Proietto Laura R, Whitley R David, Brooks Dennis E, Schultz Gregory E, Gibson Daniel J, Berkowski William M, Salute Marc E, Plummer Caryn E
a Department of Small Animal Clinical Sciences , University of Florida College of Veterinary Medicine , Gainesville , FL , USA.
b Institute for Wound Research, Department of Obstetrics and Gynecology , University of Florida College of Medicine , Gainesville , FL , USA.
Curr Eye Res. 2017 Jun;42(6):813-821. doi: 10.1080/02713683.2016.1262428. Epub 2017 Jan 27.
To develop a novel ex vivo extended culture model of canine corneal epithelial cell wound healing.
Canine corneoscleral rims (CSR) were obtained and, after preparation for culture, were placed on a nutating scaffold and incubated in physiological conditions. In experiment 1, eight CSR in a serum-containing antimicrobial-fortified medium were monitored for epithelial integrity and bacterial infection up to 28 days in culture. CSR were assessed histologically at the end of the culture period end points 0, 7, 14, and 28 days with accompanying scanning electron microscopic (SEM) and transmission electron microscopic (TEM) evaluation. Samples for microbial culture were obtained at days 0, 3, 7, 14, and 28. In experiment 2, uniform 8-mm-diameter superficial corneal epithelial wounds were created and monitored for re-epithelialization in the same culture conditions or in a serum-free protein equivalent medium, with four CSR per group. Standardized digital images were obtained with cobalt filter at the time of fluorescein staining and media change every six hours. Image J imaging software was used to measure the area of fluorescein retention. Re-epithelialization rates were calculated and CSR then fixed for immunohistochemistry (IHC).
All corneas survived to end points as described in experiment 1 with no evidence of contamination or compromised epithelial integrity. Histologically, a multilayered epithelium was maintained and corneal edema was not appreciated until day 14. SEM examination revealed epithelial cell layer confluence and migrating epithelial cells of normal cellular morphology with normal cell-cell interactions on TEM. In experiment 2, all eight corneas healed with a healing rate of 0.702 ± 0.130 mm/h (1.25 mm/day epithelial cell migration rate) and were positive in IHC evaluation for markers of corneal fibrosis.
This ex vivo canine corneal wound healing model is an appropriate and clinically relevant tool for assessment and modulation of epithelial wound healing.
建立一种新型的犬角膜上皮细胞伤口愈合的体外延长培养模型。
获取犬角膜缘组织（CSR），经培养准备后，置于旋转支架上，在生理条件下进行培养。在实验1中，将8个CSR置于含血清的抗菌强化培养基中，监测其上皮完整性和细菌感染情况，培养时间长达28天。在培养期结束时，即第0、7、14和28天的终点，对CSR进行组织学评估，并进行扫描电子显微镜（SEM）和透射电子显微镜（TEM）评估。在第0、3、7、14和28天采集微生物培养样本。在实验2中，制作直径8毫米的均匀浅表角膜上皮伤口，在相同培养条件下或无血清蛋白等效培养基中监测其再上皮化情况，每组4个CSR。在荧光素染色时用钴滤光片获取标准化数字图像，每6小时更换培养基。使用Image J成像软件测量荧光素保留面积。计算再上皮化率，然后将CSR固定用于免疫组织化学（IHC）检测。
在实验1中，所有角膜均存活至所述终点时间点；未发现污染迹象或上皮完整性受损。组织学检查显示，多层上皮得以维持，直到第14天才出现角膜水肿。SEM检查显示上皮细胞层融合，TEM显示迁移的上皮细胞形态正常，细胞间相互作用正常。在实验2中，所有8个角膜均愈合，愈合速率为0.702±0.130毫米/小时（上皮细胞迁移速率为1.25毫米/天），IHC评估显示角膜纤维化标志物呈阳性。
这种体外犬角膜伤口愈合模型是评估和调节上皮伤口愈合的合适且与临床相关的工具。
