3D-Reconstruction of the Human Conventional Outflow System by Ribbon Scanning Confocal Microscopy

TLDR: In this high-resolution, volumetric RSCM analysis, human eyes had far fewer outflow tract vessels than porcine eyes, which may point to factors downstream of the TM that increase the authors' vulnerability to glaucoma.

Abstract: Purpose: The risk for glaucoma is driven by the microanatomy and function of the anterior segment. We performed a computation-intense, high-resolution, full-thickness ribbon-scanning confocal microscopy (RSCM) of the outflow tract of two human eyes. We hypothesized this would reveal important species differences when compared to existing data of porcine eyes, an animal that does not spontaneously develop glaucoma. Methods: After perfusing two human octogenarian eyes with lectin-fluorophore conjugate and optical clearance with benzyl alcohol benzyl benzoate (BABB), anterior segments were scanned by RSCM and reconstructed in 3D for whole-specimen rendering. Morphometric analyses of the outflow tract were performed for the trabecular meshwork (TM), limbal, and perilimbal outflow structures and compared to existing porcine data. Results: RSCM provided high-resolution data for IMARIS-based surface reconstruction of outflow tract structures in 3D. Different from porcine eyes with an abundance of highly interconnected, narrow, and short collector channels (CCs), human eyes demonstrated fewer CCs which had a 1.5x greater cross-sectional area (CSA) and 2.6x greater length. Proximal CC openings at the level of Schlemm9s canal (SC) had a 1.3x larger CSA than distal openings into the scleral vascular plexus (SVP). CCs were 10.2x smaller in volume than the receiving SVP vessels. Axenfeld loops, projections of the long ciliary nerve, were also visualized. Conclusion: In this high-resolution, volumetric RSCM analysis, human eyes had far fewer outflow tract vessels than porcine eyes. Human CCs spanned several clock-hours and were larger than in porcine eyes. These species differences may point to factors downstream of the TM that increase our vulnerability to glaucoma.

Figures

Fig 1. Macroscopic view of a BABB-cleared human eye. Human anterior segment pre and post BABB clearing.

Fig 2. Volumetric limbal reconstruction by ribbon scanning confocal microscopy. RSCM reconstruction of eye 1 (1) and eye 2 (2) lectin-fluorophore perfused human eyes. Frontal (A) and sagittal (B) view of volumetric RSCM reconstructions. Frontal and sagittal views of labeled outflow tracts surfaces (C and D, respectively) with TM/SC in yellow, CC in blue, and SVP in red.

Fig 3. Radial charts with volumes. Frontal view of outflow tract structures. Volume measurements are plotted to correspond to their anatomical locations. TM/SC in yellow, CC in blue, SVP in red.

Fig 5. Collector channel morphology. Top panels show a volumetric reconstruction of eyes with CCs in blue. Bottom panels SN-IN show magnified CC morphology in each quadrant. SN: supranasal, ST: supratemporal, IT: infratemporal, IN: infranasal.

Fig 6. Cross-section areas and ellipticities of CC orifices at their proximal points of origin and at their distal points of connection with the SVP. A) Cross-section areas (CSA) of CC openings at their proximal points of connection with SC and their distal points of connection with the SVP. SN: supranasal, ST: supratemporal, IT: infratemporal, IN: infranasal. B) CC opening ellipticities of the proximal and distal ends. 1.0: perfect circle, 0.0: perfect line (averages with error bars using standard deviation).

Fig 4. Outflow tract volumes. Volumes of outflow structures by anatomic location for eye 1 (top) and eye 2 (bottom) eye. SN: supranasal, ST: supratemporal, IT: infratemporal, IN: infranasal (averages with error bars using standard deviation).

Frequently asked questions

Q1. What are the contributions in this paper?

In this paper, the authors used ribbon scanning confocal microscopy ( RSCM ) to reconstruct the outflow tract virtually. 

Q2. What are the future works in this paper?

In the future, fluid dynamics studies that use https: //doi. org/10. 1371/journal. pone. The experiments were not meant to measure or compare outflow function Such structure-function correlation or computational fluidics simulation studies will be necessary in the future. While Imaris surfaces created informative approximations of outflow structures, this automated segmentation may need to be supplemented with manual segmentation for data that might be used in future fluid dynamics studies. Future studies will need to assess glaucoma specific changes of morphology and glycocalyx composition of the conventional outflow tract.