Fuchs Heiko, Hu Xiaonan, Meister Roland, Huang Yuqing, Bartram Martin C, Pielen Amelie, Junker Bernd, Tode Jan, Framme Carsten
Department of Ophthalmology, University Eye Hospital, Hannover Medical School, Hannover, Germany.
Maximilians-Augenklinik, Nürnberg, Germany.
PLoS One. 2025 Apr 2;20(4):e0320558. doi: 10.1371/journal.pone.0320558. eCollection 2025.
Glaucoma, a leading cause of blindness, is often driven by elevated intraocular pressure (IOP), which damages the optic nerve. Transscleral filtration surgery reduces IOP but is frequently complicated by excessive wound healing from Tenon fibroblasts (TFs), impeding aqueous humor absorption. Mitomycin C (MMC), used for over 30 years in ophthalmic surgeries, inhibits TF proliferation but carries significant side effects, including hypotony, blebitis, and endophthalmitis, due to its non-selective cytotoxicity. MMC's inability to entirely prevent fibrosis increases surgical failure risk, often necessitating further interventions like bleb needling. This study investigates whether ascorbic acid (AA) and hydrogen peroxide (H2O2) can selectively target TFs without damaging scleral fibroblasts (SFs) in vitro, using MMC as a benchmark.
Primary human TFs and SFs were cultured from patient trabeculectomy tissues. Cells were treated with various concentrations of MMC, AA, or H2O2. Cytotoxic effects were analyzed via live-cell imaging. Immunocytochemistry and Western Blot assessed catalase expression in both cell types and recombinant catalase was used to validate its protective effect against AA- and H2O2-induced cell death.
Short-term exposure (5 min) to 0.02%-0.04% MMC or long-term exposure to 0.00025%-0.001% MMC caused cytotoxicity in TFs and SFs, with SFs dying significantly earlier. In contrast, AA (6-8 mM) selectively induced cell death in TFs without harming SFs. H2O2 also showed selective cytotoxicity towards TFs. Lower catalase expression in TFs compared to SFs was determined via Western blot and immunocytochemistry, highlighting a mechanism for this selective effect. Recombinant catalase neutralized the cytotoxic effects of AA and H2O2 on TFs.
Unlike MMC, Ascorbic acid and hydrogen peroxide exhibit selective cytotoxicity towards Tenon fibroblasts, which may provide a safer, more targeted approach for preventing fibrosis in glaucoma surgery. Additional in vivo studies are needed to explore the clinical applicability of these findings.
青光眼是导致失明的主要原因，通常由眼内压（IOP）升高引起，IOP升高会损害视神经。经巩膜滤过手术可降低IOP，但常因Tenon成纤维细胞（TFs）过度愈合而导致并发症，阻碍房水吸收。丝裂霉素C（MMC）在眼科手术中已使用30多年，可抑制TFs增殖，但由于其非选择性细胞毒性，会带来包括低眼压、睑缘炎和眼内炎等显著副作用。MMC无法完全预防纤维化增加了手术失败风险，常常需要进一步干预，如房水囊肿针刺术。本研究以MMC为对照，调查抗坏血酸（AA）和过氧化氢（H2O2）在体外能否选择性地作用于TFs而不损伤巩膜成纤维细胞（SFs）。
从患者小梁切除组织中培养原代人TFs和SFs。用不同浓度的MMC、AA或H2O2处理细胞。通过活细胞成像分析细胞毒性作用。免疫细胞化学和蛋白质印迹法评估两种细胞类型中过氧化氢酶的表达，并使用重组过氧化氢酶验证其对AA和H2O2诱导的细胞死亡的保护作用。
短期（5分钟）暴露于0.02%-0.04%的MMC或长期暴露于0.00025%-0.001%的MMC会导致TFs和SFs出现细胞毒性，SFs死亡明显更早。相比之下，AA（6-8 mM）可选择性诱导TFs细胞死亡而不损害SFs。H2O2对TFs也表现出选择性细胞毒性。通过蛋白质印迹法和免疫细胞化学确定TFs中过氧化氢酶表达低于SFs，这突出了这种选择性作用的机制。重组过氧化氢酶可中和AA和H2O2对TFs的细胞毒性作用。
与MMC不同，抗坏血酸和过氧化氢对Tenon成纤维细胞具有选择性细胞毒性，这可能为青光眼手术中预防纤维化提供一种更安全、更具针对性的方法。需要进一步的体内研究来探索这些发现的临床适用性。
