Using quantitative intravital multiphoton microscopy to dissect hepatic transport in rats

Dunn, Kenneth W.; Ryan, Jennifer

doi:10.1016/j.ymeth.2017.04.015

Cited by 13 publications

(8 citation statements)

References 73 publications

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“…However, imaging techniques are constantly evolving to produce accurate three-dimensional reconstructions of tissues with high spatial resolution (Arganda-Carreras et al, 2010 ; Hoehme et al, 2010 ). Intra-vital imaging techniques for visualizing molecular dynamics in live animals (Benechet et al, 2017 ; Dunn and Ryan, 2017 ) could further augment our modeling efforts at small spatial scales. However, these methods would introduce new challenges such as lack of control over distribution of stimulus in the immediate microenvironment of hepatocytes.…”

Section: Discussionmentioning

confidence: 99%

Causality Analysis and Cell Network Modeling of Spatial Calcium Signaling Patterns in Liver Lobules

et al. 2018

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Dynamics as well as localization of Ca2+ transients plays a vital role in liver function under homeostatic conditions, repair, and disease. In response to circulating hormonal stimuli, hepatocytes exhibit intracellular Ca2+ responses that propagate through liver lobules in a wave-like fashion. Although intracellular processes that control cell autonomous Ca2+ spiking behavior have been studied extensively, the intra- and inter-cellular signaling factors that regulate lobular scale spatial patterns and wave-like propagation of Ca2+ remain to be determined. To address this need, we acquired images of cytosolic Ca2+ transients in 1300 hepatocytes situated across several mouse liver lobules over a period of 1600 s. We analyzed this time series data using correlation network analysis, causal network analysis, and computational modeling, to characterize the spatial distribution of heterogeneity in intracellular Ca2+ signaling components as well as intercellular interactions that control lobular scale Ca2+ waves. Our causal network analysis revealed that hepatocytes are causally linked to multiple other co-localized hepatocytes, but these influences are not necessarily aligned uni-directionally along the sinusoids. Our computational model-based analysis showed that spatial gradients of intracellular Ca2+ signaling components as well as intercellular molecular exchange are required for lobular scale propagation of Ca2+ waves. Additionally, our analysis suggested that causal influences of hepatocytes on Ca2+ responses of multiple neighbors lead to robustness of Ca2+ wave propagation through liver lobules.

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Section: Discussionmentioning

confidence: 99%

Causality Analysis and Cell Network Modeling of Spatial Calcium Signaling Patterns in Liver Lobules

et al. 2018

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“…Following delivery, animals were acclimated at least 4 days before studies. Surgical preparations were conducted as previously described 2,3 . All animal experiments were approved and conducted according to Institutional Animal Care and Use Committee guidelines of Indiana University, and adhered to the NRC guide for care and use of animals.…”

Section: Experimental Animal Model and Surgical Proceduresmentioning

confidence: 99%

“…Since the STAFF macro measures microvascular flow in a fixed set of regions, organ stability is crucial. Organ immobilization methods for intravital microscopy are described in our previous publications 2,4 . Field of view may shift briefly during respiration, but returns to its original position after each respiration, allowing reliable measurements.…”

Section: Intravital Microscopymentioning

confidence: 99%

A simple automated method for continuous fieldwise measurement of microvascular hemodynamics

Clendenon

Hoene

et al. 2019

Microvascular Research

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Microvascular perfusion dynamics are vital to physiological function and are frequently dysregulated in injury and disease. Typically studies measure microvascular flow in a few selected vascular segments over limited time, failing to capture spatial and temporal variability. To quantify microvascular flow in a more complete and unbiased way we developed STAFF (Spatial Temporal Analysis of Fieldwise Flow), a macro for FIJI open-source image analysis software. Using highspeed microvascular flow movies, STAFF generates kymographs for every time interval for every vascular segment, calculates flow velocities from red blood cell shadow angles, and outputs the data as color-coded velocity map movies and spreadsheets. In untreated mice, analyses demonstrated profound variation even between adjacent sinusoids over seconds. In acetaminophen-treated mice we detected flow reduction localized to pericentral regions. STAFF is a powerful new tool capable of providing novel insights by enabling measurement of the complex spatiotemporal dynamics of microvascular flow.

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“…Intravital Microscopy Studies of Fluorescent Bile Salt Transport. Fluorescent bile salt transport was characterized using intravital multiphoton microscopy, using an approach similar to that previously applied (Babbey et al, 2012;Ryan et al, 2014) and described in detail elsewhere (Dunn and Ryan, 2017). Multiphoton microscopy was conducted with an Olympus Fluoview 1000 MPE confocal/multiphoton microscope system (Olympus, Tokyo, Japan) mounted on an Olympus IX-81 inverted stand, using an Olympus 25X, NA1.05 water immersion objective, with 830 nm excitation provided by a Mai Tai DeepSee laser (Spectra-Physics, Santa Clara, CA).…”

Section: Downloaded Frommentioning

confidence: 99%

“…In previous studies, we have demonstrated that quantitative multiphoton microscopy can be used to quantify organic anion and bile acid transport in the liver of living rats at subcellular resolution (Babbey et al, 2012;Ryan et al, 2014;Dunn and Ryan, 2017). Here we extend this approach to assay transporter inhibition in rats, using cholylglycyl amidofluorescein (CGamF) and cholyl-lysyl-fluorescein (CLF) as fluorescent bile salt probes (de Waart et al, 2010;Kruglov et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

Intravital Multiphoton Microscopy with Fluorescent Bile Salts in Rats as an In Vivo Biomarker for Hepatobiliary Transport Inhibition

et al. 2018

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The bile salt export pump (BSEP) is expressed at the canalicular domain of hepatocytes, where it mediates the elimination of monovalent bile salts into the bile. Inhibition of BSEP is considered a susceptibility factor for drug-induced liver injury that often goes undetected during nonclinical testing. Although in vitro assays exist for screening BSEP inhibition, a reliable and specific method for confirming Bsep inhibition in vivo would be a valuable follow up to a BSEP screening strategy, helping to put a translatable context around in vitro inhibition data, incorporating processes such as metabolism, protein binding, and other exposure properties that are lacking in most in vitro BSEP models. Here, we describe studies in which methods of quantitative intravital microscopy were used to identify dose-dependent effects of two known BSEP/Bsep inhibitors, 2-[4-[4-(butylcarbamoyl)-2-[(2,4-dichlorophenyl)sulfonylamino]phenoxy]-3-methoxyphenyl]acetic acid (AMG-009) and bosentan, on hepatocellular transport of the fluorescent bile salts cholylglycyl amidofluorescein and cholyl-lysyl-fluorescein in rats. Results of these studies demonstrate that the intravital microscopy approach is capable of detecting Bsep inhibition at drug doses well below those found to increase serum bile acid levels, and also indicate that basolateral efflux transporters play a significant role in preventing cytosolic accumulation of bile acids under conditions of Bsep inhibition in rats. Studies of this kind can both improve our understanding of exposures needed to inhibit Bsep in vivo and provide unique insights into drug effects in ways that can improve our ability interpret animal studies for the prediction of human drug hepatotoxicity.

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Using quantitative intravital multiphoton microscopy to dissect hepatic transport in rats

Cited by 13 publications

References 73 publications

Causality Analysis and Cell Network Modeling of Spatial Calcium Signaling Patterns in Liver Lobules

Causality Analysis and Cell Network Modeling of Spatial Calcium Signaling Patterns in Liver Lobules

A simple automated method for continuous fieldwise measurement of microvascular hemodynamics

Intravital Multiphoton Microscopy with Fluorescent Bile Salts in Rats as an In Vivo Biomarker for Hepatobiliary Transport Inhibition

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