Yale University School of Medicine, New Haven, Connecticut.
Invest Ophthalmol Vis Sci. 2014 Feb 18;55(2):935-40. doi: 10.1167/iovs.13-12890.
To create an in vivo model of vector-mediated trabecular meshwork (TM) ablation and replacement.
We generated a conditionally cytotoxic, trackable vector, HSVtkiG, that expressed herpes simplex virus 1 thymidine kinase (HSVtk) and enhanced green fluorescent protein (eGFP). We optimized HSVtkiG ablation in vitro with ganciclovir (GCV) in comparison to eGFP control vector GINSIN and investigated the mechanism. Right eyes of 24 rats were then injected intracamerally with either HSVtkiG or GINSIN, before intraperitoneal GCV was administered 1 week later. Intraocular pressure, central corneal thickness (CCT), and slit-lamp exams were assessed for 8 weeks. Transduction and ablation were followed by gonioscopic visualization of eGFP. Histology was obtained with TM cell counts and immunohistochemistry markers of inflammation.
Transduction and ablation parameters were established in vitro. Apoptosis was the cause of cell death. In vivo, transduction was seen gonioscopically to be targeted to the TM, followed by disappearance of eGFP marker fluorescence in HSVtkiG-transduced cells after injection of GCV. Ablation resulted in an IOP decrease of 25% in HSVtkiG-injected eyes 2 days after GCV but not in GINSIN or noninjected control eyes (P < 0.05). Trabecular meshwork cellularity was decreased at the time of lowest IOP and recovered thereafter, while CCT remained unchanged. Inflammation was absent.
A vector-based system for inducible ablation of cells of the outflow tract was developed. Trabecular meshwork ablation lowered IOP, and recovery of cellularity and IOP followed. This model may be useful to study pressure regulation by the TM, its stem cells, and migration patterns.
建立一种载体介导的小梁网（TM）消融和置换的体内模型。
我们生成了一种条件性细胞毒性、可追踪的载体 HSVtkiG，它表达单纯疱疹病毒 1 胸苷激酶（HSVtk）和增强型绿色荧光蛋白（eGFP）。我们用更昔洛韦（GCV）对 HSVtkiG 进行了体外优化消融，并与 eGFP 对照载体 GINSIN 进行了比较，研究了其机制。然后，将 24 只大鼠的右眼分别注射 HSVtkiG 或 GINSIN 后，1 周后腹腔内给予 GCV。评估 8 周内的眼压、中央角膜厚度（CCT）和裂隙灯检查。通过共焦显微镜观察 eGFP 的转导和消融。组织学检查包括 TM 细胞计数和炎症的免疫组织化学标志物。
在体外建立了转导和消融参数。细胞死亡的原因是细胞凋亡。在体内，通过共焦显微镜观察到转导靶向 TM，然后在注射 GCV 后 HSVtkiG 转导细胞中 eGFP 标记荧光消失。GCV 后 2 天，HSVtkiG 注射眼的眼压降低了 25%，而 GINSIN 或未注射对照眼则没有（P < 0.05）。在眼压最低时 TM 细胞数量减少，此后恢复，而 CCT 保持不变。没有炎症。
开发了一种用于诱导流出道细胞消融的载体系统。小梁网消融降低了眼压，细胞数量和眼压随后恢复。该模型可能有助于研究 TM、其干细胞和迁移模式对眼压的调节。
