We thank Mrs. Caroline Robertson for the voice-over and Mrs. Elena Pirovano for her assistance in video production. This study was supported by the University of Milano Bicocca, "Fondo di Ateneo 2011".

1.	How to Make the Probe Holder (Single Site or Multi-site)
<ol>
 <li>The materials required are natural rubber, small plastic tubes and a metal stylet. The probe holder can be customized to the size of the animal, the number and size of the Laser Doppler probes, and the cerebral vascular territory which needs to be monitored. </li>
 <li>Cut the natural rubber of the needed size (approximately 10 mm x 10 mm for a 300 g rat). </li>
 <li>Mark the positions of the probe(s) and the bregma on the natural rubber, according to the desired stereotaxic coordinates; for middle cerebral artery occlusion, the typical coordinates for the ischemic core are expected at bregma -1 mm, 5 mm lateral to the midline; for a peripheral ischemic borderzone territory, the expected coordinates can be at bregma +2 mm, 2 mm lateral to the midline. </li>
 <li>Mark very clearly the position of the bregma with an X, this is the landmark for attaching the probe holder to the skull. </li>
 <li>Insert the stylet into a small plastic tube (its size needs to match the size of the probe).</li>
 <li>Insert the stylet into the natural rubber at the point where the probe has to be positioned; push the stylet into the rubber until the plastic tube has been inserted into the rubber too. </li>
 <li>Withdraw the stylet. </li>
 <li>If multiple probes are needed, repeat steps 1.5-1.7 for other probe positions. </li>
 <li>Optional for multiple probes: wrap a tape around the plastic tubes to ensure a better stabilization of the probes.</li>
 <li>Optional: perform chemical sterilization of several probe holders for future uses. </li>
</ol>
2.	Presurgical Preparation
<ol>
 <li>MCAO stroke model is usually performed as a survival surgery. In this case, as shown in our video (survival time 24 hr post-surgery), the surgeon uses an aseptic technique with sterilized instruments and supplies. </li>
 <li>Anesthetize the rat with isoflurane (3% induction phase, 1.5% maintenance).</li>
 <li>Place the rat in prone position on the operating table. </li>
 <li>Gently shave the head of the rat. </li>
 <li>Apply an antiseptic solution to a gauze pad and disinfect the skin. </li>
 <li>Administer Lidocaine 2% 5 mg/kg subcutaneously in the cranial area. </li>
</ol>
3.	Probe Positioning and Securing Over the Intact Skull
<ol>
 <li>Make a right paramedian skin incision (for right MCAO) and dissect the subcutaneous tissue to reach the cranial fascia (galea aponeurotica).</li>
 <li>Make a right paramedian incision in the cranial fascia and perform blunt dissection to reach the skull bone; prepare a skull bone area which is large enough for the application of the probe holder.</li>
 <li>Please note: no need for drilling or bone thinning. </li>
 <li>Apply Merbromin solution to disinfect and dry the skull surface. </li>
 <li>Use a hair dryer (set for cold air) to accelerate drying the skull surface. </li>
 <li>At this point, the cranial sutures and the bregma are clearly visible.</li>
 <li>Customize the probe holder by cutting the edges with sterile scissors. </li>
 <li>Apply a small quantity of surgical glue (Cyanoacrylate, veterinary-approved) to the surface underneath the probe holder, carefully avoiding the inferior opening of the plastic tubes (please note: if a significant amount of glue remains between the optical surface of the probe and the skull, this may produce a low-signal and may damage the probe after multiple uses).</li>
 <li>Apply the probe holder to the skull surface, carefully matching the bregma with the X landmark. Apply gentle pressure over the probe holder. </li>
 <li>Use a hair dryer (set for cold air) to accelerate drying the surgical glue. </li>
 <li>Secure the probe holder by tying a surgical thread around the probe holder and the head of the animal; be careful to position the surgical thread over the mandibula, avoiding the submandibular region and the neck. </li>
 <li>Optional: fill the plastic tube(s) of the probe holder with an optical gel (e.g. a common ultrasound or electrocardiography gel); this will increase the quality of the LD signal.</li>
 <li>Place the probe(s) in the probe holder and verify the actual reading of the LD flowmeter. Use the LD flowmeter according to manufacturer's instructions. </li>
 <li>Secure the probe(s) tying them around head of the animal; be careful to position the surgical thread over the mandibula, avoiding the submandibular region and the neck. </li>
</ol>
4.	Cerebral Perfusion Monitoring During MCAO 
<ol>
 <li>Place the rat in the supine position, carefully avoiding tractional forces on the probe(s) or probe holder. </li>
 <li>Start cerebral perfusion monitoring during MCAO. </li>
</ol>
5.	Removal of the Probe(s) and Probe Holder
<ol>
 <li>Cut the sutures around the probe(s), the probe holder and the head of the animal.</li>
 <li>Gently blunt dissect cranial soft tissues and skin around the natural rubber base of the probe holder. </li>
 <li>Remove the probe holder. </li>
 <li>Apply an antiseptic solution to the skull surface. </li>
 <li>Suture the cranial skin. </li>
</ol>
6.	Post-operative Care
<ol>
 <li>Administer 2.5 ml of saline solution subcutaneously to prevent dehydration and keep the animal warm using a heating pad after stopping gaseous anesthesia. </li>
 <li>For survival surgery: provide preemptive analgesia with Ketoprofen 4 mg/kg subcutaneously and repeat the same dose at 12 hr post-operatively </li>
 <li>Under our experimental conditions, euthanasia was performed at 24 hr post-surgery by CO2 inhalation. </li>
</ol>

<ol>
	<li>Sutherland, B. A., Papadakis, M., Chen, R. L., Buchan, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+blood+flow+alteration+in+neuroprotection+following+cerebral+ischemia.">Cerebral blood flow alteration in neuroprotection following cerebral ischemia.</a> J. Physiol. 589, 4105-4114 (2011).</li><li>Prinz, V., Endres, M., Dirnagl, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+3+Modeling+focal+cerebral+ischemia+in+rodents:+Introduction+and+overview.">Chapter 3 Modeling focal cerebral ischemia in rodents: Introduction and overview.</a> Rodent models of stroke. , (2010).</li><li>Schmid-Elsaesser, R., Zausinger, S., Hungerhuber, E., Baethmann, A., Reulen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+reevaluation+of+the+intraluminal+thread+model+of+focal+cerebral+ischemia:+evidence+of+inadvertent+premature+reperfusion+and+subarachnoid+hemorrhage+in+rats+by+laser-Doppler+flowmetry.">A critical reevaluation of the intraluminal thread model of focal cerebral ischemia: evidence of inadvertent premature reperfusion and subarachnoid hemorrhage in rats by laser-Doppler flowmetry.</a> Stroke. 29, 2162-2170 (1998).</li><li>Riva, M., Pappad&#224;, G. B., 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=Hemodynamic+monitoring+of+intracranial+collateral+flow+predicts+tissue+and+functional+outcome+in+experimental+ischemic+stroke.">Hemodynamic monitoring of intracranial collateral flow predicts tissue and functional outcome in experimental ischemic stroke.</a> Exp. Neurol. 233, 815-820 (2012).</li><li>Menon, B. K., Smith, E. E., 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=Regional+leptomeningeal+score+on+CT+angiography+predicts+clinical+and+imaging+outcomes+in+patients+with+acute+anterior+circulation+occlusions.">Regional leptomeningeal score on CT angiography predicts clinical and imaging outcomes in patients with acute anterior circulation occlusions.</a> Am. J. Neuroradiol. 32, 1640-1645 (2011).</li><li>Bang, O. Y., 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=Collateral+flow+predicts+response+to+endovascular+therapy+for+acute+ischemic+stroke.">Collateral flow predicts response to endovascular therapy for acute ischemic stroke.</a> Stroke. 42, 693-699 (2011).</li><li>Shuaib, A., Butcher, K., Mohammad, A. A., Saqqur, M., Liebeskind, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collateral+blood+vessels+in+acute+ischaemic+stroke:+a+potential+therapeutic+target.">Collateral blood vessels in acute ischaemic stroke: a potential therapeutic target.</a> Lancet Neurol. 10, 909-921 (2011).</li><li>Liu, S., Zhen, G., Meloni, B. P., Campbell, K., Winn, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+Stroke+Model+Guidelines+for+preclinical+stroke+trials+(1st+edition).">Rodent Stroke Model Guidelines for preclinical stroke trials (1st edition).</a> J. Exp. Stroke Transl. Med. 2, 2-27 (2009).</li><li>Shepherd, A. P., &#214;berg, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laser-Doppler Blood Flowmetry. , (1990).</li><li>Ulu&#231;, K., Miranpuri, A., Kujoth, G. C., Akt&#252;re, E., Ba&#351;kaya, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+Cerebral+Ischemia+Model+by+Endovascular+Suture+Occlusion+of+the+Middle+Cerebral+Artery+in+the+Rat.">Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat.</a> J. Vis. Exp. (48), e1978 (2011).</li><li>Garcia, J. H., Wagner, S., Liu, K. F., Hu, X. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurological+deficit+and+extent+of+neuronal+necrosis+attributable+to+middle+cerebral+artery+occlusion+in+rats.+Statistical+validation.">Neurological deficit and extent of neuronal necrosis attributable to middle cerebral artery occlusion in rats. Statistical validation.</a> Stroke. 26, 627-634 (1995).</li></ol>

The funding for the production of this video-article was provided by Moor Instruments Ltd (Axminster, Devon, UK), which produces the Laser Doppler instruments shown in our experimental study.
The experimental protocol was approved by the Committee on Animal Care of the University of Milano Bicocca, in accordance with the national guidelines on the use of laboratory animals (D.L. 116/1992) and the European Union Directive for animal experiments (2010/63/EU), under project license (n. 219/2011-B) from the Italian Ministry of Health.

We developed in-house a simple and low-cost system for a secure attachment of one or multiple twin-fibre (deep penetration) LD probes to the intact skull of rats during the MCAO procedure. Albeit apparently a trivial problem, obtaining a reliable attachment of the LD probe to the skull is actually a major issue in this experimental setting, since it is the prerequisite for a smooth signal detection and a successful monitoring of cerebral perfusion. 
Invasive procedures, such as burr holes and bone screws, usually prolong the surgical time and introduce more experimental variables related to craniotomy, and this may discourage researchers and refrain them from using LD monitoring. On the other hand, the use of single fibre (low penetration) probes, which are thinner and relatively easier to be glued directly to the skull surface, gives low-quality signal and cannot be used reliably with adult rats without drilling or thinning the skull.
We used simple and low-cost materials, such as natural rubber, plastic tubes and a metal stylet. A custom-made probe holder can be produced in few minutes and adapted to the experimental conditions. These probe holders can accommodate one or more deep-penetration LD probes, for classical single site monitoring on the ischemic core or for multiple-site monitoring in different arterial territories in the same hemisphere or across the two hemispheres. Many probe holders could be produced, chemically sterilized, and stored for future use. Veterinary-approved surgical glue (cyanoacrilate), accelerated by cold air, is used to attach the probe holder to the intact surface of the rat skull, according to the desired cranial coordinates. Finally, the probe set-up is further secured in place by common sutures. 
The overall time of this LD probe set-up, after mastering this technique, is about 10 min. 
As shown in this video, we routinely monitor cerebral perfusion in the central MCA territory (LD probe 1: ischemic core) and in the peripheral MCA territory (LD probe 2: mainly a penumbral area). In our recent study we showed that the variability of blood flow changes in LD probe 2 (mean 52% &plusmn; 16% SD, compared to the baseline) is higher compared to LD probe 1 (mean 31% &plusmn; 6% SD, compared to the baseline) and may be used to predict stroke outcome4.
We may provide some trouble-shooting advices for researchers which would like to use our in-house developed system. At the beginning of the experiment, be careful to dry very well the skull surface (with Merbromin and cold air) before attaching the probe holder to prevent premature detachment. Moreover, be sure to apply the glue on the natural rubber, avoiding contact with the open end of the plastic tube and the optical surface of the LD probe, to prevent poor signal and potential damage to the probe. When tying the suture around the head of the animal, be careful in order to avoid airway obstruction (this is prevented by positioning the suture over the mandibular bone). After positioning and securing the probes, be careful not to traction probe cables when turning the animal in the supine position for cervical surgery; this step usually requires two people, one person holding the animal and a second person holding the probe cables and gently positioning them to the desired position. Finally, eventual blood contamination of the twin-fibre LD probe is easily managed following the cleaning instructions provided by the manufacturer. 
Our optimized system for cerebral perfusion monitoring, as shown in this video, could provide an easier, more rapid and more reliable alternative to the probe set-up systems which are currently sold by commercial companies in this field. Moreover, we believe that the use of this system by other researchers may enhance the study of cerebral hemodynamics in the experimental stroke field, leading to the development of a new generation of cerebral collateral therapeutics.

Translational research on hemodynamic factors affecting stroke pathophysiology and therapy needs to be implemented, since this important issue is often paradoxically neglected by basic research studies1. 
Cerebral perfusion monitoring is an essential, but underused, tool for accurate ischemic stroke modeling2. Aside from the confirmation of arterial vessel occlusion and exclusion of subarachnoid hemorrhage3, continuous cerebral perfusion monitoring may provide useful data about the degree and consistency of perfusion deficit, the functional status of intracranial collateral vessels and the hemodynamic effect of new therapeutic approaches. 
A recent study from our group indicate that multi-site hemodynamic monitoring may be used to assess intracranial collateral circulation and can predict infarct size and functional deficit4. These experimental findings are consistent with clinical studies which showed that the functional performance of cerebral collateral circulation is predictive of clinical outcome in ischemic stroke patients 5, 6. For this reason, cerebral collaterals have been advocated as a potential therapeutics target in the acute phase of ischemic stroke7.
Laser-Doppler (LD) instruments are the most common tool used to measure cerebral perfusion in experimental ischemic stroke and their use is recommended by recent guidelines on this theme8. LD instruments measure microvascular perfusion in a small cortical volume, the depth of the recorded signal being dependent on the width of fibre separation, with twin fibre LD probes allowing a deeper penetration compared to single fibre LD probes9. Blood flow values are expressed as arbitrary perfusion units (P.U.) which indicate relative rather than absolute cerebral blood flow. Calibration of P.U. is usually performed using motility standards, according to manufacturer's instructions. LD flowmetry allows a continuous dynamic monitoring and generation of quantitative data within the same session. 
Among the technical problems that currently limit the use of LD, a major issue is obtaining a secure and prolonged attachment of a deep-penetration Laser Doppler probe to the animal skull. This is essential for prolonged monitoring and if multiple probes are used for different cerebral arterial territories, as we are performing in our laboratory. 
In particular, prolonged surgical time is required if probes are attached to the skull using burr holes or cranial screws, whereas poor signal and unsecure attachment occurs if single fibre (low-penetration) LD probes are attached to the skull by simple surgical glue. Twin fibre (deep-penetration) LD probes provide a higher and more consistent signal, but they are larger than single fibre probes and cannot be attached to the skull using surgical glue only.
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 describe a simple method to obtain an efficient, custom made, low cost holder for single or multiple twin-fibre (deep-penetration) LD probes, to be used for prolonged monitoring of cerebral perfusion over the intact skull. 
The surgical procedure for transient MCAO in the rat could be seen in the video-article by Ulu&ccedil; and coworkers10 and is not shown in this video.

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>MoorVMS-LDF 2-channel Laser Doppler Monitor</td>
 <td>Moor Instruments</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>VP12 probe</td>
 <td>Moor Instruments</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent/Material</td>
 </tr>
 <tr>
 <td>Doccol silicon-coated filament size 4-0, diameter with coating 0.39mm</td>
 <td>Doccol Corporation</td>
 <td>403956PK10</td>
 <td></td>
 </tr>
 <tr>
 <td>Natural rubber, e.g. common pacifiers for newborns</td>
 <td></td>
 <td></td>
 <td>Multiple suppliers</td>
 </tr>
 <tr>
 <td>Metal stylet, e.g. from spinal needle 18 GA x 90 mm </td>
 <td></td>
 <td></td>
 <td>Multiple suppliers</td>
 </tr>
 <tr>
 <td>Plastic tubes, e.g. from vein set for infusion 25 GA x 20 mm</td>
 <td></td>
 <td></td>
 <td>Multiple suppliers</td>
 </tr>
 <tr>
 <td>Nonabsorbable suture, coated, braided silk</td>
 <td></td>
 <td></td>
 <td>Multiple suppliers</td>
 </tr>
 <tr>
 <td>Cyanoacrylate surgical glue</td>
 <td></td>
 <td></td>
 <td>Multiple suppliers</td>
 </tr>
 <tr>
 <td>Isoflurane (100% v/v) for veterinary use</td>
 <td></td>
 <td></td>
 <td>Multiple suppliers</td>
 </tr>
 </tbody>
 </table>

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.

Transient MCAO (60 min) was induced by inserting a silicone-coated filament in the external carotid artery. The filament was then pushed through the terminal end of the internal carotid artery up to the origin of the MCA, under LD monitoring. Common carotid artery and pterygopalatin artery were transiently occluded during the surgical insertion of the filament. A schematic representation of the surgical procedure is shown in Figure 1A.
The cranial coordinates for positioning the two LD probes were chosen according to the underlying arterial territory. Preliminary experiments with gelatin-ink perfusion (Figure 1B) showed that the ischemic core is expected in the central MCA territory (bregma -1 mm, 5 mm from midline; Probe 1), while the collateral flow is expected in the borderzone territory between the cortical branches of middle and anterior cerebral arteries (bregma +2 mm, 2 mm from midline; Probe 2).
Cerebral hemodynamics was studied using multi-site Laser Doppler probes during the entire period of the surgical procedure, i.e. before, during and after MCAO (Figure 2). The cerebral perfusion deficit during MCAO was smaller and showed a higher degree of variability in Probe 2 compared to Probe 1, suggesting inter-individual differences in the functional performance of intracranial collaterals under ischemic conditions. The multi-site Laser Doppler monitoring also allows to study the cerebral hemodynamic changes during the occlusion of proximal extra-cranial cerebral arteries (common carotid artery, internal carotid artery, pterygopalatin artery).
Stroke outcome was assessed 24 hr after reperfusion by infarct volume, calculated on 19 consecutive sections stained with Cresyl violet (Figure 3), and Garcia functional neuroscore11. Immunohistochemistry for specific markers associated with ischemic brain injury was performed, in order to obtain a topographical distribution of neuronal loss (microtubule associated protein 2, MAP2) and ischemic penumbra (heat shock protein-70, Hsp70) in relation to the multi-site hemodynamic monitoring of intracranial circulation (Figure 4). 
<img src="/files/ftp_upload/50214/50214fig1.jpg" alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/50214/50214fig1highres.jpg"/> 
Figure 1. Cerebral perfusion monitoring during intraluminal MCAO in the rat. A. Schematic representation of the surgical procedure for transient MCAO. A silicone-coated filament was used to occlude the origin of the MCA, after being introduced in the external carotid artery and pushed through the internal carotid artery. Proximal cervical arteries were either ligated (external carotid artery) or transiently occluded (pterygopalatin artery and common carotid artery) during the procedure. B. A representative brain is shown after gelatin-ink staining. Transcardiac perfusion of gelatin-ink solution were performed 60 min after the onset of ischemia, without reperfusion. The normally perfused brain was stained by gelatin-ink and appeared as grey-colored with black stained vessels, while the ischemic (not-perfused) area remained unstained (pink-colored). Cranial coordinates for positioning the two LD probes are shown. Probe 1 = -1 mm from bregma, 5 mm from midline; Probe 2 = +2 mm from bregma, 2 mm from midline. 
<img src="/files/ftp_upload/50214/50214fig2.jpg" alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/50214/50214fig2highres.jpg"/> 
Figure 2. Cerebral hemodynamic recordings using multi-site Laser Doppler probes. A typical hemodynamic pattern which suggests functionally active intracranial collaterals under ischemic conditions is shown. In this animal, LD tracings showed a smaller perfusion deficit in Probe 2 channel, compared to Probe 1 channel, during both CCA occlusion and MCA occlusion. MCA-O=middle cerebral artery occlusion. CCA-O=common carotid artery occlusion. P.U.= perfusion units. 
<img src="/files/ftp_upload/50214/50214fig3.jpg" alt="Figure 3" fo:content-width="6in" fo:src="/files/ftp_upload/50214/50214fig3highres.jpg"/> 
Figure 3. Representative brain sections for the calculation of infarct volume. Histological coronal sections (50 &mu;m; n = 19 with 250 &mu;m interval; bregma +2.5 mm to &#x2212;3.0 mm) are fixed in 4% paraformaldehyde and stained with Cresyl violet 0.1%. Infarct volume is calculated using ImageJ image processing software, corrected for inter-hemispheric asymmetries due to cerebral edema, and expressed in mm3. <a href="https://www.jove.com/files/ftp_upload/50214/50214fig3large.jpg" target="_blank"> Click here to view larger figure</a>
<img src="/files/ftp_upload/50214/50214fig4.jpg" alt="Figure 4" fo:content-width="6in" fo:src="/files/ftp_upload/50214/50214fig4highres.jpg"/> 
Figure 4. Immunostaining of molecular markers of neuronal loss and penumbra. Representative consecutive brain sections are shown, which were stained with Cresyl violet 0.1% (A) or immunostained with markers of neuronal loss (microtubule associated protein 2, MAP2; B) and ischemic penumbra (heat shock protein-70, Hsp70; C).

Watch this Scientific Journal Video about Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion at JoVE.com

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

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 ...

optimized-system-for-cerebral-perfusion-monitoring-rat-stroke-model

Research

JoVE Journal

Medicine

23.0K Views.  University of Milano Bicocca. 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.

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

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

Mouse Model of Middle Cerebral Artery Occlusion

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the brain (ischemic stroke) 2. Since most ischemic strokes (~80%) occur in the territory of middle cerebral artery (MCA) 3, many animal stroke models that have been developed have focused on this artery. The intraluminal monofilament model of middle cerebral artery occlusion (MCAO) involves the insertion of a surgical filament into the external carotid artery and threading it forward into the internal carotid artery (ICA) until the tip occludes the origin of the MCA, resulting in a cessation of blood flow and subsequent brain infarction in the MCA territory 4. The technique can be used to model permanent or transient occlusion 5. If the suture is removed after a certain interval (30 min, 1 h, or 2 h), reperfusion is achieved (transient MCAO); if the filament is left in place (24 h) the procedure is suitable as a model of permanent MCAO. This technique does not require craniectomy, a neurosurgical procedure to remove a portion of skull, which may affect intracranial pressure and temperature 6. It has become the most frequently used method to mimic permanent and transient focal cerebral ischemia in rats and mice 7,8. To evaluate the extent of cerebral infarction, we stain brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) to identify ischemic brain tissue 9. In this video, we demonstrate the MCAO method and the determination of infarct size by TTC staining.

We demonstrate in the video a method for producing a middle cerebral artery occlusion in adult mice using an intraluminal monofilament. We also show how to evaluate the extent of cerebral infarction by 2,3,5-triphenyltetrazolium chloride (TTC) staining.

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the ...

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 ...

The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to develop suitable animal models to study the events that unfold during ischemic insult. These techniques include transient or permanent, focal or global ischemia models using many different animal models, with the most common being rodents.
The permanent MCA ligation method which is also referred as pMCAo in the literature is used extensively as a focal ischemia model in rodents 1-6. This method was originally described for rats by Tamura et al. in 1981 7. In this protocol a craniotomy was used to access the MCA and the proximal regions were occluded by electrocoagulation. The infarcts involve mostly cortical and sometimes striatal regions depending on the location of the occlusion. This technique is now well established and used in many laboratories 8-13. Early use of this technique led to the definition and description of &#x201C;infarct core&#x201D; and &#x201C;penumbra&#x201D; 14-16, and it is often used to evaluate potential neuroprotective compounds 10, 12, 13, 17. Although the initial studies were performed in rats, permanent MCA ligation has been used successfully in mice with slight modifications 18-20 .
This model yields reproducible infarcts and increased post-survival rates. Approximately 80% of the ischemic strokes in humans happen in the MCA area 21 and thus this is a very relevant model for stroke studies. Currently, there is a paucity of effective treatments available to stroke patients, and thus there is a need for good models to test potential pharmacological compounds and evaluate physiological outcomes. This method can also be used for studying intracellular hypoxia response mechanisms in vivo. 
Here, we present the MCA ligation surgery in a C57/BL6 mouse. We describe the pre-surgical preparation, MCA ligation surgery and 2,3,5 Triphenyltetrazolium chloride (TTC) staining for quantification of infarct volumes.

Middle cerebral artery (MCA) ligation is a technique to study focal cerebral ischemia in animal models. In this method, the middle cerebral artery is exposed by craniotomy and ligated by cauterization. This method gives highly reproducible infarct volumes and increased post-operative survival rates compared to other methods available.

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to ...

Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We have established bilateral common carotid artery occlusion as a preconditioning stimulus to induce early ischemic tolerance to transient focal cerebral ischemia in C57Bl6/J mice. In this video, we will demonstrate the methodology used for this study.

There is accumulating evidence, that ischemic preconditioning (PC) &#x2013; a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We established bilateral common carotid artery occlusion (BCCAO) as a preconditioning stimulus to induce early ischemic tolerance (IT) to transient focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) in C57Bl6/J mice.

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a ...

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 ...

Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be performed in many locations overlying the rat cerebrum, but is most easily carried out by creating a craniectomy over the readily accessible frontal or parietal bones. Most frequently, we have used this technique in combination with the endothelin-1 middle cerebral artery occlusion model of ischemic stroke to quantify the changes in middle cerebral artery vessel diameter that occur with injection of endothelin-1 into the brain parenchyma adjacent to the proximal MCA4, 5. In order to visualize the proximal portion of the MCA during endothelin -1 induced MCAO, we use a technique to create a cranial window through the temporal bone on the lateral aspect of the rat skull (Figure 1). Cerebral arteries can be visualized either with the dura intact or with the dura incised and retracted. Most commonly, we leave the dura intact during visualization since endothelin-1 induced MCAO involves delivery of the vasoconstricting peptide into the brain parenchyma. This bypasses the need to incise the dura directly over the visualized vessels for drug delivery. This protocol will describe how to create a cranial window to visualize cerebral arteries in a step-wise fashion, as well as how to avoid many of the potential pitfalls pertaining to this method.

This article describes a method for visualizing rat cerebral arteries through a cranial window using temporal craniectomy in order to view proximal portions of the middle cerebral artery (Figure 1). This versatile method can be combined with various techniques of drug delivery to measure cerebral artery reactivity in vivo.

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic stroke. However, the use of tPA is limited by its narrow therapeutic window and by increased risk of hemorrhagic transformation. There is an urgent need to develop suitable stroke models to study new thrombolytic agents and strategies for treatment of ischemic stroke. At present, two major types of ischemic stroke models have been developed in rats and mice: intraluminal suture MCAO and embolic MCAO. Although MCAO models via the intraluminal suture technique have been widely used in mechanism-driven stroke research, these suture models do not mimic the clinical situation and are not suitable for thrombolytic studies. Among these models, the embolic MCAO model closely mimics human ischemic stroke and is suitable for preclinical investigation of thrombolytic therapy. This embolic model was first developed in rats by Overgaard et al.1 in 1992 and further characterized by Zhang et al. in 19972. Although embolic MCAO has gained increasing attention, there are technical problems faced by many laboratories. To meet increasing needs for thrombolytic research, we present a highly reproducible model of embolic MCAO in the rat, which can develop a predictable infarct volume within the MCA territory. In brief, a modified PE-50 tube is gently advanced from the external carotid artery (ECA) into the lumen of the internal carotid artery (ICA) until the tip of the catheter reaches the origin of the MCA. Through the catheter, a single homologous blood clot is placed at the origin of the MCA. To identify the success of MCA occlusion, regional cerebral blood flow was monitored, neurological deficits and infarct volumes were measured. The techniques presented in this paper should help investigators to overcome technical problems for establishing this model for stroke research.

Embolic Middle Cerebral Artery Occlusion MCAO for Ischemic Stroke with Homologous Blood Clots in Rats

In this paper, we demonstrate a standard method for producing an embolic middle cerebral artery occlusion with homologous blood clots (fibrin-rich) in adult rat. This model closely mimics human ischemic stroke and is suitable for preclinical study of thrombolytic therapy for ischemic stroke.

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic ...

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 ...