Loewen, R. | Search bioRxiv

bioRxiv2020

Dose-dependent effects of netarsudil, a Rho-kinase inhibitor, on the distal outflow tract

PurposeTo characterize the effects of netarsudil on the aqueous humor outflow tract distal to the trabecular meshwork (TM). We hypothesized that netarsudil increases outflow facility in eyes with and without circumferential ab interno trabeculectomy (AIT) that removes the TM. Methods64 porcine anterior segment cultures were randomly assigned to groups with (n=32) and without circumferential AIT (n=32). Cultures were exposed to 0.1, 1, and 10 M netarsudil (N= 8 eyes per concentration). For each concentration, IOP and vessel diameters were compared to their respective pretreatment baselines. Outflow tract vessel diameters were assessed by spectral-domain optical coherence tomography (SDOCT) and rendered in 4D (XYZ time-series). ResultsNetarsudil at 1 M reduced IOP in both eyes with TM (-0.60{+/-}0.24 mmHg, p = 0.01) and in eyes without TM (-1.79{+/-}0.42 mmHg, p<0.01). At this concentration, vessels of the distal outflow tract dilated by 72%. However, at 0.1 M netarsudil elevated IOP in eyes with TM (1.59{+/-}0.36 mmHg, p<0.001) as well as in eyes without TM (0.23{+/-}0.32 mmHg, p<0.001). Vessels of the distal outflow tract constricted by 31%. Similarly, netarsudil at a concentration of 10 M elevated IOP both in eyes with TM (1.91{+/-}0.193, p<0.001) and in eyes without TM (3.65{+/-}0.86 mmHg, p<0.001). At this concentration, outflow tract vessels constricted by 27%. ConclusionIn the porcine anterior segment culture, the dose-dependent IOP changes caused by netarsudil matched the diameter changes of distal outflow tract vessels. Hyper- and hypotensive properties of netarsudil persisted after TM removal.

physiology↗

bioRxiv2020

3D-Reconstruction of the Human Conventional Outflow System by Ribbon Scanning Confocal Microscopy

PurposeRisk for glaucoma is driven by the microanatomy and function of the anterior segment. We performed a computation-intense, high-resolution, full-thickness ribbon-scanning confocal microscopy (RSCM) of the outflow tract of two human eyes. We hypothesized this would reveal important species differences when compared to existing data of porcine eyes, an animal that does not spontaneously develop glaucoma. MethodsAfter perfusing two human octogenarian eyes with lectin-fluorophore conjugate and optical clearance with benzyl alcohol benzyl benzoate (BABB), anterior segments were scanned by RSCM and reconstructed in 3D for whole-specimen rendering. Morphometric analyses of the outflow tract were performed for the trabecular meshwork (TM), limbal, and perilimbal outflow structures and compared to existing porcine data. ResultsRSCM provided high-resolution data for IMARIS-based surface reconstruction of outflow tract structures in 3D. Different from porcine eyes with an abundance of highly interconnected, narrow, and short collector channels (CCs), human eyes demonstrated fewer CCs which had a 1.5x greater cross-sectional area (CSA) and 2.6x greater length. Proximal CC openings at the level of Schlemms canal (SC) had a 1.3x larger CSA than distal openings into the scleral vascular plexus (SVP). CCs were 10.2x smaller in volume than the receiving SVP vessels. Axenfeld loops, projections of the long ciliary nerve, were also visualized. ConclusionIn this high-resolution, volumetric RSCM analysis, human eyes had far fewer outflow tract vessels than porcine eyes. Human CCs spanned several clock-hours and were larger than in porcine eyes. These species differences may point to factors downstream of the TM that increase our vulnerability to glaucoma. Grant informationNational Eye Institute K08EY022737 (NAL); Initiative to Cure Glaucoma of the Eye and Ear Foundation of Pittsburgh (NAL); Wiegand Fellowship of the Eye and Ear Foundation of Pittsburgh (YD); P30-EY08098 (NAL); Department grant by Research to Prevent Blindness (NAL); an unrestricted fellowship grant from the Xiangya Hospital of Central South University (SC).

physiology↗