Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2020.01.17.910257

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

Loewen, R.·Waxman, S.·Wang, C.·Atta, S.·Chen, S.·Watkins, S. C.·Watson, A. M.·Loewen, N. A.
View original source ↗Export metadata ↓

Abstract

PurposeRisk 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. MethodsAfter 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. ResultsRSCM 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 Schlemms 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. ConclusionIn 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. Grant informationNational Eye Institute K08EY022737 (NAL); Initiative to Cure Glaucoma of the Eye and Ear Foundation of Pittsburgh (NAL); Wiegand Fellowship of the Eye and Ear Foundation of Pittsburgh (YD); P30-EY08098 (NAL); Department grant by Research to Prevent Blindness (NAL); an unrestricted fellowship grant from the Xiangya Hospital of Central South University (SC).

Source connections

Explore related subjects

physiologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Loewen, R., Waxman, S., Wang, C., Atta, S., Chen, S., Watkins, S. C., Watson, A. M., Loewen, N. A.. 2020-01-24. 3D-Reconstruction of the Human Conventional Outflow System by Ribbon Scanning Confocal Microscopy. https://doi.org/10.1101/2020.01.17.910257

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

IBD-Derived Colonic Fibroblasts Exhibit an Osteopontin-Enriched Secretome, and Osteopontin Restrains Human Colonic Organoid Maturation

Background: Intestinal fibroblasts are extensively remodeled in inflammatory bowel disease (IBD), yet the soluble stromal signals that directly influence epithelial maturation remain incompletely understood. We examined whether fibroblasts derived from inflamed IBD colon display an osteopontin (OPN; SPP1)-enriched secretory phenotype and whether extracellular OPN directly modifies non-neoplastic human colonic epithelium. Methods: Conditioned media from 5 noninflamed-associated fibroblast (NAF) and 4 inflammatory-associated fibroblast (IAF) cultures were analyzed in the validated multi-donor cytokine-array matrix, with orthogonal SPP1 RT-qPCR validation in a complementary fibroblast cohort. Recombinant OPN was then tested in human colonic organoids from 3 donors using donor-resolved molecular and functional analyses under standard, fibroblast-conditioned, and WNT-modified culture conditions. Donor identity defined biological replication. Results: OPN showed the strongest positive rank-based separation between IAF and NAF cultures: all 4 IAF values were higher than all 5 NAF values (Cliff's delta=1.00; exact Mann-Whitney P=0.0159; median ratio=3.64; Benjamini-Hochberg q=.19). Fibroblast RT-qPCR showed approximately 10-fold higher mean SPP1 expression in IAF than NAF cultures (P<.05). In organoids, OPN consistently reduced KRT20, FABP1, CA2, and MUC2 from Day 5 to Day 9. SOX9, HES1, and NOTCH1 increased at Day 9, whereas LGR5 and ALDH provided no evidence of canonical stem-cell expansion. Organoid-area and EdU responses were modest and donor dependent. Conclusions: IBD-derived colonic fibroblasts can display an OPN-enriched secretory phenotype. In human colonic organoids, OPN is sufficient to impair epithelial maturation, whereas its effects on growth and proliferation are variable and depend on the surrounding niche.

physiology↗
bioRxiv2026

A multiscale analysis of liver lobule fibrosis and its impact on drug propagation and metabolism - a DLA approach

Employing DLA methods, this paper explores the self-assembly of collagen fibers and resulting fibrosis at three scales up to the scale of regular lobule models. This allows a mechanistic exploration of the effects of collagen on drug transport (flow and diffusion) and metabolism. In addition, this method permits an analysis of fiber growth characteristics. First, variations of the DLA method of Parkinson et al (1994) will be used to generate multiple explicit collagen microfibril self-assembly using DLA particles in one dimension using cubic grid blocks of (4 mm)3 in a 240 x 20 x 20 grid model. The second stage will be to assess the consequences of various densities of these fibers in three dimensions on flow reductions at a higher scale. Here we utilize DLA methods in cubic grid blocks of (80 nm)3 to mimic 3D collagen self-assembly of fibrils. We then apply a pressure gradient or specified flow rates across a spatially gridded version of these models to quantify flow effects. This region represents a local zone of liver tissue affected by fibrosis. Analytic models of fibrotic effects on flow are employed for comparison. A third stage explores the implications of fibrosis in a liver lobule model using multiple grid blocks of size 3200 mm to represent the lobule tissue. Here, a continuum model of fiber density is employed, based on the previous two scales. The model also includes the effects of additional grid blocks representing sinusoidal flow paths found in the lobule. We contrast and quantify drug propagation and metabolism of molecular dissolved versus nanoparticle delivery vehicles in fibrotic media, achieved by upscaling explicit collagen distributions to appropriate average values.

physiology↗
bioRxiv2026

Pulmonary pressure load shapes right ventricular molecular remodelling in dilated cardiomyopathy

Right ventricular (RV) adaptation to pulmonary hypertension determines outcome in dilated cardiomyopathy (DCM), but the molecular mechanisms of the transition to decompensation remain unclear. We analysed RV tissue from explanted hearts of patients with end-stage DCM using single-nucleus RNA sequencing (n=21), mass spectrometry and Olink Reveal proteomics (both n=44), and integrated these molecular profiles with echocardiographic and right-heart catheterisation measures to identify molecular correlates of RV dysfunction. Mean pulmonary arterial pressure was the dominant correlate of RV transcriptional remodelling, particularly in cardiomyocytes, where higher pressure was associated with contractile remodelling, autophagy, vesicle trafficking and glucose metabolism. In contrast, RV decompensation was characterised by immune activation and reduced oxidative phosphorylation exclusively at the proteomic level. Integrative multi-omics factor analysis (MOFA) further identified fibrosis as the dominant molecular program shared across transcriptomic and proteomic layers. Together, these findings indicate molecular adaptation to pressure load and tissue fibrosis during progression towards RV failure.

physiology↗