We next evaluated the effect of liquid formulation on SCS thickness, as well as the SCS collapse rate over time in the living rabbit (
Fig. 5A). We chose solutions of CMC at different concentrations in HBSS and the commercial viscoelastic product Discovisc (Alcon, Fort Worth, TX, USA) (which contains 1.65 MDa hyaluronic acid) as liquid formulations for this study, because these liquid formulations were previously shown to distribute differently in the SCS, compared with HBSS.
19
The initial SCS thickness at the injection site varied greatly with choice of liquid formulation from 0.43 ± 0.06 mm with HBSS to 2.1 ± 0.1 mm with 5% CMC in HBSS (
Figs. 5B,
6). The value for HBSS found here in the living rabbit eye is larger than what was found as explained above in the rabbit eye ex vivo. This could be because the in vivo measurement was made at the injection site, which was the site of maximum SCS thickness, whereas the ex vivo measurement was reported as the average SCS thickness through the expanded SCS. Use of Discovisc, which had previously been reported to initially remain near the site of injection in the SCS,
19 showed a SCS thickness of 1.5 ± 0.4 mm, which was significantly larger than the value for HBSS (
P < 0.01, Sidak's multiple comparison test). Suprachoroidal space injection of solutions containing 1%, 3%, and 5% CMC in HBSS (viscous solutions that have also been reported to localize at the injection site
19) had initial SCS thicknesses of 0.7 ± 0.1, 1.6 ± 0.2, and 2.1 ± 0.1 mm, respectively (
Fig. 5B). These data indicate that changing the formulation (to increase viscosity) had a larger effect on SCS thickness than increasing injection volume for a given formulation.
We next monitored SCS thickness over time at eight positions around the globe for all the formulations tested (
Supplementary Fig. S5). After injection of HBSS, the SCS thickness over the injection site achieved its peak value immediately after injection and then decreased according to a roughly first-order exponential decay; that is, there was no significant difference between SCS thickness immediately post injection (
θ0) and the maximal SCS thickness (
θmax) (
P > 0.99, Sidak's multiple comparison test,
Fig. 5B). Measurements at other locations around the globe behaved similarly.
There was also no difference between initial and maximal SCS thickness over the injection site for Discovisc and 1% CMC (
P ≥ 0.97, Sidak's multiple comparison test). However, the SCS thickness measured at the other sites behaved differently, which is consistent with a previous study.
19 With Discovisc, the decrease in SCS thickness at the injection site over time was accompanied by a concomitant increase in SCS thickness at adjacent sites in the SCS (
Supplementary Fig. S5A, 4 hours). By 2 days, the SCS thickness throughout the entire eye had returned to baseline. In contrast, 1% CMC expanded the SCS only at or near the injection site for the entire time course (data not shown). Because Kim et al.
19 had shown that Discovisc was able to facilitate the distribution of particles throughout the SCS and that CMC was able to localize particles near the injection site, we hypothesize that the expansion of a region of SCS was necessary for particle deposition in that region.
With 3% CMC and 5% CMC solutions,
θ0 over the injection site was different than
θmax (
P < 0.01, 2-way ANOVA) because the SCS thickness initially increased over the course of hours after the injection. This expansion of the SCS could be explained by an osmotic and hydration effect of the CMC within the SCS, which could draw in water from the surrounding tissue to dilute the CMC and cause swelling of the gel. Besides the swelling at the site of injection, the behavior of the SCS thickness at other positions (
Supplementary Fig. S4B) was similar to that found with 1% CMC.
To describe the time course of SCS thickness changes after injection over the injection site, we used a second-order exponential equation that could account for both the observed expansion and collapse of the SCS:
where
t is the time post injection,
θ(
t) is the SCS thickness as a function of time,
A and
B are thickness constants,
τexp is the expansion time constant, and
τcol is the collapse time constant (
Fig. 5C). This equation described the data from all the liquid formulations well (Pearson coefficient
r2 > 0.76).
Using this equation, we calculated the characteristic times associated with each of the liquid formulations. As expected, the liquid formulations that did not cause further expansion of the SCS after injection (i.e., HBSS, Discovisc, and 1% CMC) had calculated
τexp values were all on the order of seconds (
Fig. 5C, left). In contrast,
τexp values for the 3% CMC and 5% CMC liquid formulations ranged from 2.8 to 9.1 hours, and there was no significant difference among these
τexp values (
P = 0.77,
F-test).
There were significant differences in
τcol values among the liquid formulations tested (
Fig. 5C, right). With HBSS as the liquid formulation,
τcol was 19 ± 3 minutes. With the Discovisc liquid formulation,
τcol was 6 ± 2 hours, which was significantly longer than the HBSS value (
P < 0.005,
F-test). With all of the CMC liquid formulations,
τcol ranged from 2.4 to 9.2 days, which was also longer than for HBSS (
P < 0.0001,
F-test) but not different with respect to each other (
P = 0.47,
F-test). It is notable that collapse of SCS-containing 1% CMC solution (that did not swell after injection) and SCS-containing 5% CMC solution (which did swell after injection) had comparable
τcol values, which suggests that dissociation of the crosslinks found in CMC gels
28 is the rate-limiting step to CMC clearance from the SCS resulting in collapse.