Morphometric analysis of aqueous humor outflow structures with spectral-domain optical coherence tomography

Abstract

Purpose: To describe morphometric details of the human aqueous humor (AH) outflow microvasculature visualized with 360-degree virtual castings during active AH outflow in cadaver eyes and to compare these structures with corrosion casting studies.

Methods: The conventional AH outflow pathways of donor eyes (n = 7) and eyes in vivo (n = 3) were imaged with spectral-domain optical coherence tomography (SD-OCT) and wide-bandwidth superluminescent diode array during active AH outflow. Digital image contrast was adjusted to isolate AH microvasculature, and images were viewed in a 3D viewer. Additional eyes (n = 3) were perfused with mock AH containing fluorescent tracer microspheres to compare microvasculature patterns.

Results: Observations revealed components of the conventional outflow pathway from Schlemm's canal (SC) to the superficial intrascleral venous plexus (ISVP). The superficial ISVP in both our study and corrosion casts were composed of interconnected venules (10-50 μm) forming a hexagonal meshwork. Larger radial arcades (50-100 μm) drained the region nearest SC and converged with larger tortuous vessels (>100 μm). A 360-degree virtual casting closely approximated corrosion casting studies. Tracer studies corroborated our findings. Tracer decorated several larger vessels (50-100 μm) extending posteriorly from the limbus in both raw and contrast-enhanced fluorescence images. Smaller tracer-labeled vessels (30-40 μm) were seen branching between larger vessels and exhibited a similar hexagonal network pattern.

Conclusions: SD-OCT is capable of detailed morphometric analysis of the conventional outflow pathway in vivo or ex vivo with details comparable to corrosion casting techniques.

Figure 1.

A drawing showing the general organization of the aqueous and blood vessels of the ocular limbus. The superficial limbus is indicated between the upper and middle horizontal dashed lines, the mid limbus lies between the middle and lower horizontal dashed lines, and the deep limbus lies below the lower horizontal dashed line. AC, anterior chamber; ACA, anterior ciliary artery; CA, corneal arcades; BM, Bowman's membrane; CC, collector channel; CE, corneal endothelium; CLA, circular limbal artery; DM, Descemet's membrane; DIVP, deep intrascleral venous plexus; E, ocular surface epithelium; MLIP, midlimbal intrascleral plexus; PVP, perilimbal venous plexus; SC, Schlemm's canal; SL, Schwalbe's line; SS, scleral spur; TM, trabecular meshwork. Reprinted from Experimental Eye Research, Vol. 91, Issue 2, E. L. van der Merwe and S.H. Kidson, Advances in imaging the blood and aqueous vessels of the ocular limbus, pages 118–126, 2010, with permission from Elsevier.

Figure 2.

Several straight radial arcades draining vessels perpendicular to the limbal margin are shown (yellow arrows). Contrast enhancement was applied to SD-OCT images to increase image clarity. Enhancing contrast significantly increased both the contrast associated with vessels (yellow arrows) and also the contrast of noise (blue arrows). (A) An image is shown prior to contrast enhancement. (B) The same image is shown after contrast enhancement. Manual contrast adjustment followed in both SD-OCT and fluorescence images until an optimal balance was achieved. Bar = 500 μm.

Figure 3.

The superficial venous plexus visualized was composed of a series of small interconnected venules between 25 and 100 μm in diameter with many interconnecting branch points forming a dense vascular hexagonal meshwork (A). Red arrows indicate a vessel seen on the virtual casting (A) and its corresponding location in B-scan (B). Blue arrows indicate a suspected aqueous vein (C) descending from the superficial ISVP to the midlimbal ISVP and its corresponding location in B-scan (D). Yellow arrows indicate two suspected aqueous veins seen in this 180 degree rotated virtual casting image (E).

Figure 4.

The path of a large tortuous vein is shown after image contrast enhancement (yellow stars). A smaller venous structure is shown draining into the larger tortuous vein (yellow arrow). The smallest visualized structures, between 15 and 30 μm, form a dense vasculature mesh surrounding the large vessels. Light and dark striations of unknown origin are also seen (blue arrow). Bar = 500 μm.

Figure 5.

(A) Fluorescence imaging of eyes perfused with fluorescent microspheres (diameter = 0.2 μm) revealed a meshwork-like network of aqueous humor outflow veins with significant variability in tracer labeling observed near the episcleral limbus. Bar = 1 mm. (B) Local histogram equalization and background subtraction filters applied to a fluorescence image reveals a meshwork of outflow vasculature extending into the outflow vascular tree down to Schlemm's canal. Bar = 1 mm.

Figure 6.

(A) Aqueous humor outflow microvasculature perfused with fluorescent microspheres is compared with (B) a neoprene corrosion casting of Schlemm's canal. A similar network of aqueous humor outflow veins is visible extending posteriorly away from Schlemm's canal in both images. Bar = 1 mm. Adapted by permission from BMJ Publishing Group Ltd. Anatomical study of Schlemm's canal and aqueous veins by means of neoprene casts: Part I. Aqueous veins, Norman Ashton, British Journal of Ophthalmology, Vol. 35, pages 291–303, 1951.

Figure 7.

Two in vivo scans taken at separate times in the temporal quadrant of one individual are shown. The path of a tortuous vessel approximately 100 μm in diameter is shown (yellow stars). A site of anastomoses-conversion is indicated (yellow arrowhead). A venule between 20–40 μm in diameter is shown (blue arrowhead).

Figure 8.

In vivo versus ex vivo virtual casting images are shown. Direct observations did not show marked differences in vivo (A) versus ex vivo (B). In vivo castings appeared to have a smaller vessel diameter, perhaps due to engorgement of vessels during perfusion ex vivo. In vivo castings are expected to also include arterial blood vessels, which are not active in the cadaver eye. Bar = 500 μm.

Figure 9.

Doppler studies were performed on one eye in vivo to provide further evidence of venous outflow. (A) A large tortuous vein (yellow arrow) casts shadows towards the anterior chamber. Several aqueous venous shadows can be seen (brown arrows). A contact lens worn during this study is seen (purple arrows). (B) The vessel can be seen to have a characteristic laminar flow pattern, with slower flow near the edges of the vessel and more uniform flow near its center (blue arrow). Slow blood flow increases reflectance and mimics stationary tissue. (C) Color Doppler flow patterns are indicated and overlaid on top of a structural scan. Both blue and red flow patterns are seen in the same vessel due to the variable course of the vessel in relation to the SD-OCT scanner resulting in blood flowing towards and away from the probe at different areas. AC = anterior chamber.

Figure 10.

Close similarities were observed between 360-degree virtual castings (A) and previous studies using colored silicone casting agents in monkey eyes (B) and neoprene casting agents in human eyes (C). Note the dense array of vasculature in virtual castings compared to silicone and neoprene corrosion castings. Schlemm's canal is seen as a discontinuous ring. OS = oculus sinister (left eye). Bar = 1 mm. Image (B) adapted with permission from Jocson VL, Sears ML. Channels of aqueous outflow and related blood vessels. II. Cercopithecus ethiops (Ethiopian green or green vervet). Arch. Ophthalmol. 1969;81:244–253; Image (C) adapted by permission from BMJ Publishing Group Ltd. Anatomical Study of Schlemm's Canal and Aqueous Veins by means of Neoprene Casts: Part II. Aqueous Veins (continued), Norman Ashton, British Journal of Ophthalmology, Vol. 36, pages 265–267, 1952.

Figure 11.

A high power view of a neoprene corrosion casting (A) is compared to a virtual casting using SD-OCT (B). All three major vessel types identified in this study were also seen in previous corrosion castings. A large tortuous vein, possibly an aqueous vein, is seen in both images draining vessels posterior from the limbus margin (yellow arrows). A midsized radial vessel is visualized (blue arrow). Small interconnecting venules (purple arrows) form a “hexagonal” meshwork (brackets) in both the neoprene casting and the SD-OCT virtual cast. Bar = 500 μm. Image adapted by permission from BMJ Publishing Group Ltd. Anatomical study of Schlemm's canal and aqueous veins by means of neoprene casts: Part I. Aqueous veins, Norman Ashton, British Journal of Ophthalmology, Vol. 35, pages 291–303, 1951.

Figure 12.

(A, B) A quantitative assessment of the number of vessels present was attempted using a function that skeletonized the images and made counting vessels manually easier, but a more precise algorithm is necessary. We have not yet determined the magnitude of the effect of processing on vessel diameter compared to raw images. (C, D) Noise that is removed in 2D cross-section cannot be distinguished definitely from vessel diameter. Three-dimensional data is necessary to confirm the identity of vessels and remove a sampling of cross-sectional measurements that include noise.