Visualization of conventional outflow tissue responses to netarsudil in living mouse eyes

Abstract

Visual impairment due to glaucoma currently impacts 70 million people worldwide. While disease progression can be slowed or stopped with effective lowering of intraocular pressure, current medical treatments are often inadequate. Fortunately, three new classes of therapeutics that target the diseased conventional outflow tissue responsible for ocular hypertension are in the final stages of human testing. The rho kinase inhibitors have proven particularly efficacious and additive to current therapies. Unfortunately, non-contact technology that monitors the health of outflow tissue and its response to conventional outflow therapy is not available clinically. Using optical coherence tomographic (OCT) imaging and novel segmentation software, we present the first demonstration of drug effects on conventional outflow tissues in living eyes. Topical netarsudil (formerly AR-13324), a rho kinase/ norepinephrine transporter inhibitor, affected both proximal (trabecular meshwork and Schlemm's Canal) and distal portions (intrascleral vessels) of the mouse conventional outflow tract. Hence, increased perfusion of outflow tissues was reliably resolved by OCT as widening of the trabecular meshwork and significant increases in cross-sectional area of Schlemm's canal following netarsudil treatment. These changes occurred in conjunction with increased outflow facility, increased speckle variance intensity of outflow vessels, increased tracer deposition in conventional outflow tissues and decreased intraocular pressure. This is the first report using live imaging to show real-time drug effects on conventional outflow tissues and specifically the mechanism of action of netarsudil in mouse eyes. Advancements here pave the way for development of a clinic-friendly OCT platform for monitoring glaucoma therapy.

Keywords: Conventional outflow; Netarsudil (PubChem CID: 66599893); Ocular hypertension; Rho kinase inhibitor; Schlemm’s canal; Trabecular meshwork.

Figure 1

Netarsudil lowered intraocular pressure (IOP) in both pigmented and nonpigmented mice. Ten week-old C57 and 6-14 week-old CD1 mice were divided into 2 age and gender-matched groups (5 mice/group, 4 groups total). For each strain of mice, one group was given 10 μl of 0.04% netarsudil mesylate topically, and another group received 10 μl of a placebo (CF324-01) eye drop. All eye drops were applied to right eyes. IOP was measured in both eyes before eye drops were given. The data represent mean ± S.E.M. of ΔIOP compared between netarsudil and placebo-treated group analyzed by Mann Whitney U-test. *, P < 0.05; **, P < 0.01.

Figure 2

Netarsudil mesylate enhanced IOP recovery in living mouse eyes. Vehicle (0.001% DMSO) or 100 nM netarsudil mesylate was preloaded into one of two perfusion needles and inserted intracamerally into contralateral eyes of living mice. Both eyes were simultaneously exposed to intraocular pressure (IOP) of 15 mmHg, allowing drug/vehicle to enter eyes for 30 min. IOP was artificially raised to 40 mmHg in both eyes and held for 5 min using a fluid reservoir. Connection to the fluid reservoir was closed, but remained open to pressure transducers, which monitored IOP in both eyes over time. Panel A. Representative IOP decay curves in paired eyes exposed to vehicle (DMSO) or netarsudil mesylate. Panel B. Relative changes in α (a constant characterizing the rate of pressure decay). The data represent mean ± S.E.M., N=8, **, P < 0.01 by student t-test.

Figure 3

Netarsudil mesylate increased outflow facility in perfused mouse eyes ex vivo. Outflow facility measurement was conducted in paired enucleated eyes using an iPerfusion system. The drug (netarsudil mesylate) and vehicle (0.001% DMSO) were preloaded in microneedles and perfused for 45-60 min before exposing eyes to 9 sequential pressure steps. Panel A. Representative experiment showing flow rate (Q) vs pressure (P) that was used to calculate outflow facility in a single pair of eyes. Panel B. Cello plot (Sherwood et al., 2016) shows percentage change in facility for each pair of eyes in response to treatment with netarsudil. Dark blue error bars indicate confidence interval from the fitting of the model to the flow-pressure data and lighter error bars include additional uncertainty arising from differences between contralateral eyes of C57BL/6 mice (Sherwood et al., 2016). Shaded region shows estimated lognormal distribution of percentage change in facility. Outer white lines show 2 standard deviations and central lines shows the average increase. Dark central band shows the 95% confidence interval on the mean increase. N=8, P = 0.006 by a paired weighted t-test. Panel C. Cello plot shows percentage change in facility for each pair of eyes in response to treatment with netarsudil for CD1 mice. Lighter error bars assume that the additional uncertainty arising from differences between contralateral eyes in CD1 mice is similar to that of C57 mice. N=6, P=0.025.

Figure 4

Enhanced tracer deposition in outflow tissues of living mice subjected to netarsudil mesylate treatment. Fluorescent microbeads were loaded into microneedles in the presence or absence of netarsudil mesylate, anterior chambers were cannulated and paired eyes simultaneously perfused at a constant flow rate of 0.167 μl/min. Perfusion was stopped after one hour and the mice were maintained for another hour before being euthanized. Shown are flat mounted anterior segments of C57 and CD1 mouse eyes that were visualized using epifluorescence microscopy (panel A). Summary of comparisons between netarsudil mesylate-treated (NT) and vehicle (0.001% DMSO)-treated groups are shown in panel B. Fluorescence intensity, the width and area of fluorescence in conventional outflow region were automatically quantified. Data represent mean ± S.E.M, N=5 for both strains of mice. *, P < 0.05, **, P < 0.01 compared to their contralateral vehicle control eyes by student t-test. Panels C and D show representative fluorescence intensity and width maps of conventional outflow regions from four quadrants of paired eyes. The variation of mean intensity (of all pixels with identical angles to the center) (panels C) and the variation of width (panels D) are shown with the variation of angle from the center of the circular TM/SC regions (from −180 to 180 degrees). The 0 (180) degree is represented by the positive X-axis.

Figure 5

Netarsudil-induced changes in conventional outflow tissue morphology of living mice visualized by optical coherence tomography (OCT). Panels A shows representative averaged OCT images from 200 B-scans of iridocorneal angles (identical positions) before and 45 min after topical netarsudil or placebo treatment of living C57 mice (IR: iris; black arrows point to TM tissue). Overlayed atop averaged images are segmentations (blue) of SC using Schlemm II software. Arrow in red, shows expanded TM after netarsudil treatment. The speckle variance images (SPV) of same eyes are shown alongside averaged images. Arrows in white point out scleral vessels conducting aqueous humor outflow and asterisks indicate SC. Panels B and C show quantitative results summarizing effect of treatments on area and speckle variance intensity of SC and scleral vessels conducting aqueous humor outflow using Schlemm III software. The data represent mean ± S.E.M.. N = 5 for each treatment. *P < 0.05 (student t-test).

Figure 6

Netarsudil increased cross-sectional area of Schlemm's canal (SC) lumen in living mice with elevated intraocular pressure (IOP) visualized by optical coherence tomography (OCT). Panel A shows representative averaged images from 200 B-scans of iridocorneal angles (identical positions) of a live C57 mouse before and 40 min after netarsudil treatment, with IOP held at 10 mmHg. Semi-automatic segmentation (SEG) of SC is shown overlayed in blue. Bottom panels show OCT speckle variance (SPV) images. (IR = iris). Panels B and C show results of quantitative SC segmentation from OCT images analyzed using Schlemm II software. C57 and CD1 mouse eyes were imaged at the same location by OCT with a glass needle inserted into anterior chamber to control intraocular pressure sequentially at 10, 15, and 30 mmHg before and 30-60 min after treatment (topical netarsudil or placebo eye drops). The data show SC area (± S.D.) relative to the area of SC measured just prior to treatment at the starting IOP of 10 mmHg. Eleven mice were examined in this set of experiments. *P < 0.05; **P < 0.01 (Mann Whitney U-test).

Figure 7

Netarsudil-induced changes in flow area and intensity in scleral vessels visualized on OCT speckle variance images. Panels A show representative speckle variance images of C57 and CD1 mouse outflow tissues before and after netarsudil or placebo eye drops. Arrows show scleral vessels before and 30-60 min after netarsudil treatment. Panels B and C show a summary of quantitative segmentation data expressed as relative changes in scleral vessel cross-sectional area and intensity after netarsudil or placebo treatment in C57 pigmented versus CD1 non-pigmented mice. Speckle variance images were visualized by Schlemm II version software, and the areas of the intrascleral plexi near SC were selected for analysis. Speckle variance intensities in those areas were analyzed using Schlemm III version software. The data represent mean ± S.E.M,, N=4-5 from each group and each strain of mice, *, P < 0.05 (Student’s t-test).