The sclera is an ocular tissue rich of collagenous extracellular matrix, which is built up and maintained by relatively few, still poorly characterized fibroblast-like cells. The aims of this study are to add to the characterization of scleral fibroblasts and to examine the reaction of these fibroblasts to inflammatory stimulation in an ex vivo organotypic model. Scleras of scleraxis-GFP (SCX-GFP) mice were analyzed using immunohistochemistry and qRT-PCR for the expression of the tendon cell associated marker genes scleraxis (SCX), mohawk and tenomodulin. In organotypic tissue culture, explanted scleras of adult scleraxis GFP reporter mice were exposed to 10 ng/ml recombinant interleukin 1-ß (IL1-ß) and IL1-ß in combination with dexamethasone. The tissue was then analyzed by immunofluorescence staining of the inflammation- and fibrosis-associated proteins IL6, COX-2, iNOS, connective tissue growth factor, MMP2, MMP3, and MMP13 as well as for collagen fibre degradation using a Collagen Hybridizing Peptide (CHP) binding assay. The mouse sclera displayed a strong expression of scleraxis promoter-driven GFP, indicating a tendon cell-like phenotype, as well as expression of scleraxis, tenomodulin and mohawk mRNA. Upon IL1-ß stimulation, SCX-GFP+ cells significantly upregulated the expression of all proteins analysed. Moreover, IL1-ß stimulation resulted in significant collagen degradation. Adding the corticosteroid dexamethasone significantly reduced the response to IL1-ß stimulation. Collagen degradation was significantly enhanced in the IL1-ß group. Dexamethasone demonstrated a significant rescue effect. This work provides insights into the characteristics of scleral cells and establishes an ex vivo model of scleral inflammation.

巩膜是一种富含胶原细胞外基质的眼部组织，由相对较少、特征仍然很差的成纤维细胞样细胞构成和维持。本研究的目的是增加巩膜成纤维细胞的特征，并在离体器官模型中检查这些成纤维细胞对炎症刺激的反应。使用免疫组织化学和 qRT-PCR 分析 scleraxis-GFP (SCX-GFP) 小鼠的巩膜肌腱细胞相关标记基因scleraxis (SCX)、莫霍克和腱调节蛋白的表达. 在器官型组织培养中，成年巩膜 GFP 报告小鼠的外植巩膜暴露于 10 ng/ml 重组白细胞介素 1-ß (IL1-ß) 和 IL1-ß 与地塞米松的组合。然后通过对炎症和纤维化相关蛋白 IL6、COX-2、iNOS、结缔组织生长因子、MMP2、MMP3 和 MMP13 进行免疫荧光染色分析组织，并使用胶原杂交肽 (CHP) 分析胶原纤维降解情况) 结合测定。小鼠巩膜表现出巩膜启动子驱动的 GFP 的强烈表达，表明肌腱细胞样表型，以及巩膜、腱调节蛋白和莫霍克蛋白的表达mRNA。在 IL1-ß 刺激后，SCX-GFP+ 细胞显着上调了所有分析蛋白质的表达。此外，IL1-ß 刺激导致胶原蛋白显着降解。添加皮质类固醇地塞米松显着降低了对 IL1-ß 刺激的反应。IL1-ß 组的胶原蛋白降解显着增强。地塞米松表现出显着的拯救效果。这项工作提供了对巩膜细胞特征的见解，并建立了巩膜炎症的离体模型。