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Posted ContentDOI

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

24 Jan 2020-bioRxiv (Cold Spring Harbor Laboratory)-

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.

AbstractPurpose: 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 (2 min read)

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.

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Citations
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Journal Article
Abstract: Purpose To visualize and quantify conventional outflow directly in its anatomic location. Methods We obtained fluorescein canalograms in six porcine whole eyes and six porcine anterior segment cultures. Eyes were perfused with a constant pressure of 15 mmHg using media containing 0.017 mg/ml fluorescein. Flow patterns were visualized using a stereo dissecting microscope equipped for fluorescent imaging. Images were captured every 30 seconds for 20 minutes for time lapse analysis. Anterior chamber cultures were imaged again on day three of culture. Canalograms were first analyzed for filling time per quadrant. We then wrote a program to automatically compute focal flow fits for each macropixel and to detect convergent perilimbal flow patterns with macropixels grouped into 3 equal-radial width rings around the cornea. A generalized additive model was used to determine fluorescence changes of individual macropixels. Results The resulting imaging algorithm deployed 1024 macropixels that were fit to determine maximum intensity and time to fill. These individual fits highlighted the focal flow function. In whole eyes, significantly faster flow was seen in the inferonasal (IN) and superonasal (SN) quadrants compared to the superotemporal (ST) and inferotemporal (IT) ones (p<0.05). In anterior chamber cultures, reduced flow on day 1 increased in all quadrants on day 3 except in IT (p<0.05). Perilimbal ring analysis uncovered convergent perilimbal flow. Conclusions An algorithm was developed that analyzes regional and circumferential outflow patterns. This algorithm found flow patterns that changed over time and differ in whole eyes and anterior segment cultures.

42 citations


Posted ContentDOI
20 Sep 2021
TL;DR: A 10% reduction in intraocular pressure after trabeculopuncture can be used as predictor for the success of 180° AIT in porcine eyes in patients with an unidentified distal outflow resistance.
Abstract: Purpose: To investigate trabeculopuncture (TP) for predicting the outcome of ab interno trabeculectomy (AIT). AIT is an effective, low-risk procedure for open angle glaucoma. Despite widespread utilization, it fails in patients with an unidentified distal outflow resistance. Methods: We bisected 81 enucleated porcine eyes and perfused them for 72 hours. They were assigned to two groups: trial (n=42) and control (n=39). Intraocular pressure (IOP) was measured continuously. At 24 hours, four YAG-laser trabeculopunctures on the nasal trabecular meshwork were performed, followed by a 180° AIT at the same site at 48 hours. Eyes were divided into TP and AIT responders and non-responders; the proportion of TP responders between both AIT groups was compared. Results: Both post-TP and post-AIT IOPs were lower than baseline IOP (p=0.015 and p<0.01, respectively). The success rates of TP and AIT were 69% and 85.7%, respectively. The proportion of TP responders among AIT responders was greater than that of AIT non-responders (p<0.01). Sensitivity and specificity values of TP as predictive test for AIT success were 77.7% and 83.3%, respectively. The positive and negative predictive values were 96.6% and 38.5%, respectively. Conclusion: A 10% reduction in IOP after TP can be used as predictor for the success (>20% IOP decrease) of 180° AIT in porcine eyes.

References
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01 Dec 1980-Nature

822 citations


"3D-Reconstruction of the Human Conv..." refers background in this paper

  • ...Lectins are ​carbohydrate-binding proteins with a high specificity ​[26,27]​ and can be used to study vessels by binding to their glycocalyx ​[28–30]​....

    [...]


Book
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TL;DR: The study of interactions between lectins and immobilized the structure biology and application of documents animal lectins form function and clinical lectin from ricinus communis (castor bean) and the role of polyvalency in the mechanism of glyco recognition.
Abstract: the lectins properties functions and applications in the lectins properties functions and applications in the lectins properties functions and applications in the lectins properties functions and applications in the lectins properties functions and applications in cell structures functions study guide chapter 7 lectins: function, structure, biological properties and the study of interactions between lectins and immobilized the structure biology and application of documents animal lectins form function and clinical lectin from ricinus communis (castor bean) agglutinin lectin individual gel kit applications cosmo bio roles of polyvalency in the mechanism of glyco recognition. the natural physicians healing therapiesproven remedies b00kreviews cell book reviews a43 cell cultured human conjunctival cells secrete mucins unconjugated lectin staining kit #1 (cat. no.: lk-001) gold colloids and gold conjugates sigma-aldrich document about bibleflying saucers is available on print biological activity of purified momardica charantia lectin nucleoside phosphate sugars: syntheses on practical scales how to be a frequent flyer sbnfs algal lectins and their potential uses mission of hope imotec autism and paleodiets direct-ms case studies in community policing ceyway latelier de marie claire french edition ebook | ufcgymmatthews artocarpus integrifolia lectin (jacalin) aniara concanavalin a (con a) aniara research communications indian institute of science cross-linked leucaena seed gum mat rix: an affinity the dancing wu li masters an overview of the new physics product information general procedure biotin labeled lectin complex of zinc and lectins from seeds of vigna radiata as depression in north carolina social workers implications quarter life fling opalfs

796 citations


"3D-Reconstruction of the Human Conv..." refers background in this paper

  • ...Lectins are ​carbohydrate-binding proteins with a high specificity ​[26,27]​ and can be used to study vessels by binding to their glycocalyx ​[28–30]​....

    [...]


Journal ArticleDOI
TL;DR: Pressure reduction on fewer medications was clinically and statistically significantly better 1 year after stent plus cataract surgery versus cataracts surgery alone, with an overall safety profile similar to that of catarACT surgery alone.
Abstract: Objective To assess the safety and efficacy of the iStent trabecular micro-bypass stent (Glaukos Corporation, Laguna Hills, CA) in combination with cataract surgery in subjects with mild to moderate open-angle glaucoma. Design Prospective, randomized, open-label, controlled, multicenter clinical trial. Participants A total of 240 eyes with mild to moderate open-angle glaucoma with intraocular pressure (IOP) ≤24 mmHg controlled on 1 to 3 medications were randomized to undergo cataract surgery with iStent implantation (treatment group) or cataract surgery only (control). Fifty additional subjects were enrolled to undergo cataract surgery with iStent implantation under protocol expansion. Data in this report are based on the first 240 eyes enrolled. Intervention Implantation of the iStent trabecular micro-bypass stent in conjunction with cataract surgery or cataract surgery only. Main Outcome Measures The primary efficacy measure was unmedicated IOP ≤21 mmHg at 1 year. A secondary measure was unmedicated IOP reduction ≥20% at 1 year. Safety measures included best-corrected visual acuity (BCVA), slit-lamp observations, complications, and adverse events. Results The study met the primary outcome, with 72% of treatment eyes versus 50% of control eyes achieving the criterion ( P P = 0.003). The overall incidence of adverse events was similar between groups with no unanticipated adverse device effects. Conclusions Pressure reduction on fewer medications was clinically and statistically significantly better 1 year after stent plus cataract surgery versus cataract surgery alone, with an overall safety profile similar to that of cataract surgery alone. Financial Disclosure(s) Proprietary or commercial disclosure may be found after the references.

366 citations


OtherDOI
TL;DR: Ocular blood flow is controlled both via direct autonomic influences on the vasculature of the optic nerve, choroid, ciliary body, and iris, as well as via indirect influences on retinal blood flow.
Abstract: The autonomic nervous system influences numerous ocular functions. It does this by way of parasympathetic innervation from postganglionic fibers that originate from neurons in the ciliary and pterygopalatine ganglia, and by way of sympathetic innervation from postganglionic fibers that originate from neurons in the superior cervical ganglion. Ciliary ganglion neurons project to the ciliary body and the sphincter pupillae muscle of the iris to control ocular accommodation and pupil constriction, respectively. Superior cervical ganglion neurons project to the dilator pupillae muscle of the iris to control pupil dilation. Ocular blood flow is controlled both via direct autonomic influences on the vasculature of the optic nerve, choroid, ciliary body, and iris, as well as via indirect influences on retinal blood flow. In mammals, this vasculature is innervated by vasodilatory fibers from the pterygopalatine ganglion, and by vasoconstrictive fibers from the superior cervical ganglion. Intraocular pressure is regulated primarily through the balance of aqueous humor formation and outflow. Autonomic regulation of ciliary body blood vessels and the ciliary epithelium is an important determinant of aqueous humor formation; autonomic regulation of the trabecular meshwork and episcleral blood vessels is an important determinant of aqueous humor outflow. These tissues are all innervated by fibers from the pterygopalatine and superior cervical ganglia. In addition to these classical autonomic pathways, trigeminal sensory fibers exert local, intrinsic influences on many of these regions of the eye, as well as on some neurons within the ciliary and pterygopalatine ganglia.

268 citations


Journal ArticleDOI
TL;DR: The results indicate that a surprisingly high fraction of aqueous outflow resistance resides in the distal aspects of the outflow system at normal IOP, and that this distal resistance drops as IOP is increased.
Abstract: Aqueous outflow resistance of enucleated human eyes was measured at 7 and 25 mm Hg before and after partial and complete (12 clock hours) internal trabeculotomy. Following complete trabeculotomy, 71% of the resistance was eliminated at 25 mm Hg while only 49% was eliminated at 7 mm Hg. In contrast to published findings in eyes with intact trabecular meshwork where the resistance increased with increasing IOP (1), following complete trabeculotomy, the resistance decreased 2% per mm Hg with increasing IOP. Experiments with trabeculotomy limited to part of the circumference showed that a one hour trabeculotomy produced 41% (25 mm Hg) to 60% (7 mm Hg) of the effect of a twelve hour trabeculotomy. The results indicate that a surprisingly high fraction of aqueous outflow resistance resides in the distal aspects of the outflow system at normal IOP, and that this distal resistance drops as IOP is increased.

216 citations


"3D-Reconstruction of the Human Conv..." refers background in this paper

  • ...A growing body of evidence points to an outflow resistance distal to the TM as an essential aspect of both healthy ​[3,24,25]​ and glaucomatous ​[8–11]​ physiology....

    [...]


Frequently Asked Questions (2)
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. 

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.