Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

Summary

This article reports on a detailed method for the dynamic measurement and quantification of blood flow velocity within individual blood vessels of the mouse liver vasculature using intravital microscopy imaging in combination with a specific methodology for image acquisition and analysis.

Abstract

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various biological events in real time and in live animals. This technology has greatly advanced our understanding of physiological processes and pathogen-mediated phenomena in specific organs.

In this study, IVM is applied to the mouse liver and protocols are designed to image in vivo the circulatory system of the liver and measure red blood cell (RBC) velocity in individual hepatic vessels. To visualize the different vessel subtypes that characterize the hepatic organ and perform blood flow speed measurements, C57Bl/6 mice are intravenously injected with a fluorescent plasma reagent that labels the liver-associated vasculature. IVM enables in vivo, real time, measurement of RBC velocity in a specific vessel of interest. Establishing this methodology will make it possible to investigate liver hemodynamics under physiological and pathological conditions. Ultimately, this imaging-based methodology will be important for studying the influence of L. donovani infection on hepatic hemodynamics.

This method can be applied to other infectious models and mouse organs and might be further extended to pre-clinical testing of a drug’s effect on inflammation by quantifying its effect on blood flow.

Introduction

Organ-specific hemodynamics are important physiological features of any mammalian organ. Abnormalities in blood flow may be the consequence of inflammation and a sign of organ dysfunction¹. Thus, blood flow organization, structure and function appear as critical parameters for analysis under physiological and pathological conditions. The techniques that have been commonly used for analyzing blood flow in a specific organ contain several limitations, including the resolution limit of the technique itself (e.g. Doppler imaging of blood flow), the capacity for measurement of absolute blood flow only (volume of blood per unit serving an organ) (

Protocol

Ethics statement: All the animal studies were performed in accordance with guidelines and protocols that were approved by the Institutional Animal Care and Use Committee of the Aix-Marseille Université, France. Female C57Bl/6 mice at 8 - 10 weeks old were commercially obtained and handled according to the rules of Décret N° 8 87–848, October 19, 1987, Paris. All the experiments using L. donovani LD1S parasites were conducted in accordance with biosafety regulations from the French and European Union legislation.

1. Mouse Infection with L. donovani Promastigote Parasites

1. Prepare the ....

Results

The specific architectural organization of the sinusoids in the liver can be visualized based on the autofluorescent property of this organ (Figure 1, panel B and C, green), the intraperitoneal injection of Hoechst for the labeling of hepatocyte nuclei (Figure 1B, blue) and the intravenous injection of fluorescent BSA for the staining of the hepatic circulatory system (Figure 1C, red). The liver is composed of several different vessel subtypes with different functions an.......

Discussion

The recent development of intravital microscopy of the mouse liver opens up new possibilities for the investigation of physiological response to infection in vivo and in real time^5,9,10. Organ blood flow is a critical physiological parameter that is often altered in many diseases. However, the status of liver hemodynamics under physiological and infectious conditions remains a poorly explored area. In this study, IVM-based methods, which were previously adapted for the study of the spleen an.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Hoechst 33342	Sigma Aldrich	B2261
BSA-Alexa 647	lifetechnologies	A34785
Dextran-FITC 500 mol wt	SIGMA	46947
Ketamine	PanPharma	20434
Xylazine	Bayer	KP07KEU
Vetedine	Pharma Animal	6869029
Cyanoacrylate liquid	Cyanolit	5833300005
Coverslip frame: Membrane slide for microdissection part N°: 5013	Molecular machines	50103
Coverslip DiaPath 24x60 ep: 1.6 mm	DiaPath	61061
Confocal laser scanning microscope	Leica	TCS-SP5
LAS-AF viewer	Leica software	Version 3.1.0 buid 8587