Over the past 15 years, there has been an unprecedented explosion of outflow-targeted treatments to lower intraocular pressure (IOP) for glaucoma. In the last year, 2 new drugs targeting the conventional outflow have come to market.^1,2 In the decade prior, minimally invasive glaucoma surgeries (MIGS) exploded onto the scene as safe and easy glaucoma surgical options.¹ During this time, the unquestioned conclusion has been that targeting conventional outflow for IOP lowering is more complex than originally thought. MIGS are inconsistent,¹ and new conventional outflow drugs may do more than influence the trabecular meshwork by impacting distal outflow pathways.³ Therefore, improving current tools and developing new ones is necessary to simply understand the question of where the aqueous flows to better understand and improve modern therapeutics.

Aqueous Angiography

Aqueous humor outflow (AHO) pathways have been structurally studied using histology in the laboratory and optical coherence tomography (OCT) in live subjects.⁴ To complement structural information, the desire was to visualize where the fluid was flowing. This began in the laboratory with bead-based methods where fluorescent bead-tracers were placed into the eye, and their accumulation was observed in the trabecular meshwork (TM).⁵ These methods were limited in that they were not compatible with live subjects. Subsequently, methods like canalograms/channelograms were devised where during canaloplasty surgeries, tracers were introduced into Schlemm’s canal to visualize distal outflow pathways.⁶ However, the limitation was that outflow information did not include TM contributions. Therefore, a patient-friendly method that could visualize AHO from the anterior chamber and beyond was needed.

Aqueous angiography (AA) was developed as a method to visualize as physiologic as possible AHO using techniques suitable for live humans.^7,8 Borrowing from retina patient care where tracers (fluorescein or indocyanine green [ICG]) are delivered into peripheral veins to image retinal blood flow using angiographic cameras, we decided to add the same tracers into the eye and use the same camera pointed on the ocular surface to image AHO. Using a Heidelberg Spectralis and tubing/supplies from the operating room, aqueous angiography was developed in the laboratory using postmortem eyes from many species (pig, cow, dog, cat, and human^9-11). Then, the Spectralis was modified via installation onto an articulating arm (FLEX module; Figure 1) which allowed for camera positioning needed to image live subjects (nonhuman primate and humans) in the operating room.^12-14 From these experiments, we learned that AHO was segmental, pulsatile, and dynamic.

AHO Is Segmental

Previous laboratory experiments using fluorescent beads in animal models had already shown that AHO may be segmental. Beads could collect in one part of the TM but not the other.¹⁵ In other words, AHO was not circumferential and uniform around the limbus. Using AA, the same result was seen. After tracer delivery into the anterior chamber (AC), segmental AHO was seen with some parts of the eye showing more AHO than others (Figure 2). Consistent with MIGS literature, more AHO was seen nasally.¹⁴ These results were shown in postmortem eyes in the laboratory and in live subjects, across all species tested, and regardless of the choice of tracer. Put together, the results from AA combined with all prior laboratory publications now put to rest the question of whether AHO is uniform around the eye. It isn’t. It is segmental, and this fundamental biological description may challenge prior AHO research (ie, if TM cells were studied from high-flow vs low-flow regions) and serve as a method to discover novel therapies in the future.

AHO Is Pulsatile

When AA was brought to live subjects, additional behaviors were noted. First, angiographic AHO was pulsatile. Pulsatile AHO meant that AHO had an alternating and cyclical tempo while the angiographic outflow pattern was unchanging. This was reminiscent of 2 prior findings. First, pulsatile blood flow has been seen at aqueous and episcleral veins under high magnification.¹⁶ Second, pulsatile structural changes have been seen using phase-contrast OCT where outflow pathways alternately became structurally bigger and smaller.¹⁷ In OCT studies, careful correlation of structural pulsatility to the digital cardiac pulse showed an in-phase pulsatility with a slight temporal lag — the time that would be needed for cardiac output to reach the eye.¹⁸ In angiographic studies, while we did not have synchronized cardiac data with aqueous angiographic imaging, the aqueous angiographic pulsatility rates in nonhuman primates matched that of published average nonhuman primate heart rates.¹² Therefore, while the cause of this behavior is currently unknown, this observation becomes a potential link between the cardiovascular system and AHO/IOP. Choroidal expansion has been proposed to cause narrow-angle glaucoma via expansion, creating a piston-like effect.¹⁹ In this case, cardiac pulsatility transmitted to the choroid could cause a pulsatile posterior segment piston to influence AHO.

AHO Is Dynamic

When visualized with AA, AHO was also dynamic during live imaging. Unlike pulsatile AHO that maintained a stable pattern alternating between low and high signal, dynamic AHO meant that segmental patterns of AHO could shift across the eye. Regions of high or low flow could change, implying that in some circumstances the eye needed to open up more AHO (a positive phenomenon), and in other situations the eye needed less AHO (a negative phenomenon). This was seen in both nonhuman primates and humans.^12,13

The regulation behind dynamic AHO is unclear. Options include regulation at the TM where the eye can increase or decrease AHO. Alternatively, given that distal AHO pathways are reminiscent of vascular pathways with smooth muscle walls, dynamic AHO could be achieved by local regulation via distal AHO pathway vasoconstriction and dilation.²⁰ Yet a third option is biomechanical. Extraocular muscles insert onto the globe, and it has been long known that muscular contraction increases IOP.²¹ In our experiments, dynamic events could be seen during periods of eye motion, suggesting that forces placed on the ocular surface could direct where aqueous humor flows.

It is also important to emphasize that dynamic AHO was rare. In about 9 minutes of AHO video recording in nonhuman primates, there were only 16 dynamic events.¹² Therefore, AHO patterns are for the most part stable, and it appears that dynamic events arise at specific times due to reasons not yet appreciated. Reasons could include the need to adjust outflow to maintain a stable IOP.

AHO Imaging and Clinical Care

So how is AHO structural and fluid-flow imaging useful to patient care? First, a better understanding of the actual biology is important. Previous assumptions and an overly simplistic description of AHO (circumferential and unchanging) may have led to improper research conclusions in the past as well as therapeutic options left on the table. A fundamental description of AHO with the above metrics needs to be done, comparing normal and glaucomatous eyes. The pathology in glaucoma may be the loss of segmental high-flow regions or dynamic capacity. Also, new treatments could be envisioned. Drugs could be targeted to alter segmental, pulsatile, or dynamic AHO. Surgery could be personalized with this information as well.

The potential of improved glaucoma surgeries bears particular attention given that the recent explosion of MIGS was what drove much of the above research. Potentially, MIGS placement could be targeted with this AHO information for improved results. MIGS in high-flow regions may lead to improved IOP lowering by accessing known and stable AHO pathways. MIGS in low-flow regions could alternatively be enhanced by trying to restore AHO in low-flow regions to its full potential. To try to address some of these questions, in the laboratory and in live humans, sequential AA was developed to evaluate how AHO patterns changed with surgery. Indocyanine green angiography was performed first to establish a baseline pattern. Then TM bypass was performed, and fluorescein aqueous angiography was used to query the effect. In postmortem human eyes, it was shown that low-flow regions could be rescued.¹⁰ This was also the case in live human glaucomatous eyes.²² When surgery was performed in high-flow regions, faster and stronger angiographic AHO was seen. Now the goal is to determine which of these patterns is associated with better long-term IOP lowering.

AHO Imaging and the Future

The only certain lesson from all of this research is that AHO is not as simple as once thought. AHO is not a basic concept with one equation (the Goldman equation) modeling its behavior in all circumstances. New AHO characteristics include segmental, pulsatile, and dynamic AHO. Together, this describes a new concept of dynamic variable outflow (DVO), which means that AHO is not everywhere and it is not always stable and unchanging. New and innovative IOP-lowering therapeutic options may be developed by better studying DVO, particularly in those locations where AHO is actively changing and can be manipulated. GP

References

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Goichi Akiyama, MD, PhD, is a glaucoma specialist with the Doheny Eye Institute in Los Angeles, California. He reports no related disclosures.

Alex S. Huang, MD, PhD, is an assistant professor in the Department of Ophthalmology at the University of California, Los Angeles, and a founding member of the Doheny Eye Institute. He reports grant support from Heidelberg Engineering, Glaukos, and Diagnosys, as well as consultancy to Aerie Pharmaceuticals.