real-time cerebral blood flow monitoring with periorbital doppler probe in rodent middle cerebral artery occlusion stroke models

Periorbital Laser Doppler Flowmetry for MCAO-Induced Ischemic Stroke in Rodents

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&#39;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 Assessment Networks (SPAN) Standard Operative Procedures (SOPs)5.
The use of LDF confirms correct filament placement during the MCAO procedure and, therefore, contributes to study design rigor and subsequent results, particularly in experiments designed to explore drug efficacy. LDF use reduces surgical mistakes stemming from incorrect filament placement, which results in the dichotomous situation of either no injury when the filament is not placed far enough or animal deaths due to vessel perforation occurring when the filament is inserted too far. On the other hand, the use of LDF is not associated with infarct size variability commonly observed following the MCAO procedure6. The use of LDF in the MCAO procedure may be perceived as difficult and onerous, particularly in rats, because the skull is thicker than in mice and may require skull thinning prior to LDF placement7,8. Also, dorsal probe placement is often described with some protocols requiring specialized equipment or preparation7,8,9. With any of these barriers, implementation of the LDF to confirm filament placement may not take place.
In this protocol, we describe the placement of the laser Doppler probe at the skull and over the distal region of the middle cerebral artery-a periorbital placement-for the assessment of blood flow during the MCAO procedure in both mice and rats. Our rationale was to develop a procedure that has multiple advantages over some methods reported in the literature7,8,9,10 in that it is minimally invasive, fast, and does not require skull thinning or specialized equipment beyond the laser Doppler probe.

Author Spotlight: Advancing Stroke Research with Periorbital Doppler Monitoring of Regional Cerebral Blood Flow in Rodent Models

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

I investigated ischemic stroke, focusing on microglia, reperfusion injury, and sex differences in the brain. My work aims to understand how these factors affect brain injury and to develop targeted treatments for improved stroke treatments at acute and chronic timelines. Reproducibility and sample size are key challenges in stroke research.Variability in injury size from the middle cerebral artery occlusion procedure often requires larger sample sizes for accurate statistical testing, which may be underrecognized and impact reproducibility of science in our field. Our most significant findings and contributions revolve around microglia quantifications and the intersection with ischemic stroke from an acute and chronic perspective. Using Laser Doppler flowmetry in MCAO can be challenging, especially in rats due to thicker skulls and the perception that skull thinning and specialized equipment for dorsal probe placement are needed.These barriers may hinder LDF implementation for confirming filament placement. The protocol outlines placing the Laser Doppler probe at the skull in distal middle cerebral artery region periorbital placement to assess blood flow during MCAO in mice and rats. It offers advantages over other methods by being minimally invasive, fast, and requiring only the Laser Doppler probe.

Real-Time Cerebral Blood Flow Monitoring with Periorbital Doppler Probe in Rodent Middle Cerebral Artery Occlusion Stroke Models

To begin, place the prepared and anesthetized animal on the operating table for the procedure. Using a cotton swab, apply Betadine to the prepared skin in a spiral motion, starting from the center of the surgical area. Then wipe the area with an alcohol pad.Using small scissors, make a vertical cut between the corner of the eye and the fold of the ear to expose the superficial fascia and underlying muscle. Then employing sharp forceps and curved scissors, carefully cut through the muscle layer until reaching the skull. Employing forceps cautery tool, clot any emerging blood before widening the window as necessary to ensure a clear route to the skull.After blotting with cotton swabs to remove excess blood, place the animal supine. Use forceps to retract any skin or tissue and place the Doppler probe into the periorbital window so that it abuts the skull without any skin or muscle obstructing the laser signal. Secure the probe with laboratory tape.Monitor the relative blood flow readings using the appropriate software. A typical ischemia pattern during distal middle cerebral artery or MCA occlusion surgery was evident as there was a rapid cerebral blood flow drop when the carotid artery was tied in the filament advanced to the MCA osteo, followed by a quick CBF increase indicating reperfusion when the filament was withdrawn and the carotid artery was untied. The tracing during rat and mouse MCAO surgery showed failed ischemia due to a loosened carotid occlusion, indicated by a slow drop in CBF.

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Research

JoVE Journal

Neuroscience

27 Views. To begin, place the prepared and anesthetized animal on the operating table for the procedure. Using a cotton swab, apply Betadine to the prepared skin in a spiral motion, starting from the center of the surgical area. Then wipe the area with an alcohol pad.Using small scissors, make a vertical cut between the corner of the eye and the fold of the ear to expose the superficial fascia and underlying muscle. Then employing sharp forceps and curved scissors, carefully cut through the musc...

Video: Real-Time Cerebral Blood Flow Monitoring with Periorbital Doppler Probe in Rodent Middle Cerebral Artery Occlusion Stroke Models

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.

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.

Middle cerebral artery occlusion (MCAO) is the gold-standard method for preclinical modeling of ischemic stroke in rodents. However, successful occlusion ...