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Editors Selection IGR 12-2

Anatomical Structures: Distal Aqueous Outflow Pathways

Comment by Nils Loewen & Choa Wang on:

75468 Deep tissue analysis of distal aqueous drainage structures and contractile features, Gonzalez JM; Ko MK; Hong YK et al., Scientific reports, 2017; 7: 17071

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Gonzalez et al.¹ present a smorgasbord of images of the distal outflow tract of the mouse in a total of 14 figures with up to 20 subpanels per figure. The authors had several goals that were rather ambitious: (1) to establish 2-photon deep tissue imaging with 3D reconstruction of the imaging data; (2) to distinguish blood from lymphatic markers; and (3) to detect structures suggesting contractility. The primary technique applied in these studies has a name that is almost as long as the list of goals: 'trans-scleral multimodal 2-photon imaging with 2-photon excitation fluorescence and second harmonic generation (2P TPEF SHG)'. 2P TPEF SHG uses deep red and near infrared light to penetrate tissue deeper than visible light used in standard confocal microscopy. It has reduced scattering which allows the fluorescent target to be well identified. The principal behind 2P TPEF SHG is that when two photons with the same frequency interact with a non-linear material they are combined in a process called second harmonic generation which generates a new photon with twice the energy of the initial photons. The difference between the exciting and the emitting light allows for easy separation of the second harmonic generation signal and a high axial and lateral resolution that is comparable to that of confocal microscopy but without having to use pinholes.

This technique is impressive and useful because it allows imaging tissues in vivo up to 100 to 200 microns² with a resolution that allows to distinguished structures several microns in size. The practically achievable depth depends on the amount of light scatter, however. A downside of 2P TPEF SHG is that fluorophores are necessary which requires either the use of transgenic animals with fluorescent proteins or ex vivo stains or antibodies. In contrast to confocal microscopy, that can achieve up to 365 nm resolution at 2 mm depth,^3,4 the tissue does not have to be made transparent.

If a bit overwhelming at first, Gonzalez et al.'s manuscript is a treasure trove for outflow researchers that convincingly demonstrates that features are present in the distal outflow tract of the mouse that should allow it to actively contract and dilate. Recent studies show that the distal outflow tract can indeed regulate outflow and IOP by contracting and dilating in human and porcine eyes.^5,6 Interestingly, the distal outflow tract endothelium was Prox1-positive, CD31-positive but LYVE-1-negative, giving it a signature different from blood and true lymphatic vessels. Schlemm's canal is a VEGF-C/VEGFR3-responsive lymphatic-like vessel⁷ that develops from transscleral veins⁸ and emerges together with the collector channels. While disappointing that not more lymphatic features were seen in the distal outflow tract of the mouse, they may play a more prominent role in larger primate eyes. Lymphatic features in direct proximity to these structures are already known to get activated by epibulbar glaucoma surgery where aqueous is shunted into an artificial conjunctival fluid pocket and absorbed by lymphatics.⁸ Recent discoveries on prostaglandin analogs, arguably the most successful eye drops in glaucoma, showed that in addition to enhancing uveoscleral outflow, they increase contraction and propulsion of lymphatics.⁹

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