Imaging the pathway

Anatomists used to draw the aqueous humor outflow pathway. Then OCT imaging came along.

Much of our knowledge of the aqueous humor outflow pathway stems from the work of gifted anatomists.1 While providing a basic understanding of the components of outflow pathway, their drawings and histological sections of the limbus fail to convey the rich vascular density and characteristic vascular morphology of this most fascinating system.

Physical castings of the vasculature provided the first true visualizations of the outflow pathway. For the first time, we saw the distinct morphology and density of the rich vascular network distal to Schlemm’s canal. Modern imaging technology has created the opportunity to view these structures within cadaveric and living eyes. Leveraging the differences in scattering characteristics of clear fluid and turbid tissue, we demonstrated the ability to isolate portions of the functioning outflow pathway within volumetric scans of the limbus.2 This work has been reproduced in other institutions3 and spurred a growing interest in visualization of the outflow pathway in-situ. Work is underway using tracers and dyes in animal eyes, human cadaveric eyes and even in living eyes during invasive procedures.

This article will provide a brief update on the present status of efforts to visualize the aqueous humor outflow pathway.


In a series of groundbreaking publications, Norman Henry Ashton, cbe, dsc (Lond), frcp, frcs, gave us our first view of the morphology of the human aqueous humor outflow vasculature.4-6 Best known for his later landmark work demonstrating the role of oxygen in retinopathy of prematurity,7-10 Dr. Ashton’s early research included the creation of physical castings of the aqueous humor outflow pathway. He injected neoprene rubber into Schlemm’s canal (Figure 1) with sufficient pressure to fill the distal vasculature. Surrounding tissue was dissolved with acid, leaving a cast of Schlemm’s canal and the distal vasculature.5 This was the first visualization of the dense vascular network of aqueous humor vessels surrounding Schlemm’s canal.

Figure 1. Dr. Ashton’s neoprene castings of the aqueous humor outflow pathway reveal a characteristic morphology comprised of a vascular network of interconnected linear segments surrounding the canal — both anteriorly into the outer limbus and posteriorly toward the trabecular meshwork.

Focusing on Schlemm’s canal, we can observe in Figure 2 portions of the canal that separate into multiple parallel passages (yellow arrows) which then come back together into a single channel.5 These observations of local splitting and convergence of the canal challenge the dogma of a single intrascleral passage, which remains pervasive to this day. Also note that the presentation of Schlemm’s canal in Figure 2 is that of a large passageway. High pressure was needed to push the viscous neoprene through the canal and into the distal vasculature. It is possible that the pressure required to make these castings distended the canal to nonphysiological proportions.

Figure 2. Despite distortions associated with the high pressures required to push neoprene through the outflow vasculature, Dr. Ashton’s castings revealed the splitting of Schlemm’s canal into multiple parallel pathways, which converged again into a single pathway.

More recent resin castings of the outflow pathway in owl and cynomolgus monkey eyes reveals Schlemm’s canal at physiological pressures (Figure 3).11 It is possible that this is a more physiological representation of Schlemm’s canal. We will see that this presentation is consistent with that obtained in “virtual castings” of the same structures in living human eyes.12

Figure 3. Resin castings out the outflow pathway provide visualization of Schlemm’s canal (bottom) as a nodular structure.


Utilizing advances in optical coherence tomography (OCT) imaging hardware and the sophistication of applied computer vision techniques to OCT imagery, we have demonstrated the ability to map the functioning aqueous humor outflow pathway in cadaveric and living eyes.2,13-15 Aqueous humor produces almost no signal within an OCT image. Thus, aqueous humor within the outflow vasculature creates a series of dark voids within the sclera. The black regions within the processed limbal OCT B-scan are comprised of aqueous humor displacing scleral tissue within the outflow vasculature and Schlemm’s canal (arrow). When a series of these scans is obtained through a volume of the limbus and image processing is used to display only the black regions (displayed as white in a black background), the living Schlemm’s canal (Figure 4) and distal vasculature (Figure 5) are visualized.2,13,15,16

Expanding the view to the entire OCT scan volume, the similarities between the living system and the casts created by Dr. Ashton become apparent. Since first published, the work has been repeated using newer swept-source OCT devices.3

Figure 4. This virtual casting of a living human cast of Schlemm’s canal (the author’s) reveals a nodular and irregular structure. The arrows identify collector channels protruding from the canal into the surrounding vascular network.12

Figure 5. Noninvasive “vascular casting” (right) provides a visualization of the outflow pathway remarkably similar to that produced by a physical neoprene cast (left). The yellow arrows identify Schlemm’s canal.


Staining of the endothelial cells lining Schlemm’s canal is a powerful technique providing visualization of its structure.17,18 Van Der Merwe and colleagues have led the field in this area, providing unprecedented images of the morphology of Schlemm’s canal and the distal outflow pathway. (Figure 6) Their work confirms the nodular appearance of the canal as well as the intermittent splitting and convergence of the canal as observed in the techniques mentioned above.

Figure 6. The nodular morphology of Schlemm’s canal observed in both resin and “virtual castings” is confirmed in an endothelial staining in a mouse eye.

Several labs have applied a labeling approach to visualization of outflow and, additionally, to the quantification of the effect of surgical interventions on filling of the outflow vasculature distal to Schlemm’s canal. Huang, et al. have demonstrated increased filling of previously quiescent outflow channels in human cadaver eyes after implant of a trabecular meshwork bypass shunt by aqueous outflow angiography (Figure 7).19 Our group, lead by Nils Loewen, MD, has demonstrated increased filling of the outflow vasculature in a porcine flow model after ab interno trabeculoplasty, using fluorescein outflow angiography.20 We combined this technique with the “virtual casting” technique to visualize the newly opened pathways (Figure 8). Applying an automated technique allows quantification of the filling times and fluorescent intensities in these flow models, yielding a useful research tool.21

Figure 7. A large outflow vessel revealed by outflow angiography in a cadaveric outflow model.

Figure 8. Outflow angiography (left) combined with virtual casting (right) demonstrates the recruitment of previously quiescent outflow vasculature after ab interno trabeculoplasty.

The angiography technique has recently been demonstrated in living human eyes. Huang et al. have published the first series of outflow angiograms in human subjects.22 While the images provide excellent visualization of gross structures in the outflow pathway, this invasive technique does not appear to capture greater detail than that provided by the noninvasive technique of “virtual casting” or mapping of scleral voids. (Figure 9) However, the mapping of scleral voids cannot ensure that only outflow vasculature is imaged. Angiography of the outflow pathway provides a high degree of confidence that the resultant imagery contains only those vessels distal to Schlemm’s canal.

Figure 9. A side-by-side comparison of invasive outflow angiography (left) and non-invasive “virtual casting” (right) suggests that a similar level of structure detail may be obtained by either technique.3,22


The aqueous humor outflow tract has been imaged in a variety of species and by a range of techniques: from physical to virtual castings, from endothelial labeling to angiography, from mouse to man. Minor technique-related distortions inherent to the early casting techniques created false perceptions of Schlemm’s canal, perpetuating the dogmatic belief of a large continuous structure. These misperceptions were corrected with subsequent improvements in casting techniques and confirmed by non-invasive “virtual casting” as well as endothelial staining. These newer techniques showed a nodular structure with intermittent splitting into multiple parallel channels that converge again into a single passage.

From the earliest castings from Dr. Ashton to the most recent virtual casts and angiograms, the vascular distal to Schlemm’s canal has consistently presented with a characteristic morphology comprised of a network of interconnected linear segments completely surrounding Schlemm’s canal — both anteriorly into the outer limbus and posteriorly toward the trabecular meshwork. Parallel, continued development in noninvasive “virtual casting” and aqueous humor outflow angiography promise ever-nearer transition of outflow imaging from research to clinical use. OM

Imaging References:

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  4. Ashton N. Anatomical study of Schlemm’s canal and aqueous veins by means of neoprene casts. Part I. Aqueous veins. The British journal of ophthalmology. 1951;35:291-303.
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  7. Ashton N, Cook C. Direct observation of the effect of oxygen on developing vessels: preliminary report. The British journal of ophthalmology. 1954;38:433-440.
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  18. van der Merwe EL, Kidson SH. Wholemount imaging reveals abnormalities of the aqueous outflow pathway and corneal vascularity in Foxc1 and Bmp4 heterozygous mice. Experimental eye research. 2016;146:293-303.
  19. Huang AS, Saraswathy S, Dastiridou A, et al. Aqueous Angiography-Mediated Guidance of Trabecular Bypass Improves Angiographic Outflow in Human Enucleated Eyes. Investigative ophthalmology & visual science. Sep 01 2016;57:4558-4565.
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