This research was funded by NINDS 1R41NS124450. Biorender: KT26JWLYF6

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 survival surgery, necessitating the use of either an aseptic or tips-only surgical technique, as outlined in an institution-specific IACUC protocol. In this case, the surgeon uses a tips-only technique with sterilized instruments and supplies.
<ol>
	<li>Disinfect the surgical work surface with commercial disinfectant and prepare sterile surgical packs of instruments, drapes, gauze, swabs, sutures, and scalpel blades by autoclaving. Maintain sterile conditions during survival surgery according to IACUC-approved techniques, such as covering microscope knobs and draping using food-grade plastic wrap.</li>
	<li>Use a germinator dry bead sterilizer to re-sterilize surgical instruments between procedures if multiple rodent surgeries are carried out during one session.</li>
	<li>Place the rat or mouse into an induction chamber and induce anesthesia with 3%-5% isoflurane (1 L/min using combined 80% air and 20% oxygen mixture).</li>
	<li>Move the animal from the chamber to the bench and nosecone for anesthesia (mouse: 0.5 L/min 80%/20% oxygen/air mixture; rat: 1.5 L/min 80%/20% oxygen/air mixture). Apply ophthalmic ointment to both eyes and carefully shave the space between the ear and eye socket on the side of the MCAO surgery (commonly the right), so as not to cut any skin using clippers. In the rat, when shaved, the whisker dimple will be visible approximately midway between the eye socket and the ear. Continue to shave regions as needed for the MCAO procedure9 (not in the scope of this paper).</li>
	<li>Return the animal to the chamber as needed or move it to a nosecone on the prepared surgical table warmed to 37 &#176;C. Insert a rectal thermometer. The animal should be kept at approximately 36 &#176;C &#177; 1 &#176;C throughout the procedure and according to the approved IACUC protocol.</li>
	<li>Apply betadine to a cotton swab and disinfect the skin, spiraling outward from the center of the surgical region. Wipe with an alcohol prep pad. Repeat both steps for a total of three cycles. Confirm proper anesthetization and assess every 15 min during the procedure using the toe pinch reflux.</li>
</ol>
2. Periorbital Doppler probe placement
<ol>
	<li>With small scissors, make a vertical cut (mouse: ~0.5 cm; rat: ~0.7 cm) between the corner of the eye and the fold of the ear, as shown in Figure 2A (mouse) and Figure 3A (rat) to expose super facia and underlying muscle. 
	NOTE: Treat for pain preoperatively according to the IACUC protocol approval.</li>
	<li>Using sharp forceps and curved scissors, carefully cut through the muscle layer until the skull is reached. The skull will be flat and hard to touch with the tip of the forceps. This is the window in which the laser Doppler probe will sit, as visualized in Figure 2B.</li>
	<li>Use a forceps cautery tool to clot emerging blood, widen the window, and ensure the route to the skull.</li>
	<li>Once the window is open, blot with cotton swabs to remove excess blood as necessary. 
	Place the mouse or rat in the supine position.</li>
	<li>Use forceps to retract any skin or tissue and place the Doppler probe into the periorbital window so that it abuts the skull perpendicular with no skin or muscle present to impede the laser signal. This region is over the distal MCA as illustrated in Figure 1A,B. Secure the probe placement with laboratory tape. In this protocol, the use of a straight probe is described. 
	NOTE: We acknowledge that surgery protocols will vary among laboratories. In the literature (when reported), some grind the skull thin, and others do not9. It may be because the skull is thinner in the periorbital region, but this is just speculation. In our experience, direct placement without grinding can be achieved in mice and rats and is demonstrated in this protocol.</li>
	<li>Monitor the relative blood flow readings on the monitor with appropriate software (Figure 2D, Figure 3C,D). LDF readings will vary. However, the optimal range for detection of blood flow drops is &#62;200 perfusion units (PU).</li>
	<li>Proceed to the MCAO surgery9 (not in the scope of this paper). During the MCAO surgery, test probe placement by briefly tying the common carotid artery, which should cause a sudden drop in blood flow (~50%).</li>
</ol>
3. Doppler probe removal and post-operative care
<ol>
	<li>At the conclusion of the MCAO or whenever blood flow readings are completed, remove the laser Doppler probe from the surgical window. The Doppler probe typically remains in place for the duration of the mouse MCAO surgery because ischemia times are often less than 90 min. On the other hand, in the rat MCAO procedure, the Doppler probe may be removed after the filament is placed and during ischemia (typically &#62; 90 min) and then placed again in the periorbital window to confirm reperfusion by repeating steps 2.6 and 2.7. 
	NOTE: A typical LDF continuous tracing from the mouse MCAO may be observed in Figure 4A. Typical LDF tracings from a rat MCAO procedure are illustrated in Figure 4B.</li>
	<li>Inject IACUC-approved local anesthetic subcutaneously along the prospective incision site.</li>
	<li>Close the skin with 1-2 sutures using a standard surgical knot with absorbable suture thread or surgical glue, according to approved IACUC protocol. Treat for post-surgical pain according to IACUC protocol approval (e.g. bupivacaine HCL 0.5% &#38; lidocaine 2%, once intra-incision for mice and rats). Animals should not be left unattended until they are able to maintain sternal recumbency, and no animals are returned to the company of other animals until they are fully recovered.</li>
</ol>

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

<ol>
	<li>Koizumi, J., Yoshida, Y., Nakazawa, T., Ooneda, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+studies+of+ischemic+brain+edema.+I.+A+new+experimental+model+of+cerebral+embolism+in+rats+in+which+recirculation+can+be+introduced+in+the+ischemic+area.">Experimental studies of ischemic brain edema. I. A new experimental model of cerebral embolism in rats in which recirculation can be introduced in the ischemic area.</a> Japanese J Stroke. 20 (1), 84-91 (1986).</li><li>Longa, E. Z., Weinstein, P. R., Carlson, S., Cummins, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+middle+cerebral+artery+occlusion+without+craniectomy+in+rats.">Reversible middle cerebral artery occlusion without craniectomy in rats.</a> Stroke. 20 (1), 84 (1989).</li><li>Hata, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reproducible+model+of+middle+cerebral+artery+occlusion+in+mice:+hemodynamic,+biochemical,+and+magnetic+resonance+imaging.">A reproducible model of middle cerebral artery occlusion in mice: hemodynamic, biochemical, and magnetic resonance imaging.</a> J Cereb Blood Flow Metab. 18 (4), 367-375 (1998).</li><li>Dirnagl, U., Kaplan, B., Jacewicz, M., Pulsinelli, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+measurement+of+cerebral+cortical+blood+flow+by+laser-Doppler+flowmetry+in+a+rat+stroke+model.">Continuous measurement of cerebral cortical blood flow by laser-Doppler flowmetry in a rat stroke model.</a> J Cereb Blood Flow Metab. 9 (5), 589-596 (1989).</li><li>Lyden, P. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+stroke+preclinical+assessment+network:+Rationale,+design,+feasibility,+and+stage+1+results.">The stroke preclinical assessment network: Rationale, design, feasibility, and stage 1 results.</a> Stroke. 53 (5), 1802-1812 (2022).</li><li>Ingberg, E., Dock, H., Theodorsson, E., Theodorsson, A., Str&#246;m, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+laser+Doppler+flowmetry+and+occlusion+time+on+outcome+variability+and+mortality+in+rat+middle+cerebral+artery+occlusion:+inconclusive+results.">Effect of laser Doppler flowmetry and occlusion time on outcome variability and mortality in rat middle cerebral artery occlusion: inconclusive results.</a> BMC Neurosci. 19 (1), 24 (2018).</li><li>Spratt, N. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+of+the+method+of+thread+manufacture+improves+stroke+induction+rate+and+reduces+mortality+after+thread-occlusion+of+the+middle+cerebral+artery+in+young+or+aged+rats.">Modification of the method of thread manufacture improves stroke induction rate and reduces mortality after thread-occlusion of the middle cerebral artery in young or aged rats.</a> J Neurosci Methods. 155 (2), 285-290 (2006).</li><li>Watcharotayangul, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-ischemic+vascular+adhesion+protein-1+inhibition+provides+neuroprotection+in+a+rat+temporary+middle+cerebral+artery+occlusion+model.">Post-ischemic vascular adhesion protein-1 inhibition provides neuroprotection in a rat temporary middle cerebral artery occlusion model.</a> J Neurochem. 123 Suppl 2 (Suppl 2), 116-124 (2012).</li><li>Beretta, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+system+for+cerebral+perfusion+monitoring+in+the+rat+stroke+model+of+intraluminal+middle+cerebral+artery+occlusion.">Optimized system for cerebral perfusion monitoring in the rat stroke model of intraluminal middle cerebral artery occlusion.</a> J Vis Exp. (72), e50214 (2013).</li><li>Ritter, L. S., Stempel, K. M., Coull, B. M., McDonagh, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte-platelet+aggregates+in+rat+peripheral+blood+after+ischemic+stroke+and+reperfusion.">Leukocyte-platelet aggregates in rat peripheral blood after ischemic stroke and reperfusion.</a> Biol Res Nursing. 6 (4), 281 (2005).</li><li>National Research Council. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide for the Care and Use of Laboratory Animals.. , (2011).</li><li>Ingberg, E., Dock, H., Theodorsson, E., Theodorsson, A., Str&#246;m, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+parameters'+impact+on+mortality+and+variability+in+mouse+stroke+experiments:+a+meta-analysis.">Method parameters' impact on mortality and variability in mouse stroke experiments: a meta-analysis.</a> Sci Rep. 6, (2016).</li><li>Morais, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embracing+heterogeneity+in+the+multicenter+stroke+preclinical+assessment+network+(SPAN)+trial.">Embracing heterogeneity in the multicenter stroke preclinical assessment network (SPAN) trial.</a> Stroke. 54 (2), 620-631 (2023).</li><li>Str&#246;m, J. O., Ingberg, E., Theodorsson, A., Theodorsson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+parameters'+impact+on+mortality+and+variability+in+rat+stroke+experiments:+a+meta-analysis.">Method parameters' impact on mortality and variability in rat stroke experiments: a meta-analysis.</a> BMC Neurosci. 14, 41 (2013).</li></ol>

The authors have nothing to disclose.

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...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>curved spring scissors</td>
			<td>Castroviejo</td>
			<td>1501710</td>
			<td></td>
		</tr>
		<tr>
			<td>forceps #5</td>
			<td>Fine science tools</td>
			<td>11250-20</td>
			<td></td>
		</tr>
		<tr>
			<td>forceps #5/45</td>
			<td>Fine science tools</td>
			<td>1151-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Forcepts Cautery tool</td>
			<td>Conmed</td>
			<td>M18019-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory tape</td>
			<td>Fisherbrand Labeling Tape</td>
			<td>15-950</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Doppler Monitor</td>
			<td>Moore Instruments&#160;</td>
			<td>MOORVMS-LDF</td>
			<td></td>
		</tr>
		<tr>
			<td>LDF software</td>
			<td>Perisoft for Windows or moorSOFT</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse clippers</td>
			<td>Philips Norelco</td>
			<td>MG7910</td>
			<td></td>
		</tr>
		<tr>
			<td>Periflux System 4000, probe 407</td>
			<td>Perimed</td>
			<td>equipment no longer available</td>
			<td></td>
		</tr>
		<tr>
			<td>plastic wrap</td>
			<td>Glad</td>
			<td>press n seal</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat clippers</td>
			<td>oster</td>
			<td>A5 or similar</td>
			<td></td>
		</tr>
		<tr>
			<td>Small rodent anesthesia</td>
			<td>JD Medical</td>
			<td>custom order</td>
			<td></td>
		</tr>
		<tr>
			<td>small scissors</td>
			<td>excelta</td>
			<td>362 Sissors or similar</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature monitor system with probe</td>
			<td>Physitemp</td>
			<td>TCAT-2AC Controller</td>
			<td></td>
		</tr>
	</tbody>
</table>

periorbital placement of a laser doppler probe for cerebral blood flow monitoring prior to middle cerebral artery occlusion in rodent 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.

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.

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...

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 ...

periorbital-placement-laser-doppler-probe-for-cerebral-blood-flow

Research

JoVE Journal

Neuroscience

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.

129 Views.  University of Arizona.  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.

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

Özet