Periorbital Placement of a Laser Doppler Probe for Cerebral Blood Flow Monitoring Prior to Middle Cerebral Artery Occlusion in Rodent Models

Summary

A minimally invasive surgical procedure is shown here, which involves placing the laser Doppler probe onto the skull over the distal region of the middle cerebral artery (MCA), a periorbital location suitable for rats and mice, to assess blood flow during transient MCA occlusion.

Abstract

Middle cerebral artery occlusion (MCAO) is the gold-standard method for preclinical modeling of ischemic stroke in rodents. However, successful occlusion is not guaranteed by even the most skilled surgical hands. Errors primarily occur when the filament is not placed at the correct depth and include instances of either no infarction or vessel perforation, which can cause death. Laser Doppler flowmetry (LDF) is a reliable technique that provides real-time feedback on regional cerebral blood flow (CBF) during the MCAO procedure. Here we demonstrate a rapid technique for periorbital placement of a laser Doppler probe for measurement of CBF in both mice and rats. Our rationale was to simplify LDF implementation, encouraging widespread usage for improved surgical reliability. The technique eliminates the need for skull thinning and specialized equipment, with placement at the periorbital region rather than dorsal placement, promoting efficiency and ease of adoption. The protocol described here encompasses presurgical preparations, periorbital Doppler probe placement, and post-operative care. Representative results include visual depictions of procedural elements along with representative LDF tracings illustrating successful MCAO surgeries, with instances of unsuccessful filament placement leading to complications. The protocol illustrates LDF in confirming proper filament placement and offers a simplified procedure compared to alternative methods.

Introduction

The middle cerebral artery occlusion (MCAO) method has been widely used in rodents since introduced to the scientific community for application to rats in 1986¹ with the Longa adaption described in 1990², and adaptations for mice soon following³. Although not described in Longa's publication, the use of laser Doppler flowmetry (LDF) signal to confirm the filament placement was soon described in the literature⁴. LDF employment during the MCAO procedure is prominently featured in the literature but is designated as an optional step in the current Stroke Preclinical Assess....

Protocol

Adult mice and rats were used to illustrate this protocol (25 g, C57BL/6J, Jackson Laboratories; 250 g, Sprague Dawley, Envigo). Animal handling and experimental procedures were performed with approval and in compliance with the University of Arizona Institutional Animal Care and Use Committee, national laws, and according to the principles of laboratory animal care¹¹. Rats and mice were housed with a 12 h light/dark schedule (7 am-7 pm) with food and water available ad libitum.

1. Presurgical preparation

NOTE: Typically, MCAO surgery in a rodent is performed as a s....

Results

The placement of the laser Doppler probe at the MCA region is visually depicted in Figure 1, offering a pictograph of vasculature and serving as a visual guide from sagittal and dorsal perspectives. Figure 2 summarizes the critical steps for laser Doppler probe placement and outcomes in the mouse. Figure 2A presents an image of an anesthetized and prepared mouse with a dashed marking at the site of the vertical incision necessary fo.......

Discussion

The MCAO is the gold-standard procedure for modeling cerebral artery occlusion and reperfusion in rodents and has been the cornerstone of preclinical stroke research, enabling the induction of focal ischemia in rodents to mimic human stroke pathophysiology. It is an exacting surgical procedure with significant inter- and even intra-surgeon variability. While there is no evidence that the application of LDF reduces variability, it may improve scientific rigor and study outcomes in some designs. This is accomplished becaus.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
curved spring scissors	Castroviejo	1501710
forceps #5	Fine science tools	11250-20
forceps #5/45	Fine science tools	1151-35
Forcepts Cautery tool	Conmed	M18019-01
Laboratory tape	Fisherbrand Labeling Tape	15-950
Laser Doppler Monitor	Moore Instruments	MOORVMS-LDF
LDF software	Perisoft for Windows or moorSOFT	NA
Mouse clippers	Philips Norelco	MG7910
Periflux System 4000, probe 407	Perimed	equipment no longer available
plastic wrap	Glad	press n seal
Rat clippers	oster	A5 or similar
Small rodent anesthesia	JD Medical	custom order
small scissors	excelta	362 Sissors or similar
Temperature monitor system with probe	Physitemp	TCAT-2AC Controller