Investigating the IOP effects of JV-GL1
The effects of JV-GL1 on IOP were investigated in four experiments. In all studies IOP was measured under general anaesthesia, bilaterally in each mouse between 09:00 AM and 12:00 noon using a commercial rebound tonometer (TonoLab; Icare, Helsinki, Finland). For each time point IOP was calculated as the mean of three consecutive tonometer readings, each containing 6 rebound events, as previously described (Overby et al. , 2014). We designed 4 experiments to test the effect of JV-GL1 on IOP.
The first experiment investigated the IOP effect of JV-GL1 in normotensive Ptger2-/- mice (11 males & 3 females, 10.7 [10-12] weeks old, mean [95% CI]) andPtger2+/+ mice (6 males & 10 females, 12.6 [12-13] weeks old). Baseline IOP was established prior to unilateral administration of either 0.01% JV-GL1 (Ptger2-/- n=7,Ptger2+/+ n=9) or 0.01% butaprost (Ptger2-/- n=7,Ptger2+/+ n=7), with contralateral eyes receiving vehicle. IOP was measured at 3 and 24 hours, with an additional measurement at 48 hours for the JV-GL1 treated group. Data were presented as the mean difference between paired treated and untreated eyes (ΔIOP).
The second experiment investigated the IOP effect of JV-GL1 in wildtype C57BL/6J mice (10 week old males, n=18) that had been rendered ocular hypertensive by subconjunctival injection of dexamethasone-eluting nanoparticles. Baseline IOP was established over two time points prior to bilateral nanoparticle injection. IOP was measured after two weeks to establish the level of ocular hypertension induced by the glucocorticoid, referred to as the pre-treatment time point. Mice were treated bilaterally with either 0.01% JV-GL1 (n=9) or 0.01% butaprost (n=9). IOP was measured in both eyes at 3 hours, followed by measurements at 2, 6 and 9 days. JV-GL1 treated eyes were additionally measured at 12 and 16 days, whereupon a second challenge with 0.01% JV-GL1 was administered to one eye, with contralateral receiving vehicle. After 19 days, IOP was measured for a final time and mice culled for outflow facility measurements. Data was presented as the mean change in IOP from the pre-treatment time point (δIOP).
The third experiment aimed to compare the IOP-lowering effect of JV-GL1 between normotensive versus hypertensive WT C57BL/6J littermates (n=8 vs 9; 10 week old males). Normotensive individuals received unloaded nanoparticles. Baseline IOP was established once prior to bilateral injection of either dexamethasone loaded or unloaded nanoparticles into the periocular tissues. IOP was measured after 1 week to establish the level of ocular hypertension. One eye was then treated with 0.01% JV-GL1 with contralateral eye receiving vehicle. IOP was measured in both eyes at 3 and 24 hours, followed by measurements at 4 and 6 days. Data were presented as the mean difference between paired treated and untreated eyes (ΔIOP).
The fourth experiment investigated the IOP effect of JV-GL1 inPtger2-/- mice (5 males & 3 females, 12.5 [11, 14] weeks old) and Ptger2+/+ mice (5 males & 5 females, 13 [12, 14.1] weeks old) that had been rendered steroid induced ocular hypertensive. Baseline IOP was established once prior to bilateral injection of dexamethasone loaded nanoparticles into the periocular tissues. IOP was measured after one week to establish the level of ocular hypertension. One eye was then treated with 0.01% JV-GL1 with contralateral eye receiving vehicle. IOP was measured in both eyes at 3 and 24 hours, followed by measurements at 4, 6 and 11 days. Data were presented as the mean difference between paired treated and untreated eyes (ΔIOP).
In all IOP studies, the following statistical analyses were applied. Normality was checked by the Kolgomorov-Smirnov test, equity of variances between groups was determined by Levene’s test, and statistical significance between groups over multiple time points was established via a two-factor ANOVA. Errors are presented as 95% confidence intervals.