Measurement of Conventional Outflow Facility
In all perfusion studies mice were culled humanely by cervical dislocation, eyes enucleated immediately after death and outflow facility measured simultaneously in paired eyes using theiPerfusion system, as previously described (Sherwood et al. , 2016). Briefly, eyes were glued to a support platform submerged in PBS regulated at 35°C. The ocular anterior chambers were cannulated with 33-gauge bevelled needles (NanoFil, NF33BV-2, World Precision Instruments) attached to micro-manipulators and equilibrated for 30 minutes at 9 mmHg. Perfusate comprised 0.2 μm filtered DBG (PBS including divalent cations and 5.5 mM glucose). Flow was measured at pressure steps from 5 to 17 mmHg in 7 steps. The steady state criterion per step was 1 minute of <0.1 nl/min/mmHg/min variation in ratio of flow rate to pressure. Pressure steps that failed to reach steady state were excluded from further analysis, paired eyes with 4 or more successful steps were analysed. A Savitzky–Golay filter (60secs, first order) was applied to the digital pressure and flow data before calculation to increase precision without distorting the signal tendencies.
Mean steady state flow \(Q\) and pressure \(P\) for each included pressure step were calculated over a 4-minute window and fit by the relationship
\begin{equation} Q=C_{r}\left(\frac{P}{P_{r}}\right)^{\beta}P\nonumber \\ \end{equation}
\(C_{r}\) represents outflow facility at a reference pressure \(P_{r}\)(8 mmHg) and \(\beta\) characterizes the non-linearity of the\(Q\)-\(P\) relationship. Average relative change in \(C_{r}\) was compared between contralateral treated and control eyes (mean ± 95% CI) using a weighted t -test of the log-transformed data as described previously (Sherwood et al. , 2016).
Acute effects on outflow facility were determined by perfusing DBG containing 100 nM de-esterified JV-GL1 directly into the anterior chamber of normotensive C57BL/6J mouse eyes (n=7) and compared to paired vehicle perfused contralateral eyes. All pairs tested met the stability criteria. Long-term effects of topical JV-GL1 treatment on outflow facility were determined in steroid induced ocular hypertensive C57BL/6J mice (n=9) following unilateral treatment with 0.01% JV-GL1, with contralateral eyes receiving vehicle, outflow facility was measured ex vivo 3 days later. All pairs tested met the stability criteria.