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 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.

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

374 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.

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

Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence

Monitoring of the spatiotemporal characteristics of cerebral blood and tissue oxygenation is crucial for better understanding of the 
neuro-metabolic-vascular relationship. Development of new pO2 measurement modalities with simultaneous monitoring of pO2 in larger fields of view with higher 
spatial and/or temporal resolution will enable greater insight into the functioning of the normal brain and will also have significant impact on diagnosis 
and treatment of neurovascular diseases such as stroke, Alzheimer's disease, and head injury.
Optical imaging modalities have shown a great potential to provide high spatiotemporal resolution and quantitative imaging of pO2 based on hemoglobin absorption in visible and near infrared range of optical spectrum. However, multispectral measurement of cerebral blood oxygenation relies on photon migration through the highly scattering brain tissue. Estimation and modeling of tissue optical parameters, which may undergo dynamic changes during the experiment, is typically required for accurate estimation of blood oxygenation. On the other hand, estimation of the partial pressure of oxygen (pO2) based on oxygen-dependent quenching of phosphorescence should not be significantly affected by the changes in the optical parameters of the tissue and provides an absolute measure of pO2. Experimental systems that utilize oxygen-sensitive dyes have been demonstrated in in vivo studies of the perfused tissue as well as for monitoring the oxygen content in tissue cultures, showing that phosphorescence quenching is a potent technology capable of accurate oxygen imaging in the physiological pO2 range. 
Here we demonstrate with two different imaging modalities how to perform measurement of pO2 in cortical vasculature based on phosphorescence lifetime imaging. In first demonstration we present wide field of view imaging of pO2 at the cortical surface of a rat. This imaging modality has relatively simple experimental setup based on a CCD camera and a pulsed green laser. An example of monitoring the cortical spreading depression based on phosphorescence lifetime of Oxyphor R3 dye was presented. In second demonstration we present a high resolution two-photon pO2 imaging in cortical micro vasculature of a mouse. The experimental setup includes a custom built 2-photon microscope with femtosecond laser, electro-optic modulator, and photon-counting photo multiplier tube. We present an example of imaging the pO2 heterogeneity in the cortical microvasculature including capillaries, using a novel PtP-C343 dye with enhanced 2-photon excitation cross section.
Click here to view the related article <a href="http://www.jove.com/Details.php?ID=1731">Synthesis and Calibration of Phosphorescent Nanoprobes for Oxygen Imaging in Biological Systems.</a>

We present an experimental procedure for measuring the partial pressure of oxygen (pO2) in cerebral vasculature based on oxygen-dependent quenching of phosphorescence. Animal preparation and imaging procedures were outlined for both large field of view CCD-based imaging of pO2 in rats and 2-photon excitation based imaging of pO2 in mice.

Monitoring of the spatiotemporal characteristics of cerebral blood and tissue oxygenation is crucial for better understanding of the 
...

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans2,3. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.

Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable ...

Assessment of Cerebral Lateralization in Children using Functional Transcranial Doppler Ultrasound (fTCD)

There are many unanswered questions about cerebral lateralization. In particular, it remains unclear which aspects of language and nonverbal ability are lateralized, whether there are any disadvantages associated with atypical patterns of cerebral lateralization, and whether cerebral lateralization develops with age. In the past, researchers interested in these questions tended to use handedness as a proxy measure for cerebral lateralization, but this is unsatisfactory because handedness is only a weak and indirect indicator of laterality of cognitive functions1. Other methods, such as fMRI, are expensive for large-scale studies, and not always feasible with children2.
Here we will describe the use of functional transcranial Doppler ultrasound (fTCD) as a cost-effective, non-invasive and reliable method for assessing cerebral lateralization. The procedure involves measuring blood flow in the middle cerebral artery via an ultrasound probe placed just in front of the ear. Our work builds on work by Rune Aaslid, who co-introduced TCD in 1982, and Stefan Knecht, Michael Deppe and their colleagues at the University of M&uuml;nster, who pioneered the use of simultaneous measurements of left- and right middle cerebral artery blood flow, and devised a method of correcting for heart beat activity. This made it possible to see a clear increase in left-sided blood flow during language generation, with lateralization agreeing well with that obtained using other methods3.
The middle cerebral artery has a very wide vascular territory (see Figure 1) and the method does not provide useful information about localization within a hemisphere. Our experience suggests it is particularly sensitive to tasks that involve explicit or implicit speech production. The 'gold standard' task is a word generation task (e.g. think of as many words as you can that begin with the letter 'B') 4, but this is not suitable for young children and others with limited literacy skills. Compared with other brain imaging methods, fTCD is relatively unaffected by movement artefacts from speaking, and so we are able to get a reliable result from tasks that involve describing pictures aloud5,6. Accordingly, we have developed a child-friendly task that involves looking at video-clips that tell a story, and then describing what was seen.

Assessment of Cerebral Lateralization in Children using Functional Transcranial Doppler Ultrasound fTCD

Functional transcranial Doppler sonography (fTCD) is a simple and non-invasive ultrasound technique which can be used to assess the lateralization of cognitive functions, especially language, and is suitable for use with children.

There are many unanswered questions about cerebral lateralization.  In particular, it remains unclear which aspects of language and nonverbal ability are ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Isolation of Cerebrospinal Fluid from Rodent Embryos for use with Dissected Cerebral Cortical Explants

The CSF is a complex fluid with a dynamically varying proteome throughout development and in adulthood. During embryonic development, the nascent CSF differentiates from the amniotic fluid upon closure of the anterior neural tube. CSF volume then increases over subsequent days as the neuroepithelial progenitor cells lining the ventricles and the choroid plexus generate CSF. The embryonic CSF contacts the apical, ventricular surface of the neural stem cells of the developing brain and spinal cord. CSF provides crucial fluid pressure for the expansion of the developing brain and distributes important growth promoting factors to neural progenitor cells in a temporally-specific manner. To investigate the function of the CSF, it is important to isolate pure samples of embryonic CSF without contamination from blood or the developing telencephalic tissue. Here, we describe a technique to isolate relatively pure samples of ventricular embryonic CSF that can be used for a wide range of experimental assays including mass spectrometry, protein electrophoresis, and cell and primary explant culture. We demonstrate how to dissect and culture cortical explants on porous polycarbonate membranes in order to grow developing cortical tissue with reduced volumes of media or CSF. With this method, experiments can be performed using CSF from varying ages or conditions to investigate the biological activity of the CSF proteome on target cells.

The ventricular cerebrospinal fluid (CSF) bathes the neuroepithelial and cerebral cortical progenitor cells during early brain development in the embryo. Here we describe the method developed to isolate ventricular CSF from rodent embryos of different ages in order to investigate its biological function. In addition, we demonstrate our cerebral cortical explant dissection and culture technique that allows for explant growth with minimal volumes of culture medium or CSF.

The CSF is a complex fluid with a dynamically varying proteome throughout development and in adulthood. During embryonic development, the nascent CSF ...

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of motor and cognitive disabilities. Its high prevalence poses a serious health burden, as stroke is among the principal causes of long-term disability and death worldwide1. Recovery of neuronal function is, in most cases, only partial. So far, treatment options are very limited, in particular due to the narrow time window for thrombolysis2,3. Determining methods to accelerate recovery from stroke remains a prime medical goal; however, this has been hampered by insufficient mechanistic insights into the recovery process. Experimental stroke researchers frequently employ rodent models of focal cerebral ischemia. Beyond the acute phase, stroke research is increasingly focused on the sub-acute and chronic phase following cerebral ischemia. Most stroke researchers apply permanent or transient occlusion of the MCA in mice or rats. In patients, occlusions of the MCA are among the most frequent causes of ischemic stroke4. Besides proximal occlusion of the MCA using the filament model, surgical occlusion of the distal MCA is probably the most frequently used model in experimental stroke research5. Occlusion of a distal (to the branching of the lenticulo-striate arteries) MCA branch typically spares the striatum and primarily affects the neocortex. Vessel occlusion can be permanent or transient. High reproducibility of lesion volume and very low mortality rates with respect to the long-term outcome are the main advantages of this model. Here, we demonstrate how to perform a chronic cranial window (CW) preparation lateral to the sagittal sinus, and afterwards how to surgically induce a distal stroke underneath the window using a craniotomy approach. This approach can be applied for sequential imaging of acute and chronic changes following ischemia via epi-illuminating, confocal laser scanning, and two-photon intravital microscopy.

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Surgical occlusion of a distal middle cerebral artery branch (MCAo) is a frequently used model in experimental stroke research. This manuscript describes the basic technique of permanent MCAo, combined with the insertion of a lateral cranial window, which offers the opportunity for longitudinal intravital microscopy in mice.

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

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 for Encephalomyosynangiosis Treatment after Middle Cerebral Artery Occlusion-Induced Stroke in Mice

There is no effective treatment available for most patients suffering with ischemic stroke, making development of novel therapeutics imperative. The brain's ability to self-heal after ischemic stroke is limited by inadequate blood supply in the impacted area. Encephalomyosynangiosis (EMS) is a neurosurgical procedure that achieves angiogenesis in patients with moyamoya disease. It involves craniotomy with placement of a vascular temporalis muscle graft on the ischemic brain surface. EMS has never been studied in the setting of acute ischemic stroke in mice. The hypothesis driving this study is that EMS enhances cerebral angiogenesis at the cortical surface surrounding the muscle graft. The protocol shown here describes the procedure and provides initial data supporting the feasibility and efficacy of the EMS approach. In this protocol, after 60 min of transient middle cerebral artery occlusion (MCAo), mice were randomized to either MCAo or MCAo + EMS treatment. The EMS was performed 3-4 h after occlusion. The mice were sacrificed 7 or 21 days after MCAo or MCAo + EMS treatment. Temporalis graft viability was measured using nicotinamide adenine dinucleotide reduced-tetrazolium reductase assay. A mouse angiogenesis array quantified angiogenic and neuromodulating protein expression. Immunohistochemistry was used to visualize graft bonding with brain cortex and change in vessel density. The preliminary data here suggest that grafted muscle remained viable 21 days after EMS. Immunostaining showed successful graft implantation and increase in vessel density near the muscle graft, indicating increased angiogenesis. Data show that EMS increases fibroblast growth factor (FGF) and decreases osteopontin levels after stroke. Additionally, EMS after stroke did not increase mortality suggesting that protocol is safe and reliable. This novel procedure is effective and well-tolerated and has the potential to provide information of novel interventions for enhanced angiogenesis after acute ischemic stroke.

The protocol aims to provide methods for encephalomyosynangiosis-grafting of a vascular temporalis muscle flap on the pial surface of ischemic brain tissue-for the treatment of non-moyamoya acute ischemic stroke. The approach's efficacy in increasing angiogenesis is evaluated using a transient middle cerebral artery occlusion model in mice.

There is no effective treatment available for most patients suffering with ischemic stroke, making development of novel therapeutics imperative. The ...

Establishing a Transcranial Middle Cerebral Artery Occlusion Mice Model

A Modified Transcranial Middle Cerebral Artery Occlusion Model to Study Stroke Outcomes in Aged Mice

In experimental stroke research, middle cerebral artery occlusion (MCAO) with an intraluminal filament is widely used to model ischemic stroke in mice. The filament MCAO model typically exhibits a massive cerebral infarction in C57Bl/6 mice that sometimes includes brain tissue in the territory supplied by the posterior cerebral artery, which is largely due to a high incidence of posterior communicating artery atresia. This phenomenon is considered a major contributor to the high mortality rate observed in C57Bl/6 mice during long-term stroke recovery after filament MCAO. Thus, many chronic stroke studies exploit distal MCAO models. However, these models usually produce infarction only in the cortex area, and consequently, the assessment of post-stroke neurologic deficits could be a challenge. This study has established a modified transcranial MCAO model in which the MCA at the trunk is partially occluded either permanently or transiently via a small cranial window. Since the occlusion location is relatively proximal to the origin of the MCA, this model generates brain damage in both the cortex and striatum. Extensive characterization of this model has demonstrated an excellent long-term survival rate, even in aged mice, as well as readily detectable neurologic deficits. Therefore, the MCAO mouse model described here represents a valuable tool for experimental stroke research.

Author Spotlight: Innovative Techniques and Future Directions in Stroke Research 

This protocol demonstrates a unique mouse stroke model with a medium-sized infarct and an excellent survival rate. This model allows preclinical stroke researchers to extend the ischemia duration, use aged mice, and assess long-term functional outcomes.

In experimental stroke research, middle cerebral artery occlusion (MCAO) with an intraluminal filament is widely used to model ischemic stroke in mice. ...