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

Summary

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).

Abstract

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.

Introduction

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....

Protocol

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

1. Antibody and dye selection
   NOTE: The number of simultaneous colors observed will be dependent on the specific microscope....

Results

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-ti.......

Discussion

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........

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Surgical Supplies
2" x 2" non-woven sponges	McKesson Med. Surg	92242000	For liver isolation
#4-0 silk braided suture with needle	SOFSILK	N/A	4-0 Softsilk coated braided black, nonabsorbable: C-1 cutting needle
#4-0 silk braided suture without needle	Ethicon	N/A	4-0 Black braided silk, nonabsorbable
21 G blunt needle (0.5 inch)	SAI Infusion Technologies	B21-50	This 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 Technologies	B23-50	This is used for the vascular catheter to allow for connection to a syringe. An alternative source is Instech
Dissecting Scissors (Pointed Tip)	Kent Scientific	INS600393-G	Micro Dissecting Scissors; Carbide Blades; Straight; Sharp Points; 24 mm Blade Length; 4 1/2" Overall Length
McPherson-Vannas Micro Scissors (Vannas)	Kent Scientific	INS600124	These are useful for creating the openings in the trachea and vessels
Polyethylene tubing 10	Instech	BTPE-10	This is used to make the intravascular portion of the catheter. An alternative source is BD Intramedic
Polyethylene tubing 50	Instech	BTPE-50	This is used to make the extravascular portion of the catheter.  An alternative source is BD Intramedic
Polyethylene tubing 90	Instech	BTPE-90	This is used to make the tracheostomy tube.  An alternative source is BD Intramedic
USP grade sterile normal saline	Coviden	8881570121	Hospira 0.0% Sodium Chloride Injection, USP
Microscopy Supplies
Isoflurane delivery system and ventilator	Kent Scientific	Somnosuite	Combination rodent ventilator and volatile anesthetic delivery system
Foam spacer for warming pad during microscopy	N/A	N/A	This 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 scanner	Olympus	FV3000	Although we describe the use of an Olympus FV3000 using a resonance head scanner, this protocol with work with most imaging systems
Liver Microscope Tray	N/A	N/A	The liver microscope tray was designed for an inverted microscope
Antibodies & Related Reagents
Brilliant Violet 421/anti-mouse Ly6G antibody	BioLegend	127628	3 µg/mouse. To label neutrophils
BV421/F4/80 antibody	BioLegend	123132	0.75 mg/kg. To label Kupffer cells
Dulbecco's phosphate buffered saline w/o calcium or magnesium	Gibco/ThermoFisher Scientific	14190144	Used as dialysate to remove sodium azide from antibodies
DyLight649/anti-GPIbβ antibody	emfret Analytics	X649	3 µg/mouse. To label platelets
DyLight488/anti-mouse GPIbβ antibody	emfret Analytics	X488	6 µg/mouse. To label platelets
Endotoxin from Escherichia coli serotype O111:B4	Sigma-Aldrich	L3024	5 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 antibody	Invitrogen	46-0626-82	4 µg/mouse. To label P-selectin
Slide-a-Lyzer 7,000 MWCO cassette	Thermo Scientific	66370	Used to dialyze antibodies to remove sodium azide
Texas Red-labeled dextran	Sigma-Aldrich	T1287	~150 kDa; 250 µg/mouse
TRITC/bovine serum albumin	Sigma-Aldrich	A2289	500 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