Pressure-independent outflow in ex vivo mouse eyes

Abstract

Purpose : Mice are commonly used for studies of aqueous humor dynamics, but it has been reported that up to 80% of outflow is pressure-independent, which would typically be attributed to non-trabecular routes. Such high non-trabecular outflow would invalidate the mouse as a model for human outflow, which is primarily trabecular. This study aims to directly measure pressure-independent outflow at zero pressure, Q₀, in ex vivo mouse eyes. As the flow-pressure relationship is often assumed to be linear, we also examined whether non-linear behaviour could contribute to the appearance of pressure-independent outflow.

Methods : 8 pairs of enucleated eyes from C57BL/6 mice aged 9-16 weeks were perfused with PBS+5.5mM glucose using the iPerfusion system. Pressure steps of 0, 3, 6, 9, 12, 15, and 18 mmHg were applied while measuring intraocular pressure (P) and the flow rate (Q) into the eye. The contralateral eye received the same pressure regimen with an artificial pressure-independent inflow of 120 nl/min infused with a syringe pump. This was chosen to mimic reported in vivo rates of aqueous production minus pressure-independent outflow. To examine the non-linearity of the Q-P relationship, 66 additional unpaired eyes were perfused at 4-20 mmHg. Q-P data were fit by linear (Q = C P + Q₀) and non-linear (Q =C_r (P / P_r)^β P + Q₀) models where C is the facility and C_r the facility at P_r = 8 mmHg. β is an index of non-linearity.

Results : Non-zero values of β (0.75 ± 0.63; mean ± 2SD, n=66) confirmed that the Q-P relationship was non-linear. For the artificial inflow cases, Q₀ was not significantly different from zero when accounting for the resolution of the flow meter (-6 ± 2 nl/min, n=8), and Q₀ was not significantly different from 120 nl/min with infusion (-115 ± 8 nl/min, n=8). There was a strong correlation between the non-linearity parameter β and Q₀ predicted by the linear model (p<10^-6, n=66), suggesting that non-linearity in the Q-P relationship introduces an artificial pressure-independent outflow.

Conclusions : There is negligible pressure-independent outflow in enucleated eyes from young C57BL/6 mice. Non-linearity in the Q-P relationship contributes to the artificial appearance of pressure-independent outflow when analysed using a linear model. The form of the Q-P relationship thus influences interpretation of the mechanism and pathway of outflow, and assumptions regarding linearity or constancy of outflow facility should be more carefully evaluated.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.