February 2019


Presentation spotlight
New imaging technique uses fluorescent markers to identify cell makeup of aqueous collecting system

by Stefanie Petrou Binder, MD, EyeWorld Contributing Writer

Two-photon image of collector channel. A significant proportion of the channel cross-sectional area comprises wall cells with filamentous actin (red), representing a contracted state. This leaves a lumen (black oval) that is smaller than expected relative to the scleral opening (sclera in cyan).
Source: James Tan, MD

New data demonstrates how the distal outflow tract in mouse eyes exhibits cells with a mixed lymphatic and blood vessel identity

New imaging data describes the distal outflow tract as not just an inert series of tubes and openings within the sclera but a vascular system of its own type. Investigator James Tan, MD, Department of Ophthalmology, University of California, Los Angeles, discussed his novel research findings on imaging aqueous outflow during a symposium at the 36th Congress of the European Society of Cataract and Refractive Surgeons.
“The distal outflow system is not static, but a dynamic system with cells that are able to self-regulate,” Dr. Tan said in his presentation. “It can regulate itself in response to all kinds of factors, which raises some questions, for instance about the effect a dynamic system might have on bypass outcomes or how the distal outflow tract might change and adapt to the new fluid dynamic coming in from the anterior chamber after MIGS. Finally, it is possible that this system might be targeted and manipulated pharmacologically, just as we would treat arteries for systemic hypertension.”

Trabecular meshwork

Understanding the aqueous outflow tract is key in working toward solving high IOP and is the subject of Dr. Tan’s research. The trabecular meshwork has been considered a tissue of high resistance that limits the exit of aqueous into the distal outflow system. Why then is it that MIGS, in bypassing the trabecular meshwork, does not reduce IOP to that of episcleral venous pressure? And why are outcomes of MIGS so varied?
According to Dr. Tan, the aqueous outflow system may be more complex than what was thought, and understanding that complexity could yield answers to many questions. “There is a conventional view we have of the distal outflow system,” he explained. “To exit the eye, the aqueous humor has to pass through the trabecular meshwork into the distal outflow tract, which is the drainage tract that lies within the sclera. It exits the meshwork into Schlemm’s canal and into the collector channels, through the intrascleral plexus, which are large lakes of aqueous within the sclera, and onto the aqueous veins to exit the eye into the episcleral veins. But is it this simple?” he said.
Improved imaging systems have added much to the knowledge of the aqueous outflow pathway; however, little has been elucidated about this tissue on the cellular level. In his research lab, Dr. Tan performed two-photon excitation fluorescence microscopy, a technology that uses long wavelength laser that allows deeper penetration of tissues. This imaging modality allows very precise optical sectioning and high resolution to image cellular and even sub-cellular compartments with minimal phototoxicity. In eyes of transgenic reporter mice engineered to allow visualization of specific mouse proteins marked with a fluorescent marker, Dr. Tan was able to come closer to the true nature of the aqueous collecting pathways.

New imaging of the distal outflow tract

“The aqueous outflow system of the reporter mouse is remarkably similar to that of humans, only smaller. By using two-photon microscopy, we were able to see that the intrascleral plexus in mice is lined by cells,” Dr. Tan explained. He used special markers to visualize both cells and collagen in mouse eyes. “We wondered, if the distal outflow tract is lined by cells, is it a type of vascular system? A vascular system would be expected to be lined by endothelium. There are different types of endothelia, however, with each peculiar to vessel type such as blood vessels or lymphatic vessels. We needed to distinguish between them. We also wondered whether the distal tract lining had muscle cells, as are present in blood vessels. If so, muscle cells could provide this system with the capacity to contract, just like blood vessels,” he said.
The type of endothelium present in a tissue will tell you the type of fluid you are dealing with, such as blood, lymph, or even aqueous humor. Dr. Tan investigated the presence of a protein that is known to be specific to lymphatic tissue in humans, called Prox1. He was able to visualize Prox1 in the engineered mouse. Prox1 is present throughout the mouse’s Schlemm’s canal, but also in the downstream vessels including collector channels and intrascleral plexus. He went on to assess if Schlemm’s canal expressed other classic markers of true lymphatic vessels.
Dr. Tan labeled the tissue for LYVE1, a protein found only in true lymphatic endothelial cells, and found that it was present in adjacent lymphatic vessels but not Schlemm’s canal itself. He concluded that Schlemm’s canal has a partial lymphatic identity due to the presence of Prox1 but it is not truly a lymphatic vessel.
Dr. Tan considered the possibility that the distal tract also showed blood vessel characteristics. CD31 is a protein found in blood vessel endothelium but barely in lymphatics. He labeled the same mouse tissues for CD31 and found that whereas blood vessels expressed CD31 and lymphatics Prox1, Schlemm’s canal endothelium expressed both CD31 and Prox1. The evidence suggests Schlemm’s canal and the distal tract carry an identity somewhere between that of blood and lymphatic vessels.
Dr. Tan searched for other similarities between the distal aqueous tract and blood vessels such as the presence of smooth muscle in vessel walls. The distal outflow tract was labeled for alpha smooth muscle actin, a smooth muscle marker. Dr. Tan established that the intrascleral plexus was lined by smooth muscle in a pattern resembling choroidal arteries from the same eye, indicating the wall organization of the intrascleral plexus resembles that of arteries.
“In a high magnification image of a collector channel taken by our team, we visualized large scleral openings, which we tend to think are open conduits for the outflow of aqueous. In fact, they are packed with cells with a smooth muscle identity. These cells have the capacity to contract and in doing so alter the lumen size of collector channels. This could be deduced in our images as the relatively small channel lumen relative to surrounding lining cells in a contracted state,” he explained.
Based on Dr. Tan’s imaging data it seems the distal outflow tract is anything but an inert series of draining pipes. “We are dealing with a vascular system of its own type,” he said. “It has an endothelium a bit like lymphatics, but not exactly, and it does not have the full characteristics of blood vessels. We think it has its own type of endothelium and is its own type of vascular system. This is not that surprising given it carries a different kind of fluid. Aqueous humor is a unique fluid that is different from other fluids in the body. But the distal tract is similar to blood vessels in having a lining of smooth muscle with capacity to contract. If it contracts, you can imagine how it could constrict the lumen of this system, creating a lower outflow, high resistance system that could affect IOP. It certainly seems more dynamic than the system we had always envisioned,” he said.

Editors’ note: Dr. Tan has no financial interests related to his comments.

Contact information

: oranghutan@aol.com

New imaging technique uses fluorescent markers to identify cell makeup of aqueous collecting system New imaging technique uses fluorescent markers to identify cell makeup of aqueous collecting system
Ophthalmology News - EyeWorld Magazine
283 110
220 156
True, 2