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.