Departamento de Biología Celular, Histología y Farmacología, Facultad de Medicina, Grupo de Investigación Reconocido: Técnicas Ópticas para el Diagnóstico, Universidad de Valladolid, Valladolid, Spain.
Departamento de Físico Teórica, Atómica y Óptica, Facultad de Ciencias, Grupo de Investigación Reconocido: Técnicas Ópticas para el Diagnóstico, Universidad de Valladolid, Valladolid, Spain.
Exp Eye Res. 2019 Sep;186:107704. doi: 10.1016/j.exer.2019.107704. Epub 2019 Jun 19.
The extracellular matrix (ECM) confers transparency to the cornea because of the precise organization of collagen fibrils and a wide variety of proteoglycans. We monitored the corneal wound healing process after alkali burns in rabbits. We analyzed the location and expression of collagens and proteoglycans, the clinical impact, and the recovery of optical transparency. After the animals received both general and ocular topical anesthesia, the central cornea of the left eye was burned by placing an 8-mm diameter filter paper soaked in 0.5 N NaOH for 60 s. The eyes were evaluated under a surgical microscope at 1, 3, and 6 months after burning. At each time point, the clinical conditions of the burned and control corneas were observed. The arrangement of collagen fibers in the corneal stroma was visualized by Picrosirius-red staining, Gomori's silver impregnation and transmission electronic microscopy. Corneal light transmittance was also measured. Myofibroblasts presence was analyzed by immunohistochemistry. mRNA expression levels of collagen types I and III, lumican, decorin, keratocan and alpha-smooth muscle actin were determined by quantitative real-time polymerase chain reaction. One month after alkali burn, the ECM was disorganized and filled with lacunae containing different types of cells and collagen type III fibers in the wound area. Corneal opacities were present with attendant loss of light transmittance. Collagen and proteoglycan mRNA expression levels were up-regulated. After three months, wound healing progress was indicated by reduced corneal opacity, increased light transmittance, reorganization of collagen fibers and only collagen type I expression levels were at control levels. After six months, the wound area ECM morphology was similar to controls, but transmittance values remained low, denoting incomplete restoration of the stromal architecture. This multidisciplinary study of the stromal wound healing process revealed changes in corneal transmittance, collagen organization, myofibroblasts presence and ECM composition at 1, 3, and 6 months after alkali burning. Documenting wound resolution during the six-month period provided reliable information that can be used to test new therapies.
细胞外基质（ECM）由于胶原纤维和多种蛋白聚糖的精确组织，赋予了角膜透明性。我们监测了兔眼碱烧伤后的角膜愈合过程。我们分析了胶原和蛋白聚糖的位置和表达、临床影响以及光学透明度的恢复情况。在动物接受全身和眼部局部麻醉后，将直径 8 毫米的滤纸浸泡在 0.5N NaOH 中 60 秒，以灼伤左眼中央角膜。在烧伤后 1、3 和 6 个月，在手术显微镜下评估眼睛。在每个时间点，观察烧伤和对照角膜的临床状况。通过苦味酸-天狼星红染色、Gomori 银浸染和透射电子显微镜观察角膜基质中胶原纤维的排列。还测量了角膜光透过率。通过免疫组织化学分析肌成纤维细胞的存在。通过实时定量聚合酶链反应确定胶原 I 和 III 型、亮氨酸、核心蛋白聚糖、角膜蛋白聚糖和α-平滑肌肌动蛋白的 mRNA 表达水平。碱烧伤后 1 个月，ECM 紊乱，伤口区域充满含有不同类型细胞和 III 型胶原纤维的腔隙。角膜混浊存在，伴随光透过率降低。胶原和蛋白聚糖 mRNA 表达水平上调。3 个月后，通过减少角膜混浊度、增加光透过率、胶原纤维重组以及仅胶原 I 表达水平恢复到对照水平，提示伤口愈合进展。6 个月后，伤口区域 ECM 形态与对照相似，但透光值仍然较低，表明基质结构的不完全恢复。这项对基质伤口愈合过程的多学科研究揭示了碱烧伤后 1、3 和 6 个月角膜透光率、胶原组织、肌成纤维细胞存在和 ECM 组成的变化。记录 6 个月期间的伤口愈合情况提供了可靠的信息，可用于测试新的治疗方法。
