Targeting Mouse Aqueous Humor Outflow Pathway By Intracameral Perfusion Of Adenovirus

Abstract

Purpose: : The aqueous humor outflow pathway plays a key role in maintaining normal intraocular pressure (IOP). The pathogenesis of high IOP involves abnormalities in the outflow pathway. To better study these mechanisms we optimized anterior chamber perfusion and injection conditions in a mouse model in order to effectively transfer genes to the aqueous humor outflow pathway.

Methods: : Adenovirus expression of GFP driven by CMV promoter (Ad.cmv.GFP) was either perfused or injected (Bolus injection) into the mouse anterior chamber. The perfusion was conducted with a glass micro-needle connected to a controlled pump while the injection was manually pushed using a 33 gauge needle connected to a Hamilton syringe. Three days post procedure, the mice were euthanized and the expression of GFP was visualized by fluorescence microscope and confirmed by electron microscopy. Changes in IOP during pefusion were monitored by insertion of an additional micro-needle connected to a transducer into the anterior chamber. The data was recorded using a PowerLab data requisition system.

Results: : The distribution of GFP expression was highly dependent on the mode of administration of viral particles. Direct manual injections resulted in low levels of GFP expression in the corneal endothelium and TM/SC and high levels in the plexus and episcleral veins. Perfusion of Ad.cmv.GFP with the micro-needle technique provided consistent GFP expression in the outflow pathway (trabecular meshwork, Schlemm’s Canal and episcleral veins) with minimal expression in the cornea. Maximal IOP levels varied with different volume perfusion (1 μl, 13.8±5.6; 2 μl, 28.5±9.2; 3 μl, 41.39±7.7; 5 μl, 71.27±5.3; 7 μl, 107.4±16.7; 8 μl, 122.4±12.3; 10 μl, 157.7±11.7 mmHg in 30μl/min rate, n=7, 3min perfusion). Increasing the time of perfusion to 20min and 40min led to higher levels of GFP expression in cornea endothelium.

Conclusions: : The mode of administration of recombinant viruses in the anterior chamber of the mouse eye appears to be a critical parameter affecting the distribution of transgene expression in the eye. Intracameral perfusion with this micro-needle technique in the mouse model is a promising method to modulate gene expression in the aqueous humor outflow pathway.

Keywords: outflow: trabecular meshwork • injection • anterior chamber