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Amardeep Dhaliwal, Sushma Kola, James Kim, Ze Zhang, Harry Tseng, Joel S Schuman, Robert N Weinreb, Nils A Loewen; A Conditionally Cytotoxic Viral Vector for Trabecular Meshwork Ablation. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5984.
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© ARVO (1962-2015); The Authors (2016-present)
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 eGFP. We first compared HSVtkiG transduction and ablation with ganciclovir (GCV) to that of eGFP control vector GINSIN in vitro and optimized amount of vector and GCV. Right eyes of 24 rats were then injected intracamerally with either HSVtkiG or GINSIN, before intraperitoneal GCV was administered one week later. IOP, 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 using anti-GFP, anti-CD3, and anti-macrophage antibodies as markers of inflammation. A terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was deployed to confirm apoptosis as the mechanism of cell death. Experiments were repeated in perfused anterior segments.
Transduction and ablation parameters were established in vitro and apoptosis as the mechanism for HSVtkiG transduced cells exposed to GCV. 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 non-injected control eyes (P<0.05, day 2 - 35). TM cellularity was decreased at the time of lowest IOP and recovered thereafter, while CCT remained unchanged. In vitro studies showed when HSVtkiG transduced cells were exposed to GCV, cells became pyknotic and started to detach at 24 hours. Inflammation was absent. Ablation in ex vivo organ culture was consistent with in vivo findings.
A vector-based system for inducible ablation of cells of the outflow tract was developed. TM ablation lowered IOP and was followed by recovery of cellularity and IOP. This model may be useful to study pressure regulation by the TM, its stem cells and migration patterns.
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