Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

Summary

An optical system was developed to visualize hepatic microcirculation with FITC-labeled erythrocytes and to measure the partial pressure of oxygen in the microvessels with laser-assisted phosphorimetry. This method can be used to investigate physiological and pathological mechanisms by analyzing microvascular structure, diameter, blood flow velocity, and oxygen tension.

Abstract

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of the hepatic blood supply is poorly oxygenated portal vein blood derived from the gastrointestinal tract and spleen. Oxygen is delivered to hepatocytes by blood flowing from a terminal branch of the portal vein to a central venule via sinusoids, and this makes an oxygen gradient in hepatic lobules. The oxygen gradient is an important physical parameter that involves the expression of enzymes upstream and downstream in hepatic microcirculation, but the lack of techniques for measuring oxygen consumption in the hepatic microcirculation has delayed the elucidation of mechanisms relating to oxygen metabolism in liver. We therefore used FITC-labeled erythrocytes to visualize the hepatic microcirculation and used laser-assisted phosphorimetry to measure the partial pressure of oxygen in the microvessels there. Noncontact and continuous optical measurement can quantify blood flow velocities, vessel diameters, and oxygen gradients related to oxygen consumption in the liver. In an acute hepatitis model we made by administering acetaminophen to mice we observed increased oxygen pressure in both portal and central venules but a decreased oxygen gradient in the sinusoids, indicating that hepatocyte necrosis in the pericentral zone could shift the oxygen pressure up and affect enzyme expression in the periportal zone. In conclusion, our optical methods for measuring hepatic hemodynamics and oxygen consumption can reveal mechanisms related to hepatic disease.

Protocol

1. Labeling Erythrocytes with Fluorescein Isothiocyanate Isomer I (FITC)

All the experimental protocols we used were approved by the Animal Care Committee of Keio University School of Medicine.

1. Anesthetize a donor mice, and after making a celiotomy incision withdraw whole blood from the inferior vena cava with a 1-ml syringe containing small amount of heparin. The mouse was immediately euthanized by sodium pentobarbital injection (100 mg/kg i.p.).
2. Wash the whole blood twice with PBS (pH 7.4) by centrifugation for 5 min at 400 g in a swinging-bucket rotor.
3. Dissolve 10 mg of FITC in 5 ml of ....

Discussion

Delivering oxygen to tissues is an essential role of the microcirculation, and a lot of papers describe diseases related to microvessel anatomy and rheology⁶. Liver blood flow is a combination of arterial and venous blood. About 30% of the blood flow to the liver is well-oxygenated blood provided by the hepatic artery and the other 70%, provided by the portal vein, is the poorly oxygenated blood venous outflow from the spleen and gastrointestinal tract⁷. Liver diseases such as fatty liver, shock, or.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Pd(II) meso-Tetra(4-carboxyphenyl)porphine	Frontier Scientific, Inc.	PdT790
Fluorescein isothiocyanate isomer I	Sigma-Aldrich Co.	F7250
Acetaminophen	Sigma-Aldrich Co.	A7085
Equipment
photomultiplier tube	hamamatsu photonics k.k	H10722-20