keratitis.

Purpose: To investigate the efficiency of amniotic membrane transplantation (AMT) combined with conjunctival flap covering surgery (CFCS) for patients with corneal perforations in fungal keratitis (FK). Methods: In this non-comparative, retrospective case series, 16 participants of corneal perforation in FK were successfully treated by a combination of multilayer AMT and bipedicle conjunctival flap with partial tenon's capsule. Corneal healing, recurrence of FK, visual acuity, and relevant complications were reported as outcome measures. Results: Sixteen patients (13 male, 3 female) had a mean age of 58.8 ± 10.3 (range 29-72) years. The mean diameter of corneal perforation was 1.9 ± 0.7 (range 0.5-2.8) mm. Corneal perforations healed and all the patients preserved their eyeballs. During the 11.0 ± 4.4 (range 6-18) months of follow-up, there was no recurrence of FK in any of these cases. Visual acuity improved in 15 eyes (93.8 %) and remained unchanged in 1 patient (6.3 %) who had no light perception when first admitted. All 6 patients who accepted secondary keratoplasty showed improved best corrected visual acuity of more than 4 lines. The most frequently found fungi were Aspergillus species (6 of 16, 37.5 %) and Fusarium species (4 of 16, 25.0 %), followed by 1 Scedosporium apiospermum (1 of 16, 6.3 %). Conclusions: Combination AMT with CFCS is a safe and effective surgery for patients with corneal perforations in FK, particularly where eye banks and fresh corneas are not available. This surgery could preserve the integrity of the eyeball and avoid the recurrence of FK. Besides, it provides a greater opportunity for further optical keratoplasty.

Isolation and Culture of Human Meibomian Gland Ductal Cells.

Purpose: Hyperkeratinization of meibomian gland (MG) ducts is currently recognized as the primary pathologic mechanism of meibomian gland dysfunction (MGD). This research figured out a method to isolate the MG ducts and established a novel system to culture the human meibomian gland ductal cells (HMGDCs) for investigating the process of MGD. Methods: The MG ducts were obtained from the eyelids of recently deceased donors and subjected to enzymatic digestion. The acini were then removed to isolate independent ducts. These MG ducts were subsequently cultivated on Matrigel-coated wells and covered with a glass plate to obtain HMGDCs. The HMGDCs were further cultivated until passage 2, and when they reached 60% confluence, they were treated with IL-1ß and rosiglitazone for a duration of 48 hours. Immunofluorescence staining and Western blot techniques were employed to identify ductal cells and analyze the effects of IL-1ß on HMGDCs in an in vitro setting. Results: Ophthalmic micro-forceps and insulin needles can be employed for the purpose of isolating ducts. Within this particular culture system, the rapid expansion of HMGDCs occurred in close proximity to the duct tissue. MG ducts specifically expressed keratin 6 (Krt6) and hardly synthesized lipids. Furthermore, the expression of Krt6 was significantly higher (P < 0.0001) in HMGDCs compared to human meibomian gland cells. Upon treatment with IL-1ß, HMGDCs exhibited an overexpression of keratin 1, which was effectively blocked by the administration of rosiglitazone. Conclusions: The present study successfully isolated human MG ducts and cultured HMGDCs, providing a valuable in vitro model for investigating the mechanism of MGD. Additionally, the potential therapeutic efficacy of rosiglitazone in treating hyperkeratinization of ducts in patients with MGD was identified.

Limbal epithelial stem/progenitor cells attract stromal niche cells by SDF-1/CXCR4 signaling to prevent differentiation.

Corneal epithelial stem cells (SCs) are an ideal model for investigating how adult lineage-committed epithelial SCs are regulated by an anatomically defined and accessible niche, that is, limbal palisades of Vogt, located between the cornea and the conjunctiva. We have used collagenase digestion to isolate the entire limbal epithelial SCs and subjacent mesenchymal cells, and we have demonstrated that their close association is crucial for promoting epithelial clonal growth, implying that the latter serves as niche cells (NCs). After their close association was disrupted by trypsin/EDTA, single SCs and NCs could reunite to generate sphere growth in three-dimensional Matrigel in the embryonic SC medium, and that such sphere growth initiated by SC-NC reunion was mediated by SDF-1 uniquely expressed by limbal epithelial progenitor cells and its receptor CXCR4, but not CXCR7, strongly expressed by limbal stromal NCs. Inhibition of CXCR4 by AMD3100 or a blocking antibody to CXCR4 but not CXCR7 disrupted their reunion and yielded separate spheres with a reduced size, while resultant epithelial spheres exhibited more corneal differentiation and a notable loss of holoclones. For the first time, these results provide strong evidence supporting that limbal SC function depends on close physical association with their native NCs via SDF-1/CXCR4 signaling. This novel in vitro model of sphere growth with NCs can be used for investigating how limbal SC self-renewal and fate decision might be regulated in the limbal niche.

Topical Chemotherapy for Treating Ocular Surface Squamous Neoplasia with a Combination of Interferon α-2b and 5-Fluorouracil.

INTRODUCTION: This study evaluated the efficacy of combined interferon α-2b (IFNα2b) and 5-fluorouracil (5-FU) as primary treatment for ocular surface squamous neoplasia (OSSN). METHODS: In this retrospective study, 27 eyes with OSSN followed by topical application of combined IFNα2b and 5-FU were examined. Reported outcome measures were tumor response, visual acuity, time to complete resolution, recurrence and treatment complications. RESULTS: Twenty-six patients (17 male, 9 female) had a mean age of 63.9 (median, 67; range 22-83) years. Complete tumor response was observed in 24 eyes (88.9%). Three eyes (11.1%) showed partial response to the chemotherapy agents and later underwent surgical tumor removal. The median time to complete resolution was 6 (mean, 6.1; range, 3-11) weeks. Of these, the patients received between one to three cycles of 5-FU therapy (median, 2; mean, 1.8). Complications noted were transient irritation at 5-FU cycle (11 eyes, 40.7%). There was no tumor recurrence at mean follow-up of 16.1 (median, 12; range 6-38) months. CONCLUSIONS: Combination therapy of IFNα2b and 5-FU was a safe and effective treatment, inducing a short duration of administration and low recurrence rate for OSSN. TRIAL REGISTRATION: Retrospectively registered, UHCT22048.

Ten-Year Follow-up of Lamellar Keratoplasty Treatment With Acellular Porcine Corneal Stroma: A Case Report.

PURPOSE: To report a 10-year follow-up case of the first lamellar keratoplasty treatment with acellular porcine corneal stroma (APCS). METHODS: A 62-year-old woman was diagnosed with a fungal corneal ulcer and received lamellar keratoplasty treatment with APCS in 2010. The 10-year follow-up results were evaluated by slit lamp biomicroscopy, anterior segment optical coherence tomography, in vivo confocal microscopy, and corneal biomechanics analysis. RESULTS: The APCS graft maintained good biocompatibility and physical properties in transparency, stromal regeneration, elasticity, and deformation resistance. However, some disadvantages were observed, including a protracted course to eventual clearing, a decreased thickness, corneal depositions, sparsely distributed neural fibers, and low stiffness. CONCLUSIONS: This case indicated that APCS remains stable over a 10-year follow-up period. APCS can serve as a functional stromal surrogate where donor human corneal tissue is unavailable.

Sandwich (Amnion/Conjunctival-Limbal Autograft/Amnion) Transplantation for Recurrent Pterygium with Restrictive Strabismus.

(1) Background: This study aimed to evaluate the clinical outcome of Sandwich (Amnion/Conjunctival-Limbal Autograft/Amnion) transplantation for recurrent pterygium with restrictive strabismus. (2) Methods: This retrospective study included 11 eyes in 11 patients diagnosed with recurrent pterygium with restrictive strabismus who received sandwich transplantation. The outcomes were measured by pterygium recurrence, best-corrected visual acuity, esotropia (prism diopters), and treatment complications. (3) Results: Eleven patients (six males, five females) had a mean age of 60.5 (range 36-80) years. The previously received pterygium excision surgery number was 1.8 ± 1.02 (range 1-4). The mean follow-up period was 19.9 ± 8.41 (range 12-36) months. All patients had a restriction of abduction in the previously operated eye, causing esotropia in the primary position. Pre-operative esotropia was 17.2 (range 10-30) prims diopter (PD). Five eyes (45.5%) had symblepharon before surgery. All patients were orthotropic until the last follow-up. Symblepharon was released in all eyes. Free ocular motility was present in all eyes. No donor site scar formation, scleral melt, or corneal ulcer was noted. (4) Conclusions: Sandwich transplantation for recurrent pterygium with restrictive strabismus is safe and effective.