Evaluation of Adenovirus-Mediated Down-Regulation of Connective Tissue Growth Factor on Postoperative Wound Healing After Experimental Glaucoma Surgery. (2nd July 2016) - Lim, Dong Hui
ABSTRACT: Purpose : This study was aimed to determine whether adenovirus-mediated down-regulation of connective tissue growth factor (CTGF) can modulate postoperative scarring in a rabbit receiving simplified glaucoma surgery. Methods: In vitro studies were performed using a replication-deficient recombinant adenovirus that transcribes a small interfering RNA (siRNA) specific to the CTGF gene under the control of the modified CMV promoter. Primary tenon cells from a New Zealand White rabbit were transduced with 10–100 plaque-forming units (pfu) per cell of the viral vector. Seventy-two hours later, CTGF expression was analyzed by Western blot analysis. In vivo studies were conducted using 10 New Zealand White rabbits, which underwent simplified glaucoma surgery and received a postoperative subconjunctival injection of 5 µl suspension of adenovirus carrying shRNA for CTGF (2 × 10 11 pfu/ml) in the right eye, and the same amount of null virus in the left eye. Eyes were enucleated 5 d after the surgery, and immunohistochemical and histological examinations of the surgical outcome were performed. Results : Western blot analysis showed that CTGF was depleted to less than 10% of its original level in cells transduced with the adenovirus expressing CTGF-specific siRNA. This demonstrates RNA interference (RNAi)-mediated CTGF inactivation in vitro . Immunohistochemical analysis also showed that CTGF was significantly depleted in eyes transduced with the adenovirus expressing CTGF siRNA. This demonstrates RNAi-mediated CTGF inactivation in vivo . In addition, less scar tissue was observed on histological evaluation in the transduced eyes, demonstrating that inhibition of CTGF expression can modulate the wound healing process after surgery. Conclusions : Down-regulation of CTGF is effective in inhibiting postoperative scarring in vivo . This suggests that RNAi with CTGF siRNA may potentially pave the road for a novel therapeutic strategy to improve glaucoma surgery results.