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Padmanabhan P. Pattabiraman, Corey Morris, Pratap Challa, Thomas Rinkoski, Eric M. Poeschla, Vasanth Rao; Characterization of Ocular Hypertensive Rat Model Developed by Augmenting the Rho GTPase Signaling in the Eye Anterior Chamber Iridocorneal Angle. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2485.
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To test the hypothesis that abnormally activated Rho/Rho kinase signaling in aqueous humor (AH) outflow pathway may lead to increased resistance to AH drainage resulting in elevated intraocular pressure (IOP).
HIV-based lentiviral vectors carrying GFP only or constitutively active RhoAV14 mutant/GFP under CMV promoter were generated and purified. Three groups of 9 weeks old Sprague Dawley female rats (n=8/group) were injected into anterior chamber of right eye with saline, GFP or RhoAV14/GFP viral vectors (1x107 Transduction Units). IOP was monitored under sedation during day and at night using a Tonolab. Post injection IOPs were recorded on days 21, 42, 68, 86, 126, 146 and 165 in both eyes. Statistical differences in IOP were evaluated by Kruskal-Wallis and Wilcoxon rank sum tests. After ~5 1/2 months of viral vector injections, animals were euthanized and enucleated eyes were processed for histological and immunohistochemical analyses.
Viral vectors used in the animal study was first confirmed by evaluating GFP fluorescence, increased actin stress fiber formation, and quantification of RhoA and phosphorylated-myosin light chain (MLC) levels in porcine TM cells infected with these vectors. Rats injected with RhoAV14 vector demonstrated a significant increase in IOP relative to saline and GFP controls, starting from post-injection day 68, with the trend being sustained till the end of the study (165days). The mean IOP during day and at night ranged between 10-12 and 13-17 mmHg, respectively. There was a significant increase in IOP in RhoAV14 injected animals of about 20% and 30% (p<0.05, n=8) during the day and night, respectively, relative to GFP controls. The IOP increase within the RhoAV14 injected group varied between strong (n=3), moderate (n=3) or weak/no effect (n=2). All animals injected with viral vectors showed different levels of GFP expression specifically in the iridocorneal angle of the eye anterior chamber. TEM-based histology of the eyes of viral vector injected animals showed no evidence of inflammatory processes. Of the limited specimens (n=2/group) analyzed so far, we noted a dense and compact JCT region in RhoAV14 group, with overall histology of the angle being similar among different groups.
This ongoing study which was aimed at determining the physiological implications of elevated Rho/Rho kinase signaling in tissues of aqueous humor outflow pathway reveals that constitutive activation of this signaling pathway leads to increased IOP in a rodent model. This in vivo study supports the importance of Rho/Rho kinase signaling in physiological and pathological regulation of IOP in glaucoma.
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