Effects of Conditional Dicer Knockdown on the Outflow Pathway in Living Mice

Abstract

Purpose: : To evaluate the biological relevance of the regulation of gene expression mediated by microRNAs in the physiology of the outflow pathway by conditional knockdown of Dicer in living mice.

Methods: : The adenoviral vector Ad.CMV.CRE (5x106 pfu) expressing cre recombinase under the CMV promoter was injected into the anterior chamber of one eye in 7 Dicer1tm1Bdh/J mice. Eight control mice were treated in parallel with a recombinant adenovirus expressing EGFP (Ad.CMV.EGFP). IOP was monitored biweekly with a TonoLab Rebound Tonometer using light anesthesia. Morphological changes were evaluated by light microscopy of semi-thin sections, and by electron microscopy. Immunogold staining was conducted with antibodies for macrophage (CD68) and melanocyte (S100) markers.

Results: : Loss of microRNA function by deletion of dicer in the cells of the outflow pathway resulted in a significant and sustained increase in IOP six weeks after viral treatment when compared to both non-treated contralateral eyes (p < 0.001, ANOVA) or Ad.CMV.EGFP control eyes (p < 0.00001, ANOVA). There was no significant difference in mice transduced with Ad.CMV.EGFP between the treated and non-treated eyes (p = 0.30, ANOVA). The observed increase in IOP was associated with a noticeable accumulation of pigmented cells (PC) in the angle, and the TM was more densely compact compared to the control mice. The PCs present in the angle showed positive immunogold staining for the melanocyte marker S100 but not for the macrophage marker CD68.

Conclusions: : Our results suggest that loss of Dicer might lead to increased IOP by impairing the ability of the TM to eliminate melanocytes that have detached from the iris and accumulate over time in the angle causing an increase in outflow resistance. The observed alteration caused by deletion of Dicer also underscore the physiological relevance of miRNAs in the outflow pathway in vivo.

Keywords: trabecular meshwork • gene modifiers • intraocular pressure