Abstract
Purpose: :
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).
Methods: :
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.
Results: :
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.
Conclusions: :
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.
Keywords: signal transduction: pharmacology/physiology • trabecular meshwork • pathobiology