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Abstract

Introduction

Protocol

Representative Results

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Acknowledgements

Materials

References

Biology

Intravital Microscopy to Study Platelet-Leukocyte-Endothelial Interactions in the Mouse Liver

Published: October 6th, 2022

DOI:

10.3791/64239

1Center for Translational Research on Inflammatory Diseases, Michael E. DeBakey Veterans Affairs Medical Center, 2Department of Medicine, Baylor College of Medicine, 3Department of Pediatrics, Baylor College of Medicine

Intravital microscopy is a powerful tool that provides insight into both the temporal and spatial relationships of rapid and/or sequential processes. Herein, we describe a protocol to assess both protein-protein interactions and platelet-neutrophil-endothelial interactions in liver sinusoids in a murine model of experimental sepsis (endotoxemia).

Inflammation and thrombosis are complex processes that occur primarily in the microcirculation. Although standard histology may provide insight into the end pathway for both inflammation and thrombosis, it is not capable of showing the temporal changes that occur throughout the time course of these processes. Intravital microscopy (IVM) is the use of live-animal imaging to gain temporal insight into physiologic processes in vivo. This method is particularly powerful when assessing cellular and protein interactions within the circulation due to the rapid and sequential events that are often necessary for these interactions to occur. While IVM is an extremely powerful imaging methodology capable of viewing complex processes in vivo, there are a number of methodological factors that are important to consider when planning an IVM study. This paper outlines the process of conducting intravital imaging of the liver, identifying important considerations and potential pitfalls that may arise. Thus, this paper describes the use of IVM to study platelet-leukocyte-endothelial interactions in liver sinusoids to study the relative contributions of each in different models of acute liver injury.

Inflammation and thrombosis are complex processes that occur primarily in the microcirculation. This protocol outlines the surgical preparation that allows for imaging of the liver microvasculature in vivo. Although standard histology may provide insight into the end pathways for both inflammation and thrombosis, it cannot show the temporal changes that occur throughout the process. Furthermore, this method is particularly powerful when assessing the transient cellular and protein interactions that occur within the microvascular circulation due to its ability to capture, via videomicroscopy, the often rapid and sequential intera....

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All animal protocols were approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine and the Research & Development Committee of the Michael E. DeBakey Veterans Affairs Medical Center. All experiments are terminal, with euthanasia performed at the end under a surgical plane of anesthesia. See the Table of Materials for details related to all materials, reagents, and equipment used in this protocol.

1. Preparation of antibodies and dyes<.......

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Assessing the effect of vimentin rod domain in leukocyte adhesion to inflamed endothelium
Leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) binding to endothelial and platelet P-selectin occurs during the acute phase of sepsis-induced liver injury inflammation. However, the recombinant human rod domain of vimentin (rhRod) has been shown to bind to P-selectin and block leukocyte adhesion to both endothelium and platelets. This protocol was utilized in a mouse model of sepsis to visualize the real-.......

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The purpose of this methods paper is to outline the necessary steps required to reliably capture high-resolution intravital images and videos of the mouse liver under homeostatic conditions and following the administration of endotoxin or APAP. While this protocol has allowed for the consistent production of data on platelet-leukocyte-endothelial interactions in the liver, there are a number of critical steps required for success, as well as potential pitfalls that are important to avoid when using this imaging paradigm........

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This work was supported by NIH/NIGMS GM-123261 (FWL) and NIH/NHLBI HL139425 (JC). Research support was also funded by NIH/NHLBI HL116524.

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NameCompanyCatalog NumberComments
Surgical Supplies
2" x 2" non-woven spongesMcKesson Med. Surg92242000For liver isolation
#4-0 silk braided suture with needleSOFSILKN/A4-0 Softsilk coated braided black, nonabsorbable: C-1 cutting needle
#4-0 silk braided suture without needleEthiconN/A4-0 Black braided silk, nonabsorbable
21 G blunt needle (0.5 inch)SAI Infusion TechnologiesB21-50This is used to attach to the end of the tracheostomy tube to allow for connection to the ventilator. An alternative source is Instech
23 G blunt needle (0.5 inch)SAI Infusion TechnologiesB23-50This is used for the vascular catheter to allow for connection to a syringe. An alternative source is Instech
Dissecting Scissors (Pointed Tip)Kent ScientificINS600393-GMicro Dissecting Scissors; Carbide Blades; Straight; Sharp Points; 24 mm Blade Length; 4 1/2" Overall Length
McPherson-Vannas Micro Scissors (Vannas)Kent ScientificINS600124These are useful for creating the openings in the trachea and vessels
Polyethylene tubing 10InstechBTPE-10This is used to make the intravascular portion of the catheter. An alternative source is BD Intramedic
Polyethylene tubing 50InstechBTPE-50This is used to make the extravascular portion of the catheter.  An alternative source is BD Intramedic
Polyethylene tubing 90InstechBTPE-90This is used to make the tracheostomy tube.  An alternative source is BD Intramedic
USP grade sterile normal salineCoviden8881570121Hospira 0.0% Sodium Chloride Injection, USP
Microscopy Supplies
Isoflurane delivery system and ventilatorKent ScientificSomnosuiteCombination rodent ventilator and volatile anesthetic delivery system
Foam spacer for warming pad during microscopyN/AN/AThis spacer should be cut from high quality foam, should fit around the liver microscope tray and specific height dimensions are dependent upon the microscope system
Laser scanning confocal microscope system with resonance head scannerOlympusFV3000Although we describe the use of an Olympus FV3000 using a resonance head scanner, this protocol with work with most imaging systems
Liver Microscope TrayN/AN/AThe liver microscope tray was designed for an inverted microscope
Antibodies & Related Reagents
Brilliant Violet 421/anti-mouse Ly6G antibodyBioLegend1276283 µg/mouse. To label neutrophils
BV421/F4/80 antibodyBioLegend1231320.75 mg/kg. To label Kupffer cells
Dulbecco's phosphate buffered saline w/o calcium or magnesiumGibco/ThermoFisher Scientific14190144Used as dialysate to remove sodium azide from antibodies
DyLight649/anti-GPIbβ antibodyemfret AnalyticsX6493 µg/mouse. To label platelets
DyLight488/anti-mouse GPIbβ antibodyemfret AnalyticsX4886 µg/mouse. To label platelets
Endotoxin from Escherichia coli serotype O111:B4Sigma-AldrichL30245 mg/kg; Potency of endotoxin may vary from lot to lot. Therefore, the same lot should be used for a series of experiments to minimize variation due to endotoxin lot
PerCP-eFluor 710/anti-mouse P-selectin antibodyInvitrogen46-0626-824 µg/mouse. To label P-selectin
Slide-a-Lyzer 7,000 MWCO cassetteThermo Scientific66370Used to dialyze antibodies to remove sodium azide
Texas Red-labeled dextranSigma-AldrichT1287~150 kDa; 250 µg/mouse
TRITC/bovine serum albuminSigma-AldrichA2289500 µg/mouse. Dilute to a stock concentration of 50 mg/mL (5%) in normal saline. Used to label the vasculature. It may leak into the interstitial space more readily than high molecular weight dextran during inflammation

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