Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Summary

Intravital microscopy can be used in animals to visualize and measure retinal vascular diameters, bloodstream velocities, and total retinal blood flow.

Abstract

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Introduction

The retina is one of the most metabolically active tissues in the body, and consequently requires a generous blood supply. Two separate circulations meet this demand: the choroidal circulation for the outer portion of the retina, and the retinal circulation for the inner portion of the retina. Investigations of retinal perfusion are imperative for understanding the pathological mechanisms and consequences of diabetic retinopathy, oxygen-induced retinopathy, retinal artery or vein occlusion, and stroke. Several methods have been employed to quantify retinal blood flow, with each technique having its advantages, disadvantages, limitations, and assumptions. Among these techniques are infusion of 7-8 μm diameter microspheres that lodge in precapillary arterioles^1,2, quantitative autoradiography^3,4, optical microangiopathy-optical coherence tomography^5,6, magnetic resonance imaging^7,8, and intravital video microscopy^9-16. Advantages of the latter include direct live visualization of retinal vessels and flow, a dependence on only a few minor assumptions, and affordability for labs having a fluorescence microscope with an attached video camera. In previous studies of intravital video microscopy^9-16, fluorescent dextran has been used as a plasma marker, and fluorescently labeled red blood cells (from a donor animal) have been used as velocity markers. In the current protocol, 1.9-μm diameter fluorescently labeled microspheres, instead of red blood cells, are used to measure velocity, with this alteration negating the need for a blood cell donor.

Protocol

The procedures involving the use of animals were reviewed and approved by the Institutional Animal Care and Use Committee of LSUHSC-S and performed according to the criteria outlined by the National Institutes of Health. 1. Preparation of Perfusion Solutions Sonicate a 1% (by weight) stock solution of 1.9 µm diameter microspheres. Withdraw ~25-30 x 106 green fluorescent microspheres (that is, ~10 µl of the stock solution) into a 300 µl …

Representative Results

Figure 1 shows single frames of video from an experiment, with panels A-D and F-I showing fluorescent microsphere streaks captured with a 4X objective and 8 msec exposure time (and binning 2 x 2 pixels to reduce the video file size). Figure 1E shows the orientation of the retinal vessels in the other panels of the figure. Not every frame of video will have a fluorescent streak in focus; however, some frames may have multiple streaks for use in the analysis (e.g. p…

Discussion

This technique of intravital video microscopy can be applied not only to mice, but also to rats. This protocol relies on only several assumptions, but is limited to use under anesthesia and with pupil dilation as we have described it. The other assumptions and limitations are as follows:

1. Assumption of minimal optical magnification error resulting from the refractive nature of the eye. As described by others^18-20, the method of filling the space between the cornea …

Materials

Name of the Reagent/Equipment	Company	Catalog Number or Product	Comments/Description
Fluorescent microspheres	Bangs Laboratories	FS04F/10584 (green)
High molecular weight fluorescent dextran	Molecular Probes	D-7137 (green); D-7139 (red)
Microscope system	Nikon	Eclipse E600FN + attachments
4X objective	Nikon	Plan Fluor 4X	numerical aperture 0.13; working distance 17.2 mm
10X objective	Nikon	Plan 10X	numerical aperture 0.25; working distance 10.5 mm
Tropicamide ophthalmic solution	Bausch & Lomb	1% Tropicamide
Hypromellose ophthalmic solution	HUB Pharmaceuticals	2.5% Goniovisc
Image processing software	University of California San Francisco Vale Lab	Micro-Manager
Digital video camera for microscopy	Photometrics	CoolSnap ES	1392 x 1040 pixel resolution; pixel size 6.45 x 6.45 μm