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

TL;DR: 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.

Summary

Introduction

• Recent experiments in ex vivo human [1] and pig [1,2] eyes confirmed an outflow resistance distal to the trabecular meshwork (TM) that can be reduced.
• Distal outflow resistance is pronounced in glaucoma patients.
• TM bypass [6,7] and ablation [8–11] procedures were expected to reduce IOP to the level of episcleral venous pressure, around 8 mmHg [12].
• The process of assembling several million confocal images is computation-intense but could be accomplished using a high-performance computer cluster [13,14].
• Canalogram studies of human eyes [15] suggested a perilimbal, proximal vessel network with a density similar to that of porcine eyes [16–19] which seemed to contrast their RSCM studies of this species [13].

Whole eye lectin perfusion

• No human subjects were involved in the investigation.
• The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
• Therefore, a consent specifically for the research conducted here by the donor or their next of kin was not required.
• The anterior chamber was cannulated with a 20-gauge needle positioned temporally and just anterior to the limbus.

Image capture

• Samples were imaged and processed as described before [13,14].
• Briefly, full-thickness perilimbal scans were acquired with a confocal microscope designed for high-speed ribbon-scanning and large scale image stitching (RS-G4, Caliber I.D., Andover, MA, USA).
• Laser percentage, high voltage (HV), and offset were held constant throughout the volume at 7, 85, and 15, respectively.

Image processing

• The resulting series of Tagged Image File Format (TIFF) volumes comprised of 422 to 520 images were converted to the Bitplane Imaris IMS format.
• To facilitate analysis, each of the IMS files was placed on a specialized high-speed solid-state file server equipped with 20 gigabits of network bandwidth.
• Surface creation parameters were refined to optimally resolve the structural details of each region.
• The authors observed different types of CC patterns of reaches at their catchment scale: meandering, braided, anabranching [21], and anastomosing.
• The authors inverted the brightness values to highlight structures not labeled with lectins.

Statistical analysis

• The parameters of volume and location were obtained for TM and SC, CC, and SVP with the Imaris statistics function.
• CC opening lengths and widths were measured in Imaris slice mode.
• Data were analyzed by location for each eye.
• Data from human eyes in this study were compared to porcine eyes from their prior study [13].
• Statistical tests were performed in Python 3.6.

Results

• Whole human eyes could be labeled and cleared in approximately seven days using their modified BABB protocol [13].
• Lectin-labeled fluorophores intensely stained the entire outflow tract from the TM to the SVP.
• The largest TM and SC volume in this eye was in the supratemporal quadrant (STq).

Discussion

• The authors assessed the similarities and differences they found between the human and porcine outflow tract.
• These methods provide a new tool to researchers working on deciphering the aspects of structure and function of the outflow tract at high 3D resolution.
• While the authors observed 13 openings towards Schlemm’s canal, these collector channels had 18 to 20 distal openings.
• Future studies will need to assess glaucoma specific changes of morphology and glycocalyx composition of the conventional outflow tract.

S2 Movie. Fly-through movie of the supranasal quadrant of eye 1.

• Axenfeld nerve loops could readily be identified in all quadrants.
• Axenfeld loops are visible as dark structures void of lectin staining (white arrowheads).
• B) Magnified view of the area in A shows Axenfeld loop that enters the sclera.
• Axenfeld loops per quadrant (averages with error bars using standard deviation).

Author Contributions

• Ralitsa T. Loewen, Susannah Waxman, Chao Wang, Sarah Atta, Si Chen, Simon C. Watkins, Alan M. Watson, Nils A. Loewen, also known as Data curation.
• Ralitsa T. Loewen, Susannah Waxman, Chao Wang, Sarah Atta, Si Chen, Simon C. Watkins, Alan M. Watson, Nils A. Loewen, also known as Formal analysis.
• Ralitsa T. Loewen, Simon C. Watkins, Alan M. Watson, Nils A. Loewen, also known as Project administration.
• Susannah Waxman, Simon C. Watkins, Alan M. Watson, Nils A. Loewen, also known as Supervision.
• Ralitsa T. Loewen, Susannah Waxman, Alan M. Watson, Nils A. Loewen, also known as Writing – original draft.

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.