Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

Summary

Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

Abstract

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, inflammatory and metabolic conditions, using in vivo fluorescence microscopy. A closed cranial window is placed over the left parieto-occipital cortex of the mice. Local microcirculation is recorded in real time through the window using epi and fluorescence illumination, and measurements of vessels diameters and red blood cell (RBC) velocities are performed. RBC velocity is measured using real-time cross-correlation and/or fluorescent-labeled erythrocytes. Leukocyte and platelet adherence to pial vessels and assessment of perfusion and vascular leakage are made with the help of fluorescence-labeled markers such as Albumin-FITC and anti-CD45-TxR antibodies. Microcirculation can be repeatedly video-recorded over several days. We used for the first time the close window brain intravital microscopy to study the pial microcirculation to follow dynamic changes during the course of Plasmodium berghei ANKA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow and eventually vascular collapse.

Protocol

1. Craniotomy

A craniotomy in 8-to-10-week old mice needs to be performed in advance as previously described¹, except that a titanium bar is not placed in the head of the animal. The chronic cranial window is a stable preparation allowing examination of the pial microcirculation even months after being implanted. Usually, we perform our studies 2-3 weeks after the cranial window implantation.

2. Intravital microscopy

1. Two-three weeks after craniotomy, body temperature is checked and then the mouse is lightly anesthetized with isoflurane (4% for induction, 1-2% for maintenance). Light anes....

Discussion

The intravital microscopy method described here provides a unique and powerful tool for detailed observation of the pial microcirculation in the mouse. It allows singling out individual arterioles and venules and measuring changes of a number of parameters such as vessel diameters, RBC velocities, blood flow, adherence and rolling of leukocytes, platelets and other blood elements, vascular leakage, tissue pH and pO2 and potentially many other applications. The in vivo vascular response can be promptly evaluated .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Isoflurane	Baxter Internationl Inc.	FDG9623
Carbocyanine dye Dil	Molecular Probes, Life Technologies	D306
Albumin-FITC	Sigma-Aldrich	A9771
Anti-CD45-TxR Ab	Invitrogen	MCD4517
P. berghei ANKA-GFP	MR4	MRA-865