Scleral bridging technique for preventing PreserFlo microshunt exposure: A case report.

INTRODUCTION: The use of the PreserFlo microshunt is gaining popularity owing to its ease of implantation and reduced need for postoperative intervention compared to conventional trabeculectomy. PATIENT CONCERNS: However, microshunt exposure remains a severe complication of PreserFlo surgery, particularly in patients with a thin Tenon capsule and conjunctiva. However, the actual thickness and intensity of the Tenon capsule or conjunctiva can be confirmed only during surgery. DIAGNOSIS: Exfoliation glaucoma with previous several glaucoma surgeries with thinner Tenon capsule or conjunctiva. INTERVENTIONS: We performed PreserFlo implantation with a surgical technique to recover a thin Tenon capsule and conjunctiva by creating a half-thickness rectangular scleral flap under the shunt and covering it over the microshunt until the distal part, similar to the bridge. OUTCOMES: The patient had better intraocular pressure control with positive cosmetic appearance using this technique. CONCLUSION: This technique will be beneficial for both preventing exposure and holding down the top, in addition to improving cosmetic appearance.

Inhibition of the rapamycin-insensitive mTORC1 /4E-BP1 axis attenuates TGF-ß1-induced fibrotic response in human Tenon's fibroblasts.

Subconjunctival fibrosis is the major cause of failure in both conventional and modern minimally invasive glaucoma surgeries (MIGSs) with subconjunctival filtration. The search for safe and effective anti-fibrotic agents is critical for improving long-term surgical outcomes. In this study, we investigated the effect of inhibiting the rapamycin-insensitive mTORC1/4E-BP1 axis on the transforming growth factor-beta 1(TGF-ß1)-induced fibrotic responses in human Tenon's fibroblasts (HTFs), as well as in a rat model of glaucoma filtration surgery (GFS). Primary cultured HTFs were treated with 3 ng/mL TGF-ß1 for 24 h, followed by treatment with 10 µM CZ415 for additional 24 h. Rapamycin (10 µM) was utilized as a control for mTORC1/4E-BP1 signaling insensitivity. The expression levels of fibrosis-associated molecules were measured using quantitative real-time PCR, Western blotting, and immunofluorescence analysis. Cell migration was assessed through the scratch wound assay. Additionally, a rat model of GFS was employed to evaluate the anti-fibrotic effect of CZ415 in vivo. Our findings indicated that both rapamycin and CZ415 treatment significantly reduced the TGF-ß1-induced cell proliferation, migration, and the expression of pro-fibrotic factors in HTFs. CZ415 also more effectively inhibited TGF-ß1-mediated collagen synthesis in HTFs compared to rapamycin. Activation of mTORC1/4E-BP signaling following TGF-ß1 exposure was highly suppressed by CZ415 but was only modestly inhibited by rapamycin. Furthermore, CZ415 was found to decrease subconjunctival collagen deposition in rats post GFS. Our results suggest that rapamycin-insensitive mTORC1/4E-BP1 signaling plays a critical role in TGF-ß1-driven collagen synthesis in HTFs. This study demonstrated that inhibition of the mTORC1/4E-BP1 axis offers superior anti-fibrotic efficacy compared to rapamycin and represents a promising target for improving the success rate of both traditional and modern GFSs.

MiR-146a reduces fibrosis after glaucoma filtration surgery in rats.

PURPOSE: To explore the impact of microRNA 146a (miR-146a) and the underlying mechanisms in profibrotic changes following glaucoma filtering surgery (GFS) in rats and stimulation by transforming growth factor (TGF)-ß1 in rat Tenon's capsule fibroblasts. METHODS: Cultured rat Tenon's capsule fibroblasts were treated with TGF-ß1 and analyzed with microarrays for mRNA profiling to validate miR-146a as the target. The Tenon's capsule fibroblasts were then respectively treated with lentivirus-mediated transfection of miR-146a mimic or inhibitor following TGF-ß1 stimulation in vitro, while GFS was performed in rat eyes with respective intraoperative administration of miR-146a, mitomycin C (MMC), or 5-fluorouracil (5-FU) in vivo. Profibrotic genes expression levels (fibronectin, collagen Iα, NF-KB, IL-1ß, TNF-α, SMAD4, and α-smooth muscle actin) were determined through qPCR, Western blotting, immunofluorescence staining and/or histochemical analysis in vitro and in vivo. SMAD4 targeting siRNA was further used to treat the fibroblasts in combination with miR-146a intervention to confirm its role in underlying mechanisms. RESULTS: Upregulation of miR-146a reduced the proliferation rate and profibrotic changes of rat Tenon's capsule fibroblasts induced by TGF-ß1 in vitro, and mitigated subconjunctival fibrosis to extend filtering blebs survival after GFS in vivo, where miR-146a decreased expression levels of NF-KB-SMAD4-related genes, such as fibronectin, collagen Iα, NF-KB, IL-1ß, TNF-α, SMAD4, and α-smooth muscle actin(α-SMA). Additionally, SMAD4 is a key target gene in the process of miR-146a inhibiting fibrosis. CONCLUSIONS: MiR-146a effectively reduced TGF-ß1-induced fibrosis in rat Tenon's capsule fibroblasts in vitro and in vivo, potentially through the NF-KB-SMAD4 signaling pathway. MiR-146a shows promise as a novel therapeutic target for preventing fibrosis and improving the success rate of GFS.

Key Roles of Conjunctiva Fornix-Bulbar Conjunctiva-Tenon Capsule in Conjoint Fascial Sheath Suspension.

BACKGROUND: Bulbar conjunctival prolapse is one of the complications of conjoint fascial sheath (CFS) suspension and has a negative impact on surgical results. To explore the prevention methods of this complication, the authors compared the incidence of it between the below-conjunctiva fornix-bulbar conjunctiva-Tenon capsule (CBT) approach and the above-CBT approach to dissecting CFS in CFS suspension and shared their experience in the treatment of bulbar conjunctival prolapse. METHODS: From January of 2020 to August of 2021, 81 patients with severe congenital ptosis who underwent CFS suspension were enrolled and divided into two groups. Forty-five patients' (group A) CFS was dissected by means of the below-CBT approach and 36 patients' (group B) CFS was dissected by means of the above-CBT approach. Data regarding the incidence and outcomes of bulbar conjunctival prolapse and the postoperative condition were collected and analyzed. RESULTS: The incidence of bulbar conjunctival prolapse was 24.44% in group A and 2.78% in group B. Of the 12 bulbar conjunctival prolapse patients, seven patients' conditions improved after conservative treatment, and five did not. All of them underwent bulbar conjunctiva resection within 1 year and were cured. No recurrent prolapse was observed within 3 months postoperatively. At the last follow-up, the mean marginal reflex distance 1 and palpebral fissure height were 4.09 ± 0.19 mm and 9.85 ± 0.62 mm, respectively. There were no complications except lagophthalmos (16 eyelids), asymmetric eyelid contour (one patient), and trichiasis (two eyelids). CONCLUSIONS: The incidence of bulbar conjunctival prolapse decreased significantly by dissecting CFS by means of the above-CBT approach. For patients with bulbar conjunctival prolapse after CFS suspension, bulbar conjunctiva resection could provide satisfactory results. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Inhibition of heat shock protein 47 suppressed collagen production in Tenon's capsule fibroblasts.

INTRODUCTION: Glaucoma is the leading cause of irreversible blindness worldwide, and conjunctival bleb scarring remains the most frequent reason for the failure of glaucoma filtration surgery. Excessive proliferation of fibroblasts from Tenon's capsule and excessive deposition of collagen contribute to the scarification of the conjunctival bleb. Heat shock protein 47 (HSP47) is assumed to act as a collagen-specific molecular chaperone, and thereby involved in the pathogenesis of fibrotic diseases. Therefore, we investigated the effect of HSP47 knockout against collagen type I (COLI) production in rat tenon's fibroblasts. MATERIAL AND METHODS: Newborn rat tenon's fibroblasts were cultured and verified by anti-vimentin antibody. Transfection efficiency of small interference RNA targeted against HSP47 was confirmed by quantitative real-time polymerase chain reaction (RT-qPCR) at 48 h after siRNA transfection and by western blot at 72 h after transfection. The mRNA and protein expression of HSP 47 and COLI were detected by RT-qPCR and western blot. The proliferation of cells was measured by cell counting kit-8 assay. RESULTS: HSP47 siRNA down-regulated the mRNA and protein levels of HSP47 in rat Tenon's fibroblasts, and suppressed the mRNA and protein expression of COLI. Moreover, HSP47 siRNA had no significant effect on proliferation of rat Tenon's fibroblasts. CONCLUSIONS: HSP47 siRNA inhibits the production of COLI in rat Tenon's fibroblasts, and may be the potential therapeutic method in bleb scarring after glaucoma filtration surgery.

Niclosamide inhibits TGF-ß1-induced fibrosis of human Tenon's fibroblasts by regulating the MAPK-ERK1/2 pathway.

Preventing postoperative bleb scar formation is an effective way of improving glaucoma filtration surgery (GFS) outcome. Use of more effective antifibrotic drugs with fewer adverse effects may be a good way to address the problem. In the present study, we use a primary cell model, consisting of Tenon's fibroblasts obtained from patients with glaucoma, which were stimulated with TGF-ß1 to induce the fibrotic phenotype. We explored the effects of niclosamide on TGF-ß1-induced fibrosis in these cells and examined its underlying mechanism of action. A transcriptome sequencing assay was used to explore possible signaling pathways involved. Niclosamide inhibited cell proliferation and migration, and decreased the levels of alpha-smooth muscle actin, type I and type III collagen in human Tenon's fibroblasts induced by TGF-ß1. Niclosamide also induced apoptosis and counteracted TGF-ß1-induced cytoskeletal changes and extracellular matrix accumulation. Moreover, niclosamide decreased TGF-ß1-induced phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) protein expression in human Tenon's fibroblasts. The results indicate that niclosamide inhibits TGF-ß1-induced fibrosis in human Tenon's fibroblasts by blocking the MAPK-ERK1/2 signaling pathway. Thus, niclosamide is a potentially promising antifibrotic drug that could improve glaucoma filtration surgery success rate.

Effects of atorvastatin on the function of Tenon's capsule fibroblasts in human eyes.

PURPOSE: This study aimed to explore the role of atorvastatin (ATO) in the prevention and treatment of the scarring of filtration channels after glaucoma surgery. METHODS: Human Tenon's capsule fibroblasts (HTFs) were co-cultured with various concentrations of ATO. First, Cell Counting Kit-8 assay was performed to evaluate the effects of various concentrations of ATO on the viability of HTFs. Then, after the ATO stimulated the HTFs for 24 h, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to evaluate the apoptosis of HTFs. Transwell assay was also performed to evaluate the migration of HTFs. Moreover, enzyme-linked immunosorbent assay (ELISA) was performed to detect the protein expression levels of transforming growth factor-ß1 (TGF-ß1) and TGF-ß2 in the cell culture supernatant of HTFs. Western blot was carried out to detect the protein expression levels of smooth muscle actin (SMA), p38, Smad3, fibronectin, collagen I and collagen III in different groups. RESULTS: The results revealed that ATO could inhibit the proliferation and migration of HTFs. Based on the TUNEL assay, 100 µM and 150 µM ATO could induce cell apoptosis. The ELISA results indicated that ATO could down-regulate the expression level of TGF-ß2, and western blot analysis revealed that the protein expression levels of SMA, p38, Smad3, fibronectin, collagen I and collagen III in the TGF-ß2 group were all up-regulated compared with the control group, whereas the addition of ATO could reverse this up-regulation. CONCLUSIONS: ATO could inhibit the proliferation and migration of HTFs and induce their apoptosis. It was preliminary proven that ATO could inhibit the signaling pathway induced by TGF-ß. It is suggested that ATO could be a basis for the treatment of the scarring of filtration channels after glaucoma surgery.

[Epidemiological impact of the COVID-19 pandemic on enucleation cases in Germany]. / Epidemiologische Auswirkungen der COVID-19-Pandemie auf Enukleationsfälle in Deutschland.

PURPOSE: To determine the rate of enucleation in Germany and the impact that the COVID-19 pandemic may have had on its characteristics. METHODS: The rates of enucleation in Germany during 2019 and 2020 were extracted from the diagnosis-related groups (DRG) registry using the operation and procedure classification system codes 5­163.0 through 5­163.23 and 5­163.x. The data were statistically analyzed. RESULTS: The number of enucleations showed a 16.6% reduction from 1295 cases in 2019 compared to 1080 cases in 2020 (p = 0.17). In both years men averaged 54.1% of cases. Patients older than 65 years represented 53% and 56% of cases in 2019 and 2020, respectively. The most common indication for enucleation in both years was phthisis bulbi (n = 373 and n = 307, respectively), accounting for 29.7% of the cases, followed by choroidal malignancies (24%). Enucleation with the simultaneous introduction of an alloplastic orbital implant into Tenon's capsule represented the most common procedure (38.7% combined 2­year average), followed by a sheathed variant (26.6%) and a bulbar implant made of nonabsorbable microporous material (16.8%), without a significant change between years. Enucleations without the introduction of an implant increased from 7.8% in 2019 to 11.1% in 2020 (p = 0.006). The proportion of patients undergoing a reoperation slightly increased from 5.6% to 8% (p = 0.018). Most procedures (65.6%) were performed in large (> 1000 beds) public hospitals. CONCLUSION: Despite the decrease in the total number of procedures performed, the rate of enucleation in Germany was not significantly altered by the COVID-19 pandemic. The rate of enucleation without implants and reoperations significantly increased.

ALK5 Inhibition of Subconjunctival Scarring From Glaucoma Surgery: Effects of SB-431542 Compared to Mitomycin C in Human Tenon's Capsule Fibroblasts.

Purpose: The gold standard for managing postoperative ocular fibrosis in glaucoma surgery is the chemotherapeutic mitomycin C (MMC) despite its association with significant adverse effects. This study compares in vitro the antifibrotic efficacy and cytotoxicity of the small-molecule TGFß1 inhibitor SB-431542 (SB) to MMC. Methods: To measure collagen contraction, human Tenon's capsule fibroblasts (HTCFs) embedded in a three-dimensional collagen lattice were exposed to 0.2 mg/mL MMC or 20 µM SB followed by incubation with 2 ng/mL TGFß1. Total protein extracted from experimentally treated HTCFs underwent immunoblotting for α-smooth muscle actin (α-SMA), matrix metallopeptidase 9 (MMP-9), and EDA splice-variant fibronectin (EDA-FN) expression. Cytotoxicity and cell metabolism were assessed using LIVE/DEAD staining, lactate dehydrogenase (LDH) assay, and methylthiazole tetrazolium (MTT) assay. Results: Collagen lattice contraction in TGFß1-induced HTCFs was significantly lowered by SB and MMC. Pretreatment with SB and MMC significantly lowered protein expression of α-SMA, MMP-9, and EDA-FN in HTCFs relative to TGFß1 alone. HTCF viability in collagen lattices was significantly reduced with MMC pretreatment but not SB pretreatment. MMC-pretreated HTCFs had a significant increase in LDH release after 3 hours and a decrease in MTT activity after 20 minutes, while SB-pretreated HTCFs showed no significant changes via MTT or LDH assay during the same treatment period. Conclusions: SB shows comparable efficacy to MMC in reducing expression of fibrosis-promoting proteins in HTCFs and in vitro scarring activity. SB distinguishes itself from MMC by exhibiting less cytotoxicity in both two-dimensional and three-dimensional in vitro assays. Translational Relevance: This study demonstrates in vitro the potential of SB as a safer alternative ocular antifibrotic agent.

Circ_0047835 Combines with miR-144-3p to Promote the Proliferation, Invasion, Migration, and Fibrosis of TGF-ß1-Treated Human Tenon's Capsule Fibroblasts by Upregulating SP1.

PURPOSE: Glaucoma is the leading cause of blindness worldwide with complex pathogenesis. Circular RNAs (circRNAs) play critical roles in various diseases, including glaucoma. The purpose of this study was to investigate the role of circ_0047835 and underlying mechanisms in the development of fibrosis after glaucoma filtration surgery. METHODS: Human Tenon's capsule fibroblasts (HTFs) were stimulated using transforming growth factor-ß1 (TGF-ß1) to mimic a cellular model of glaucoma in vitro. Cell proliferation was evaluated by Cell Counting Kit-8 (CCK-8) assay and 5-ethynyl-2'-deoxyuridine (EdU) assay. Cell invasion and migration were detected by transwell assay and wound healing assay, respectively. Western blot assay was used to measure protein levels. The expression levels of circ_0047835, microRNA-144-3p (miR-144-3p) and specific protein 1 (SP1) mRNA were determined by real-time quantitative polymerase chain reaction (RT-qPCR). The interaction between miR-144-3p and circ_0047835 or SP1 was confirmed by dual-luciferase reporter assay and RNA Immunoprecipitation (RIP) assay. RESULTS: Circ_0047835 expression was elevated in glaucoma tissues and TGF-ß1-treated HTFs. Circ_0047835 or SP1 knockdown suppressed the proliferation, migration, invasion, and fibrosis of TGF-ß1-treated HTFs. MiR-144-3p was a target of circ_0047835, and miR-144-3p inhibition reversed the effects of circ_0047835 knockdown in TGF-ß1-treated HTFs. Moreover, SP1 was identified as a target of miR-144-3p, and miR-144-3p overexpression weakened TGF-ß1-induced proliferation, migration, invasion, and fibrosis by targeting SP1 in HTFs. Furthermore, circ_0047835 combined with miR-144-3p to regulate SP1 expression. CONCLUSION: Circ_0047835 might contribute to fibrosis progression after glaucoma surgery by regulating the miR-144-3p/SP1 axis.

ZD6474 attenuates TGF-ß1-induced fibrosis in human Tenon fibroblasts and inhibits neovascularization via AKT-mTOR signaling pathway.

PURPOSE: To investigate the anti-fibrotic effect of ZD6474 (a novel inhibitor of VEGF and EGF) in TGF-ß1 stimulated human Tenon's capsule fibroblasts (HTFs) and the anti-angiogenetic role in HUVECs, compared to that of mitomycin C (MMC). METHODS: The effects of ZD6474 on cell proliferation or migration in TGF-ß1-stimulated HTFs and HUVECs were determined, using CCK8 or wound healing assay, respectively. The typical markers of fibrosis in TGF-ß1-stimuated HTFs were detected, vimentin by immunofluorescence, α-SMA and snail by western blot. Tube formation was applied to validate the anti-angiogenesis effect in HUVECs following ZD6474 treatment. Furthermore, phosphorylated AKT and mTOR (p-AKT and p-mTOR) were evaluated, compared to the standardized total AKT and mTOR, using western blot. RESULTS: There was almost no decreased cell viability in HTFs following ZD6474 (≤ 1 µM/mL) treatment, but MMC (> 50 µg/mL) significantly impaired cell viability. ZD6474 significantly inhibited TGF-ß1-stimulated proliferation and migration in HTFs, compared to control group (**P < 0.01). ZD6474 also significantly attenuated the TGF-ß1-stimulated expression of vimentin, α-SMA and snail in HTFs. Tube formation was notably interrupted in HUVECs following ZD6474 treatment (**P < 0.01). P-AKT and p-mTOR were significantly decreased in response to ZD6474 treatment in TGF-ß1- induced HTFs and HUVECs. CONCLUSIONS: ZD6474 exerts anti-proliferation and anti-fibrotic effects in TGF-ß1-stimulated HTFs perhaps via regulating AKT-mTOR signaling pathway. ZD6474 also inhibited proliferation, migration and tube formation in HUVECs via the same signaling pathway. We concluded that ZD6474 may be potentially a novel agent in preventing bleb dysfunction following glaucoma filtration surgery (GFS).

Differential effects of acetylsalicylic acid and mitomycin C on cytokine-induced Tenon's capsule myofibroblast transdifferentiation and activity: Implications for glaucoma surgery.

Inflammation-driven scarring is a major contributor to surgical failure after subconjunctival bleb forming glaucoma surgery. The current gold standard anti-scarring adjuvant mitomycin C (MMC) has variable effectiveness and is associated with significant risks. Acetylsalicylic acid (ASA), when delivered locally, repurposes the typically pro-inflammatory cyclooxygenase (COX-2) signaling for the resolution of inflammation and mitigating inflammation-mediated fibrosis. The aim of this study is to compare the effects of ASA and MMC in an in vitro model of subconjunctival scarring. Glaucoma patient-derived Tenon's capsule fibroblasts (HTCFs) were treated with TGFß1 (2 ng/mL) plus or minus ASA (1600 µg/ml), or MMC (0.05, 0.1, 0.2 mg/mL). In vitro collagen contraction, MTT, LDH, immunofluorescence, and Western blot assays were performed. To elucidate the mechanistic effects of ASA in TGFß1-induced HTCFs, liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to identify and measure pro-inflammatory and pro-resolving lipid mediator secretion. ASA was at least as effective as MMC in reducing TGFß1-induced HTCF-mediated collagen contraction, metabolic activity, and pro-fibrotic protein expression, with less cytotoxicity. Within cytokine-activated HTCFs, ASA significantly impaired secretion of pro-inflammatory lipid mediators prostaglandin E2 and 6-keto-prostaglandin F1α and significantly increased secretion of the pro-resolving mediators 5-hydroxyeicosatetraenoic acid (HETE), 15-HETE and 18-hydroxyeicosapentaenoic acid (HEPE). ASA reduces cytokine-induced myofibroblast transdifferentiation in HTCFs, being non-inferior to MMC in vitro. ASA's effects are associated with a unique lipid mediator expression profile, suggesting that the ASA-induced resolution of inflammation may be a promising strategy to mitigate inflammation-mediated scarring and could offer a novel alternative as a surgical adjuvant.

Efficacy and Safety of Amniotic Membrane Transplantation Combined with Closure of Tenon Capsule and Bulbar Conjunctival Space in the Treatment of Primary Pterygium.

Objective: The aim of the study is to evaluate the safety and effectiveness of amniotic membrane transplantation combined with the closure of the tenon capsule and bulbar conjunctival space. Methods: This study retrospectively included 100 patients with primary pterygium who received closed bulbar conjunctiva and tenon capsule space combined with amniotic membrane transplantation in our hospital from January 2020 to June 2021 as the experimental group and 100 patients with routine treatment in the same period as the control group. The postoperative efficacy evaluation and postoperative complications of the two groups were compared, so as to comprehensively evaluate the safety and effectiveness of this method. Results: The results showed that the postoperative complications of the two groups were significantly improved by Fisher's exact test (χ 2 = 14.510, P = 0.006 < 0.05). The comparison results showed that the treatment group showed significant advantages in six indexes compared with the observation group and the difference between the two groups was statistically significant (P < 0.05) of in the NRS score, Prabhasawat score, inspection of the ocular surface comprehensive analyzer, corneal fluorescein staining, conjunctival fluorescein staining in the operation area, breakup time of tear film examination of the two groups at 3, 7 and 14 days, and 1, 6 and 12 months after the operation. Conclusions: Amniotic membrane transplantation combined with the closure of the tenon capsule and bulbar conjunctival space is safer than conventional surgery in the treatment of primary pterygium. It has a shorter recovery time, higher safety, and a positive curative effect. It can be considered to popularize this operation in clinic.

Inhibitory Effects of 3',4'-Dihydroxyflavonol in a Mouse Model of Glaucoma Filtration Surgery and TGFß1-Induced Responses in Human Tenon's Fibroblasts.

Purpose: Cytotoxic agents such as mitomycin C (MMC) are part of the mainstay treatment for limiting subconjunctival scarring following glaucoma filtration surgery (GFS). However, a safer antifibrotic therapy is clinically needed. The anti-scarring properties of 3',4'-dihydroxyflavonol (DiOHF) were evaluated in a mouse model of GFS and in cultured human Tenon's fibroblasts (HTFs). Methods: GFS was performed in C57BL/6 mice receiving daily intraperitoneal injections of DiOHF or vehicle or a single intraoperative injection of MMC. Eyes were harvested on day 14 for assessment of collagen deposition, expression of alpha-smooth muscle actin (α-SMA), cluster of differentiation 31 (CD31), and 4-hydroxy-2-nonenal (4HNE) in the conjunctiva/Tenon's layer. The inhibitory effects of DiOHF on transforming growth factor ß (TGFß)-induced responses were also assessed in HTFs. Results: Treatment with DiOHF demonstrated a reduction in collagen deposition at the GFS site compared to vehicle-treated mice. The degree of 4HNE-positive fluorescence was significantly reduced in DiOHF-treated eyes compared to the other groups, indicating a decrease in oxidative stress. A reduction in expression of α-SMA and CD31 was seen in DiOHF-treated conjunctiva compared to those treated with vehicle. Concordant results were demonstrated in cultured HTFs in vitro. Furthermore, treatment of cultured HTFs with DiOHF also displayed a reduction in the proliferation, migration, and contractility of HTFs. Conclusions: Treatment with DiOHF reduces scarring and angiogenesis in the conjunctiva of mice with GFS at a level comparable to MMC. The reduction in oxidative stress suggests that DiOHF may suppress scarring via different mechanisms from MMC. Translational Relevance: DiOHF may be a safer and superior wound modulating agent than conventional antifibrotic therapy in GFS.

Conjunctiva and Tenon's Capsule Handling in the Port Delivery System with Ranibizumab Implant Insertion Procedure: Surgical Pearls.

OBJECTIVES: To describe conjunctiva and Tenon's capsule handling during the Port Delivery System with ranibizumab (PDS) implant insertion procedure including up-front assessments, planning, and instrumentation, with emphasis placed on the peritomy, scleral dissection, and closure steps. METHODS: Surgical pearls based on experience accumulated in the PDS clinical trial program in patients with retinal diseases. RESULTS: Preoperative preparation, specific instruments, and meticulous techniques are key to optimizing surgical outcomes. Before surgery, assessment of factors that affect conjunctival integrity and an in-office conjunctiva examination are conducted. Gentle, purposeful conjunctiva and Tenon's capsule handling with nontoothed forceps and suturing with a BV needle are recommended to prevent tissue damage. The peritomy is 6 mm by 6 mm, centered around the planned implant location in the superotemporal quadrant. A complete sub-Tenon's capsule dissection is achieved using a wide, robust lateral and posterior dissection technique to free tissue from the sclera and minimize tension. The globe is stabilized during scleral cutdown by grasping the sclera with fine-toothed forceps away from the incision edge to prevent tissue delamination. When closing the peritomy, both the conjunctiva and Tenon's capsule are completely captured and sutured with scleral anchoring at the apex of the peritomy to help prevent conjunctival retraction and erosion. Mitigation and detection of adverse events is critical to successful surgical outcomes. CONCLUSIONS: The PDS implant insertion procedure is straightforward, but it requires planned preoperative preparation, specific instruments, and meticulous techniques. The surgical pearls described here offer insights for optimizing outcomes. [Ophthalmic Surg Lasers Imaging Retina. 2022;53:266-273.].

The Effect of Irradiated Riboflavin in Human Tenon's Fibroblast - A Study on Cellular Viability.

PURPOSE/ AIM: The main purpose of this work is to study the cellular viability effect of irradiated riboflavin in cultured human tenon fibroblasts. MATERIALS AND METHODS: The tenon tissue was harvested from a patient undergoing strabismus surgery. The human tenon fibroblast cell culture and isolation were performed according to the standard laboratory cell culturing protocol. The cells were divided into three groups: control, treatment with irradiated and non-irradiated riboflavin. There were five different concentrations (0.00156%, 0.003125%, 0.00625%, 0.0125%, 0.025%) in each group of riboflavin. The fibroblasts were treated with riboflavin and the cellular viability was assessed at 24-hour and 48-hour post treatment with MTT 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide colorimetric assay. The absorbance values were analysed using Magellan microplate reader data analysis. A triplicate of readings was taken. The data were presented as mean ± standard deviation of the triplicates. Statistical analysis was performed with Statistical Package for Social Sciences (SPSS) analysis version 23. RESULTS: Irradiated riboflavin caused a concentration-dependent cell death in human tenon fibroblast cell culture (p < .05). The antiproliferative difference between irradiated and non-irradiated riboflavin was significant up to 48 hours (p < .05). Post hoc multiple comparisons showed higher concentrations of irradiated riboflavin (0.0125% and 0.025%) caused more reduction in cellular viability in human tenon fibroblast cells (p < .05). The duration of treatment is not a causative factor in this study. CONCLUSIONS: This pilot experiment demonstrated that irradiated riboflavin induced cell death in human tenon fibroblast culture in a concentration-dependent manner, but is not time-dependent. Further exploratory investigations should be performed to determine the mechanism of cell death. We postulate that apoptosis occurred in these irradiated riboflavin-treated cells.

Histopathology, matrix metalloproteinase-2 and alpha smooth muscle actin expression in Tenon's capsule of eyes with congenital glaucoma.

PURPOSE: To evaluate the ultrastructural features of collagen fibrils, matrix metalloproteinase (MMP) and alpha-smooth muscle actin (α-SMA) expression in the Tenon's capsule of buphthalmic eyes. METHODS: A prospective comparative case series study was conducted on 35 buphthalmic eyes vs 25 control eyes. Children with congenital glaucoma (CG) who underwent a combined trabeculectomy-trabeculotomy procedure with mitomycin C (CTTM); the Tenon's capsule was obtained during the surgical procedure. The control group included children with strabismus, the Tenon's capsule was obtained in the course of strabismus surgery. Both H&E and Masson's trichrome staining were done. The Metalloprotenease-2 (MMP-2), alpha smooth muscle actin (α-SMA) immunohistochemical staining was performed. RESULTS: Mean collagen percentage area by Masson trichrome stain in Tenons capsule was significantly higher in buphthalmic eyes (54.76% ± 0.32 vs 33.71% ± 1.4; P < 0.001).The percentage area of αSMA expression in Tenons capsule was significantly higher in buphthalmic eyes (4.93% ± 0.7 vs 2.00% ± 0.5; P < 0.001). However, MMP2 expression in Tenons capsule of the buphthalmic group was significantly lower than that of the control group (12.88% ± 2.95 vs 27.91% ± 0.2 respectively) P = 0.02. CONCLUSIONS: Tenon capsule of buphthalmic eyes have their own histopathological features and properties making them more liable for fibrosis with high rate of failure following antiglaucoma surgeries. Such detailed information has not been published before which may aid in the identification of new antifibrotic therapies in management of glaucoma.

COMPARISON OF RETROBULBAR, SUB-TENON ANESTHESIA AND MEDIAL CANTHUS EPISCLERAL ANESTHESIA FOR 25-GAUGE POSTERIOR VITRECTOMY.

PURPOSE: The aim of the study is to compare the efficacy, safety, and globe akinesia between retrobulbar anesthesia, sub-Tenon anesthesia, and medial canthus episcleral anesthesia for 25-gauge posterior vitrectomy. METHODS: A total of 340 25-gauge vitrectomy data sheets were retrospectively collected between November 2017 and June 2019. Ninety patients were included in the study. These patients were matched by sex and age to receive retrobulbar anesthesia (group 1, n = 30), sub-Tenon anesthesia (group 2, n = 30), and medial canthus episcleral anesthesia (group 3, n = 30). Globe akinesia was recorded after the injection of anesthetic at 2, 5, and 10 minute time intervals. Patients were asked to rate the pain during administration of anesthesia, during surgery, and postoperatively using the visual analog pain scale. RESULTS: For a perfect block, at 10 minutes, retrobulbar outperformed both sub-Tenon and medial canthus episcleral anesthesia which seemed quite similar. During administration, the three techniques did not show statistically different effects on pain. Regarding perioperative pain, retrobulbar outperformed medial canthus episcleral anesthesia. CONCLUSION: All three techniques allowed for safe surgery. Retrobulbar obtained the best results, although sub-Tenon proved to be a valid alternative. Medial canthus episcleral anesthesia obtained mostly good and fair blocks and acceptable pain levels during surgery. Further studies should investigate whether optimal anesthetic efficacy can be obtained with sub-Tenon and medial canthus episcleral techniques when higher volumes are used.

Long Noncoding RNA LINC01518 Modulates Proliferation and Migration in TGF-ß1-Treated Human Tenon Capsule Fibroblast Cells Through the Regulation of hsa-miR-216b-5p.

In this study, we investigated the expression and functions of long noncoding RNAs (LncRNAs) of LINC01518 in an in vitro model of TGF-ß1-treated human Tenon capsule fibroblast (HTF) cells. qRT-PCR was used to examine LINC01518 expression in in situ human glaucoma tissues, and in vitro HTF cells treated with TGF-ß1. Lentivirus-mediated LINC01518 knockdown was performed in HTF cells to investigate its effect on TGF-ß1-induced cell proliferation, migration and autophagy signaling pathway. The potential ceRNA candidate of LINC01518, hsa-miR-216b-5p, was probed by dual-luciferase assay and qRT-PCR. Hsa-miR-216b-5p was also knocked down in LINC01518-downregulated HTF cells to investigate the function of this lncRNA-miRNA epigenetic axis in TGF-ß1-treated HTF cells. LINC01518 was upregulated in human glaucoma tissues and cultured HTF cells. LINC01518 downregulation significantly suppressed TGF-ß1-induced cell proliferation, migration and autophagy signaling pathway in HTF cells. Hsa-miR-216b-5p was confirmed to be a ceRNA target of LINC01518. Knocking down hsa-miR-216b-5p reversed the suppressing effects of LINC01518 downregulation in TGF-ß1-treated HTF cells. Our study demonstrated that LINC01518 is a functional factor in regulating proliferation and migration in TGF-ß1-treated HTF cells, and hsa-miR-216b -5p may also be involved. Targeting the epigenetic axis of LINC01518/hsa-miR-216b-5p may provide new insight into the pathological development of human glaucoma.

The Effect of CHIR 99021, a Glycogen Synthase Kinase-3ß Inhibitor, on Transforming Growth Factor ß-Induced Tenon Fibrosis.

Purpose: This study investigated the effect of glycogen synthase kinase-3ß (GSK-3ß) inhibition on the fibrosis of human Tenon's fibroblasts (HTFs) induced by transforming growth factor-ß (TGF-ß). Methods: Quantitative real-time PCR and Western blot analyses were performed to determine the expression levels of molecules associated with the fibrosis of HTFs by TGF-ß (fibronectin, collagen Iα, and α-smooth muscle actin) and GSK-3ß. The levels of phosphorylated Smad2 and Smad3 were also analyzed in the presence of the GSK-3ß inhibitor CHIR 99021. The wound healing assay was performed to determine the effect of CHIR 99021 on the migration of HTFs. All experiments were conducted using primary cultured HTFs or human tenon tissues obtained from normal subjects and patients with glaucoma. Results: Treatment with TGF-ß resulted in an increase in the levels of molecules associated with the fibrosis of HTFs. The expression levels of these molecules were higher in the tenon tissues obtained from patients with glaucoma than those from normal subjects. When the HTFs were treated with TGF-ß, a significant increase in the active form of GSK-3ß (Y216) was observed. A significant decrease in the active form of GSK-3ß and molecules associated with fibrosis by TGF-ß was noted in HTFs treated with CHIR 99021. CHIR 99021 treatment reduced the phosphorylated Smad2/Smad2 and phosphorylated Smad3/Smad3 ratios in HTFs and attenuated HTF migration. Conclusions: Our results demonstrated the effect of GSK-3ß inhibition on the regulation of TGF-ß-mediated fibrosis of HTFs, suggesting GSK-3ß to be a potential target for maintaining bleb function after glaucoma filtration surgery.