Physical factors affecting outflow facility measurements in mice

Mice are a common animal model for studies of aqueous humor dynamics and outflow physiology. The anatomy of the conventional outflow pathway in mice is similar to that of humans with a continuous Schlemm's canal and lamellated trabecular meshwork.1 Like primates, mice possess a ciliary muscle that forms tendinous connections to the elastic fiber net of the trabecular meshwork and the inner wall endothelium of Schlemm's canal.2 Compounds that affect outflow facility in humans, including pilocarpine,2,3 TGF-β2,4,5 latanoprost6–8 prostaglandin EP4 receptor agonist,9–11 and sphingosine 1-phosphate,11,12 similarly affect outflow facility in mice. Recently, mice have been used to validate novel compounds that increase outflow facility based on hits from screening assays of cellular contractility.13 Numerous investigators have measured outflow facility in mice.2,5,6,8,11,14–26 However, on account of the small dimensions of the mouse eye and the low flow rates involved, there is greater potential for physical factors to influence ocular perfusion measurements in mice compared with larger species. For example, evaporation from the surface of the eye, which is more pronounced in smaller eyes that have a larger surface to volume ratio, may lead to dehydration of the corneoscleral shell and artifactually increase the apparent outflow rate. Importantly, this effect would manifest as a pressure-independent outflow during perfusion. Posterior bowing of the iris, known as anterior chamber (AC) deepening, artificially increases outflow facility by applying traction to the trabecular meshwork.27,28 Anterior chamber deepening typically occurs during ocular perfusion via the AC, when the pressure in the AC exceeds that in the posterior chamber (PC). The pressure difference causes the iris–lens channel to collapse like a 1-way valve, preventing pressure equilibration across the iris. Anterior chamber deepening can be prevented by perfusion via the PC, by creating a fluidic shunt across the iris,29 or by iridectomy.30 Note that AC deepening is not synonymous with ‘AC depth,' which represents the distance between the posterior cornea and anterior lens. Because mice have a relatively large crystalline lens, it has been proposed that AC deepening may be negligible in mice,18 but this has not been specifically examined. To address these gaps in knowledge, this study examined the influence of hydration and AC deepening on pressure-dependent and pressure-independent outflow in enucleated mouse eyes. We also examined the effect of temperature that could account for up to 40% variation in apparent outflow facility due to changes in water viscosity between room and physiological temperature.