High-Resolution, Three-Dimensional Reconstruction of the Outflow Tract Demonstrates Segmental Differences in Cleared Eyes

Purpose:​​ ​The​ ​rate​ ​of​ ​conventional​ ​aqueous​ ​humor​ ​outflow​ ​is​ ​the​ ​highest​ ​nasally.​ ​We hypothesized​ ​that​ ​this​ ​is​ ​reflected​ ​in​ ​regionally​ ​different​ ​outflow​ ​structures​ ​and​ ​analyzed​ ​the​ ​entire limbus​ ​by​ ​high-resolution,​ ​full-thickness​ ​ribbon-scanning​ ​confocal​ ​microscopy​ ​(RSCM). Methods:​​ ​We​ ​perfused​ ​pig​ ​eyes​ ​by​ ​anterior​ ​chamber​ ​cannulation​ ​with​ ​eight​ ​lectin-fluorophore conjugates,​ ​followed​ ​by​ ​optical​ ​clearance​ ​with​ ​benzyl​ ​alcohol​ ​benzyl​ ​benzoate​ ​(BABB).​ ​RSCM​ ​and​ ​an advanced​ ​analysis​ ​software​ ​(Imaris)​ ​were​ ​used​ ​to​ ​reconstruct​ ​a​ ​three-dimensional,​ ​whole-specimen rendering​ ​of​ ​the​ ​perilimbal​ ​outflow​ ​structures.​ ​We​ ​performed​ ​morphometric​ ​analyses​ ​of​ ​the​ ​outflow tract​ ​from​ ​the​ ​level​ ​of​ ​the​ ​trabecular​ ​meshwork​ ​(TM)​ ​to​ ​the​ ​scleral​ ​vascular​ ​plexus​ ​(SVP). Results:​ ​​Except​ ​for​ ​pigmented​ ​structures,​ ​BABB​ ​cleared​ ​the​ ​entire​ ​eye.​ ​Rhodamine-conjugated Glycine​ ​max​​ ​agglutinin​ ​(soybean,​ ​SBA)​ ​labeled​ ​the​ ​outflow​ ​tract​ ​evenly​ ​and​ ​retained​ ​fluorescence​ ​for months.​ ​RSCM​ ​produced​ ​terabyte-sized​ ​files​ ​allowing​ ​for​ ​in​ ​silico​ ​dissection​ ​of​ ​outflow​ ​tract​ ​vessel​ ​at​ ​a high​ ​resolution​ ​and​ ​in​ ​3D.​ ​Networks​ ​of​ ​interconnected​ ​lumina​ ​were​ ​traced​ ​from​ ​the​ ​TM​ ​to downstream​ ​drainage​ ​structures.​ ​The​ ​collector​ ​channel​ ​(CC)​ ​volumes​ ​were​ ​ten​ ​times​ ​smaller​ ​than​ ​the receiving​ ​SVP​ ​vessels,​ ​the​ ​largest​ ​of​ ​which​ ​were​ ​in​ ​the​ ​inferior​ ​limbus.​ ​Proximal​ ​CC​ ​diameters​ ​were​ ​up to​ ​four​ ​times​ ​the​ ​size​ ​of​ ​distal​ ​diameters​ ​and​ ​more​ ​elliptical​ ​at​ ​their​ ​proximal​ ​ends.​ ​The​ ​largest​ ​CCs were​ ​found​ ​in​ ​the​ ​superonasal​ ​and​ ​inferonasal​ ​quadrants​ ​where​ ​the​ ​highest​ ​outflow​ ​occurs. Conclusions:​ ​​RSCM​ ​of​ ​cleared​ ​eyes​ ​enabled​ ​high-resolution,​ ​volumetric​ ​analysis​ ​of​ ​the​ ​outflow tract.​ ​The​ ​proximal​ ​structures​ ​had​ ​greater​ ​diameters​ ​nasally​ ​while​ ​the​ ​SVP​ ​was​ ​larger​ ​in​ ​the​ ​inferior