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...

<a href="https://app.jove.com/t/6629">This corrects</a> the article <a href="https://dx.doi.org/10.3791/66839">10.3791/66839</a>

An erratum was issued for:&#160;<a href="https://www.jove.com/t/66839">Periorbital Placement of a Laser Doppler Probe for Cerebral Blood Flow Monitoring Prior to Middle Cerebral Artery Occlusion in Rodent Models</a>. The Authors&#160;section was&#160;updated.

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

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><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&amp;nbsp;</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.

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...

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

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 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 ...

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

Research

JoVE Journal

Neuroscience

517 Views.  University of Arizona. Ik onderzocht ischemische beroerte, met de nadruk op microglia, reperfusieletsel en sekseverschillen in de hersenen. Mijn werk is erop gericht te begrijpen hoe deze factoren hersenletsel beïnvloeden en om gerichte behandelingen te ontwikkelen voor verbeterde behandelingen van beroertes op acute en chronische tijdlijnen. Reproduceerbaarheid en steekproefomvang zijn belangrijke uitdagingen bij onderzoek naar beroertes.Variabiliteit in letselgrootte van ...

Video: Auteur in de schijnwerpers: onderzoek naar beroertes bevorderen met periorbitale doppler-monitoring van regionale cerebrale bloedstroom in knaagdiermodellen

Intraluminal Middle Cerebral Artery Occlusion (MCAO) Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United States in 20091,2. The intraluminal middle cerebral artery occlusion (MCAO) model was developed by Koizumi4 in 1986 to simulate this 
impactful human pathology in the rat. A modification of the MCAO method was later presented by Longa3. Both techniques have been widely used to identify 
molecular mechanisms of brain injury resulting from ischemic stroke and potential therapeutic modalities5. This relatively noninvasive method in rats has been 
extended to use in mice to take advantage of transgenic and knockout strains6,7. To model focal cerebral ischemia, an intraluminal suture is advanced via 
the internal carotid artery to occlude the base of the MCA. Retracting the suture after a specified period of time mimics spontaneous reperfusion, but the 
suture can also be permanently retained. This video will be demonstrating the two major approaches for performing intraluminal MCAO procedure in mice in a 
stepwise fashion, as well as providing insights for potential drawbacks and pitfalls. The ischemic brain tissue will subsequently be stained by 
2,3,5-triphenyltetrazolium chloride (TTC) to evaluate the extent of cerebral infarction8.

Intraluminal Middle Cerebral Artery Occlusion MCAO Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

The intraluminal middle cerebral artery occlusion (MCAO) model is the most frequent used model among experimental ischemic stroke models. Here we will demonstrate the entire model in detail with the guide of Laser Doppler flowmetry, and its representative results.

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United ...

Medicine

Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in 20091, 2. Several models of cerebral ischemia have been developed to mimic the human condition of stroke. It has been suggested that up to 80% of all strokes result from ischemic damage in the middle cerebral artery (MCA) area3. In the early 1990s, endothelin-1 (ET-1) 4 was used to induce ischemia by applying it directly adjacent to the surface of the MCA after craniotomy. Later, this model was modified 5 by using a stereotaxic injection of ET-1 adjacent to the MCA to produce focal cerebral ischemia. The main advantages of this model include the ability to perform the procedure quickly, the ability to control artery constriction by altering the dose of ET-1 delivered, no need to manipulate the extracranial vessels supplying blood to the brain as well as gradual reperfusion rates that more closely mimics the reperfusion in humans5-7. On the other hand, the ET-1 model has disadvantages that include the need for a craniotomy, as well as higher variability in stroke volume8. This variability can be reduced with the use of laser Doppler flowmetry (LDF) to verify cerebral ischemia during ET-1 infusion. Factors that affect stroke variability include precision of infusion and the batch of the ET-1 used6. Another important consideration is that although reperfusion is a common occurrence in human stroke, the duration of occlusion for ET-1 induced MCAO may not closely mimic that of human stroke where many patients have partial reperfusion over a period of hours to days following occlusion9, 10. This protocol will describe in detail the ET-1 induced MCAO model for ischemic stroke in rats. It will also draw attention to special considerations and potential drawbacks throughout the procedure.

Several animal models of cerebral ischemia have been developed to simulate the human condition of stroke. This protocol describes the endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAO) model for ischemic stroke in rats. In addition, important considerations, advantages, and shortcomings of this model are discussed.

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in ...

Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal models of acute ischemic stroke allows to confirm successful arterial occlusion and exclude subarachnoid hemorrhage. Cerebral perfusion monitoring can also be used to study intracranial collateral circulation, which is emerging as a powerful determinant of stroke outcome and a possible therapeutic target. Despite a recognized role of Laser Doppler perfusion monitoring as part of the current guidelines for experimental cerebral ischemia, a number of technical difficulties exist that limit its widespread use. One of the major issues is obtaining a secure and prolonged attachment of a deep-penetration Laser Doppler probe to the animal skull. In this video, we show our optimized system for cerebral perfusion monitoring during transient middle cerebral artery occlusion by intraluminal filament in the rat. We developed in-house a simple method to obtain a custom made holder for twin-fibre (deep-penetration) Laser Doppler probes, which allow multi-site monitoring if needed. A continuous and prolonged monitoring of cerebral perfusion could easily be obtained over the intact skull.

Cerebral perfusion monitoring has been demonstrated to improve accuracy in ischemic stroke models. Technical difficulties often limit the use of this essential tool for cerebrovascular research. In this video, an optimized system is shown to obtain a single or multi-site hemodynamic monitoring during intraluminal middle cerebral artery occlusion in rats.

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal ...

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2 (i.e. room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier (BBB). Rats with induced ischemic stroke were established and used as in vivo models to investigate the functional integrity of the BBB. Spectrophotometric detection of Evans blue (EB) in the brain samples with ischemic injury could provide reliable justification for the research and development of novel therapeutic modalities. This method generates reproducible results, and is applicable in any laboratory without a need for special equipment. Here, we present a visualized and technical guideline on the detection of the extravasation of EB following induction of ischemic stroke in rats.

An In Vivo Assessment of Blood-Brain Barrier Disruption in a Rat Model of Ischemic Stroke

The overall goal of this procedure is to provide a highly reproducible technique for in vivo assessment of the blood-brain barrier disruption in rat models of ischemic stroke.

Ischemic stroke leads to vasogenic cerebral edema and subsequent primary brain injury, which is mediated through destruction of the blood-brain barrier ...

Middle Cerebral Artery Occlusion Allowing Reperfusion via Common Carotid Artery Repair in Mice

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue plasminogen activator (tPA) as an approved drug treatment to target ischemic strokes. Current research in the field of ischemic stroke focuses on better understanding the pathophysiology of stroke, to develop and investigate novel pharmaceutical targets. Reliable experimental stroke models are crucial for the progression of potential treatments. The middle cerebral artery occlusion (MCAO) model is clinically relevant and the most frequently used surgical model of ischemic stroke in rodents. However, the outcomes of this model, such as lesion volume, are associated with high levels of variability, particularly in mice. The alternative MCAO model described here allows the reperfusion of the common carotid artery (CCA) and the increased perfusion of the middle cerebral artery (MCA) territory, using a tissue pad with fibrinogen-based sealant to repair the vessel, and the improved welfare of the mice by avoiding external carotid artery (ECA) ligation. This reduces the reliance on the Circle of Willis, which is known to be highly anatomically variable in mice. Representative data show that using this alternative surgical approach decreases the variability in lesion volumes between the traditional MCAO approach and the alternative approach described here.

Intraluminal filament occlusion of the middle cerebral artery is the most frequently used in vivo model of experimental stroke in rodents. An alternative surgical approach to allow common carotid artery repair is performed here, which allows the reperfusion of the common carotid artery and a full reperfusion to the middle cerebral artery territory.

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue ...

Evaluation of Cerebral Blood Flow Autoregulation in the Rat Using Laser Doppler Flowmetry

When investigating the body&#39;s mechanisms for regulating cerebral blood flow, a relative measurement of microcirculatory blood flow can be obtained using laser Doppler flowmetry (LDF). This paper demonstrates a closed skull preparation that allows cerebral blood flow to be assessed without penetrating the skull or installing a chamber or cerebral window. To evaluate autoregulatory mechanisms, a model of controlled blood pressure reduction via graded hemorrhage can be utilized while simultaneously employing LDF. This enables the real time tracking of the relative changes in the blood flow in response to reductions in arterial blood pressure produced by the withdrawal of circulating blood volume. This paradigm is a valuable approach to study cerebral blood flow autoregulation during reductions in arterial blood pressure and, with minor modifications in the protocol, is also valuable as an experimental model of hemorrhagic shock. In addition to evaluating autoregulatory responses, LDF can be used to monitor the cortical blood flow when investigating metabolic, myogenic, endothelial, humoral, or neural mechanisms that regulate cerebral blood flow and the impact of various experimental interventions and pathological conditions on cerebral blood flow.

This article demonstrates the use of laser Doppler flowmetry to evaluate the ability of the cerebral circulation to autoregulate its blood flow during reductions in arterial blood pressure.

When investigating the body&#39;s mechanisms for regulating cerebral blood flow, a relative measurement of microcirculatory blood flow can be obtained ...

Modeling Stroke in Mice: Transient Middle Cerebral Artery Occlusion via the External Carotid Artery

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options are limited to a small proportion of stroke patients within the first hours after stroke. Novel therapeutic strategies are being extensively investigated, especially to prolong the therapeutic time window. These current investigations include the study of important pathophysiological pathways after stroke, such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Over the last decade, there has been increasing concern about the poor reproducibility of experimental results and scientific findings among independent research groups. To overcome the so-called &#8220;replication crisis&#8221;, detailed standardized models for all procedures are urgently needed. As an effort within the &#8220;ImmunoStroke&#8221; research consortium (https://immunostroke.de/), a standardized mouse model of transient middle cerebral artery occlusion (MCAo) is proposed. This model allows the complete restoration of the blood flow upon removal of the filament, simulating the therapeutic or spontaneous clot lysis that occurs in a large proportion of human strokes. The surgical procedure of this &#8220;filament&#8221; stroke model and tools for its functional analysis are demonstrated in the accompanying video.

Different models of middle cerebral artery occlusion (MCAo) are used in experimental stroke research. Here, an experimental stroke model of transient MCAo via the external carotid artery (ECA) is described, which aims to mimic human stroke, in which the cerebrovascular thrombus is removed due to spontaneous clot lysis or therapy.

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options ...

A Model of Transient Cerebral Ischemia by Occlusion of Middle Cerebral Artery

Transient Middle Cerebral Artery Occlusion Model of Stroke

Stroke stands as a major cause of death or chronic disability globally. Nevertheless, existing optimal treatments are limited to reperfusion therapies during the acute phase of ischemic stroke. To gain insights into stroke physiopathology and develop innovative therapeutic approaches, in vivo rodent models of stroke play a fundamental role. The availability of genetically modified animals has particularly propelled the use of mice as experimental stroke models.
In stroke patients, occlusion of the middle cerebral artery (MCA) is a common occurrence. Consequently, the most prevalent experimental model involves intraluminal occlusion of the MCA, a minimally invasive technique that doesn't require craniectomy. This procedure involves inserting a monofilament through the external carotid artery (ECA) and advancing it through the internal carotid artery (ICA) until it reaches the branching point of the MCA. After a 45 min arterial occlusion, the monofilament is removed to allow reperfusion. Throughout the process, cerebral blood flow is monitored to confirm the reduction during occlusion and subsequent recovery upon reperfusion. Neurological and tissue outcomes are evaluated using behavioral tests and magnetic resonance imaging (MRI) studies.

Author Spotlight: Assessing Ischemic Stroke Damage Through Middle Cerebral Artery Occlusion Model 

This protocol describes the model of transient focal cerebral ischemia in mice through intraluminal occlusion of the middle cerebral artery. Additionally, examples of outcome assessment are shown using magnetic resonance imaging and behavioral tests.

Stroke stands as a major cause of death or chronic disability globally. Nevertheless, existing optimal treatments are limited to reperfusion therapies ...

Filament Insertion-based Transient Stroke Model: A Surgical Procedure to Perform Middle Cerebral Artery Occlusion in Murine Brain

Source: Llovera, G., et al. <a href="https://www.jove.com/v/62573">Modeling Stroke in Mice: Transient Middle Cerebral Artery Occlusion via the External Carotid Artery.</a> J. Vis. Exp (2021).
This video describes the procedure to develop transient stroke in a murine model using silicon-coated filament-based occlusion of the middle cerebral artery.&#160;The developed model mimics human stroke, thus helping to investigate novel therapeutic strategies for treating stroke.

Source: Llovera, G., et al. Modeling Stroke in Mice: Transient Middle Cerebral Artery Occlusion via the External Carotid Artery. J. Vis. Exp (2021).
This ...

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

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