This work was supported by grants from the Spanish Ministry of Economy and Competitiveness CSD2010-00045 (MAM), SAF2012-33216 (MAM), SAF2011-23354 (IL) and RENEVAS RD06/0026/0005 (IL), and from the Local Government of Madrid S2010/BMD-2336 (MAM) and S2010/BMD-2349 (IL). IB and MIC are fellows of the Spanish Ministry of Economy and Competitiveness.

NOTE: All experimental protocols adhered to the guidelines of the Animal Welfare Committee of the Universidad Complutense (following EU directives 86/609/CEE and 2003/65/CE).
1. Cerebral Ischemia Model
NOTE: The cerebral ischemia model in this paper involves permanent or transient occlusion of both CCA (common carotid artery) and MCA (middle cerebral artery) by ligation with a nylon suture.
<ol>
	<li>Pre-surgical preparations
	<ol>
		<li>Sterilize all surgical instruments by autoclaving or with a glass bead sterilizer. Disinfect the surgical area with 70% ethanol as well.</li>
		<li>Anesthetize mouse with appropriate anesthetics. Achieve induction by isoflurane 2.5% and maintain with by isoflurane 1.5% in a mixture of 80% air / 20% oxygen using a vaporizer. Apply artificial tears to the mice eyes to prevent dryness while under anesthesia.</li>
		<li>Place the mouse on a heating pad that is linked to a feedback device with a temperature probe placed in the rectum and set temperature at physiological levels (36.5 &#177; 0.5 &#186;C) during the surgical procedure.</li>
	</ol>
	</li>
	<li>Common carotid artery occlusion by ligation
	<ol>
		<li>Place the mouse in dorsal recumbency and immobilize with tape. Disinfect skin with 70% ethanol or povidone-iodide and cut the hair of the upper ventral part.</li>
		<li>Under a surgical microscope, make a 1 cm midline incision around the ventral surface of the neck.</li>
		<li>Pull apart all soft tissues (glandular tissue including sub-maxillary, sublingual and parotid glands) and identify the left Common Carotid Artery (CCA), which is localized lateral to the trachea. Retract the muscles and expose left CCA. Carefully dissect from vagus nerve.</li>
		<li>Once dissected, occlude the left CCA by a ligature using a 6/0 nylon suture. Ligate with a permanent knot or a slipknot (to allow reperfusion if a transient cerebral ischemia model is desired).</li>
		<li>Place the glandular tissue into its original position and close the incision on the neck skin with a 6/0 nylon suture. 
		NOTE: Sham animals are subjected to the same surgical procedure as MCAO animals but the CCA is not occluded.</li>
	</ol>
	</li>
	<li>Distal middle cerebral artery occlusion by ligation
	<ol>
		<li>Position the mouse on its right side and immobilize with tape. Disinfect the head skin with 70% ethanol or povidone-iodide and cut the hair of the mouse head (remove hair after cutting).</li>
		<li>Under a surgical microscope, make a skin incision between the eye and the ear. Move the skin away and hold it with tape.</li>
		<li>Make a horizontal incision on the temporal muscle from the right to the left. Then, make a second vertical incision on the right side of the temporal muscle (be careful to avoid cut temporal vein). Pull apart the temporal muscle from the skull and hold it with a suture to maintain a clean skull surface.</li>
		<li>Clean the skull surface with cold sterile saline to detect the exact position of the left Middle Cerebral Artery (MCA) via skull transparency. Perform a round craniotomy (1 mm in diameter) with a stainless steel burr in the frontal lobe between the zygomatic arch and the squamosal bone. Remove carefully the skull and expose the MCA.</li>
		<li>Apply a small amount of cold sterile saline solution to the surface of the brain and remove the meninges (dura and arachnoid mater) by using forceps.</li>
		<li>Perform ligation of the left MCA in the distal trunk just before the bifurcation between the frontal and posterior MCA branches. Occlude the left MCA by a ligature using a 9/0 nylon suture. Ligate with a permanent knot or a slipknot (to allow reperfusion if a transient cerebral ischemia model is desired). The period of time between CCA and MCA occlusion is approximately 10-20 minutes depending on the operator&#39;s experience.</li>
		<li>Place the temporal muscle into its original position and close the incision on the neck skin with a 6/0 nylon suture.</li>
		<li>Return the mouse to the cage to recover from the anesthesia (generally 5-10 min) and inject the mouse with buprenorphine intraperitoneal (I.P.) (0.3 mg/kg).
		<ol>
			<li>Monitor the mouse for several hr to detect any discomfort (do not leave the mouse unattended until it has regained sufficient consciousness to maintain sternal recumbency). Since post-surgical weight lost is generally observed, place mashed food in a Petri dish to facilitate eating.</li>
		</ol>
		</li>
		<li>When the animal is fully recovered, return to the company of other animals.</li>
		<li>If a transient MCAO model is desired, anesthetize the mouse again and remove the slipknot of the CCA and the MCA to allow reperfusion (usually between 0.5-2 hr (Figure 1)). 
		NOTE: Sham animals are subjected to the same surgical procedure as MCAO animals but the MCA is not occluded.</li>
	</ol>
	</li>
</ol>
2. Perfusion and Sectioning of Brain Tissue
<ol>
	<li>24 or 48 hr after MCAO, sacrifice the mouse with a lethal dose of anesthetic (e.g., sodium pentobarbital, I.P. 86 mg/kg or an overdose with isoflurane).</li>
	<li>Perfuse the mouse with phosphate buffer 0.1 M followed by 4% paraformaldehyde (PFA) in phosphate buffer (pH 7.4).</li>
	<li>Remove the brain as follows:
	<ol>
		<li>Decapitate the animal just above the spinal cord.</li>
		<li>Make a posterior-anterior incision on the head&#39;s skin to expose the skull. Make two lateral cuts at the junction of the lateral walls and the base of the skull and remove the small piece of bone.</li>
		<li>Make a cut through the skull along the sagittal suture.</li>
		<li>Remove the skull overlying each hemisphere by using a forceps to expose the brain. Use a spatula to separate the brain from the skull and transfer it into 4% PFA solution.</li>
	</ol>
	</li>
	<li>Post-fix for 3 hr in 4% PFA solution at 4 &#186;C.</li>
	<li>Remove PFA solution and incubate the brain for 48 hr in 30% sucrose solution to cryoprotect the brain.</li>
	<li>Remove sucrose solution and freeze the brain quickly in cold isopentane (-40 &#186;C). Store frozen brain at &#8211;80 &#186;C.</li>
	<li>Obtain coronal brain sections (30 &#181;m) with a freezing microtome. Collect ten serial sets in a cryopreservative solution. Each serial set is composed of around 14-16 coronal slices with an interslice distance of 300 &#956;m which adequately sample the mouse brain between 1.94 and -2.46 posterior to bregma.</li>
</ol>
3. Nissl Staining and Infarct Volume Estimation
<ol>
	<li>Nissl staining by cresyl violet
	<ol>
		<li>Mount brain sections into a superfrost microscope slide. For infarct volume determination by the Cavalieri method in Nissl-stained sections, use a 1/20 slice-sampling fraction (1/2 sections of one of the ten serial sets collected in step 2.7; approximately, a total number of 7-8 sections with an interslice distance of 600 &#956;m are used). Be careful to maintain proper anterior-posterior orientation (infarcted area is placed in the right side).</li>
		<li>Perform conventional Nissl staining as follows:
		<ol>
			<li>Allow slide-mounted sections to dry for several hr.</li>
			<li>Rehydrate slide-mounted sections and stain with 0.1% cresyl violet solution for 5 min.</li>
			<li>Dehydrate with a graded series of EtOH (70%, 90%, and 100%; 5 min per change). Clean the slide-mounted sections with xylene, add distrene-80 plasticizer xylene (DPX) mounting media and cover with a glass coverslip.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Using stereological software to estimate the infarct volume by the Cavalieri method in Nissl-stained serial sections
	<ol>
		<li>Use parameters shown in Table 1 to quantify infarct volume by the Cavalieri method14 and a stereology system, which consists of a microscope fitted with a camera, a XYZ motorized computer stage, and stereological software (The main directions provided below are related to PCs but the directions could differ for other operating systems).</li>
		<li>Turn on PC, microscope, stage controller and camera in the appropriate order. Start the stereological software.</li>
		<li>Move the 10X objective into place and select the 10X from the objective drop down menu.</li>
		<li>Move around the first Nissl-stained section and find a suitable reference point. Click the mouse to establish the reference point (to move around it is necessary to active the joy free button).</li>
		<li>Estimate the &#8220;mounted section thickness&#8221; (the actual section thickness taking into account the shrinkage). Press the &#8220;Focus Position Meter&#8221; button and calculate the thickness from the bottom to the top of the section.</li>
		<li>Create a contour tool for the contralesional and ipsilesional areas and the infarcted area.
		<ol>
			<li>Click on the menu &#8220;Display&#8221; and select &#8220;Display Settings&#8221;. This opens the Display Settings Dialog.</li>
			<li>Select the &#8220;Contours&#8221; tab and click the button &#8220;add Contour type&#8221;.</li>
			<li>Create a contour for each region to quantify and make them visible in the contour down menu. Click OK.</li>
		</ol>
		</li>
		<li>Create a Marker tool for the contralesional and ipsilesional hemispheres and the infarcted area.
		<ol>
			<li>Click on the menu &#8220;Display&#8221; and select &#8220;Display Settings&#8221; to open the Display Settings dialog.</li>
			<li>Select &#8220;Markers&#8221; tab and change the markers name by double clicking over the available markers. Make them visible in the Marker Bar. Click OK.</li>
		</ol>
		</li>
		<li>Activate the section manager.
		<ol>
			<li>Click on the menu &#8220;Tools/Serial Section Manager&#8221;. Add a new section with the &#8220;new section&#8221; button. This button opens the &#8220;Serial Section Setup dialog&#8221;.</li>
			<li>Set the &#8220;Block Advance&#8221; as 30 &#181;m. Set the &#8220;mounted section thickness&#8221; with the value estimated in the step 3.2.5.</li>
			<li>Set the &#8220;Evaluation Interval&#8221; as 20. Use a 1/20 slice sampling fraction.</li>
			<li>Set &#8220;the starting section number&#8221; as 1. Set the &#8220;Z axis value for the top of the first section&#8221; as 0. Click OK.</li>
		</ol>
		</li>
		<li>Draw a contour to outline the contralateral hemisphere.
		<ol>
			<li>Select the Contralesional hemisphere tool (previously created in the step 3.2.6) from the contour down menu.</li>
			<li>Draw a contour to outline the contralateral hemisphere.</li>
			<li>To finish tracing the contralesional hemisphere, right click the mouse and select &#8220;close contour&#8221;.</li>
		</ol>
		</li>
		<li>Estimate the contralesional hemisphere contour by Cavalieri.
		<ol>
			<li>Click on the menu &#8220;Probes&#8221; and select Cavalieri Estimator. Set &#8220;Grid Spacing&#8221; as 100 &#181;m. Set &#8220;Grid Rotation&#8221; as 0. Set the &#8220;Block Advance&#8221; as 30 &#181;m. Click OK.</li>
			<li>Select &#8220;The contralesional hemisphere&#8221; button from the Markers Bar. Right click inside of the Contralesional hemisphere contour.</li>
			<li>Select &#8220;Paint Cavalieri Markers Mode&#8221;. Right click inside the contralesional hemisphere contour again and select &#8220;Paint Markers into Contour&#8221;.</li>
			<li>Deactivate Cavalieri Estimator by clicking on probes and Cavalieri Estimator and deactivate the contralesional marker button from the marker bar.</li>
		</ol>
		</li>
		<li>Repeat the same procedure for the ipsilesional hemisphere and the infarcted area (steps 3.2.7 and 3.2.8). Save the data after the estimation of the area of each section.</li>
		<li>Calculate the contralateral, ipsilesional and infarcted areas in the rest of the Nissl-stained section.
		<ol>
			<li>Create a new section as in step 3.2.6. Do not modify the values entered in the &#8220;Serial section Setup&#8221; dialog. Click Ok.</li>
			<li>Repeat steps 3.2.7-3.2.8 in the new section.</li>
		</ol>
		</li>
		<li>Export the results of the quantification to a spreadsheet file.
		<ol>
			<li>Click &#8220;Probes&#8221; in the Stereo Investigator Menu. Select &#8220;Display Probe Run List&#8221;. This opens the &#8220;Previous Stereological Runs Dialog&#8221;.</li>
			<li>Select all the sections by clicking over them. Press the &#8220;View Results&#8221; Button. This opens &#8220;the Cavalieri Estimator Results&#8221; where the estimated area and volume for each region are displayed.</li>
			<li>To export the results to a spreadsheet file, click the &#8220;Copy all Results to Clipboard&#8221; and paste to an excel file with the main results obtained from each region (Table 2).</li>
		</ol>
		</li>
		<li>Express infarct volume as mm3 or as % of the hemisphere that is infarcted (%IH) using the formula15 %IH = InfVol/ContrVol*100, where</li>
	</ol>
	</li>
</ol>
InfVol (Infarcted Tissue Volume) = &#931;InfAreai, 
ContrVol (Contralesional Hemisphere Volume) = &#931;ContrAreai
4. Stereological Quantification of Infiltrated Neutrophils After Cerebral Ischemia by the Optical Fractionator
<ol>
	<li>Staining of the brain sections:
	<ol>
		<li>For estimating the total number of infiltrated neutrophils after MCAO by the optical fractionator16, use a 1/10 slice sampling fraction. Immunostain neutrophils by using conventional immunofluorescence techniques with the rat anti-Ly6G antibody (1A8) and the right secondary antibody on free-floating sections. In addition, a counterstaining with a nuclear maker like DAPI or TOPRO, although it is not necessary for neutrophil identification, can make easier to detect the infarcted area.</li>
		<li>Mount brain sections into a superfrost microscope slide with the infarcted area placed in the right side.</li>
	</ol>
	</li>
	<li>Quantification of infiltrated leukocytes in the ischemic brain by the optical fractionator
	<ol>
		<li>Use parameters shown in Table 3 to quantify infiltrated leukocytes in the ischemic brain by the optical fractionator approach with a stereology system. Repeat the steps 3.2.1-3.2.5 from section 3.</li>
		<li>Create a contour tool for the infarcted area as it has been described in the step 3.2.6 of the section 3.</li>
		<li>Create a marker tool for neutrophils as it has been described in the step 3.2.7 of the section 3.</li>
		<li>Activate the section manager and create a new section as in the step 3.2.8. In this case, set the &#8220;Evaluation Interval&#8221; as 10 (a 1/10 slice sampling fraction is used for estimating neutrophil number).</li>
		<li>Select the &#8220;Infarcted Area&#8221; contour tool (previously created in the step 4.2.1) from the contour down menu. Draw a contour to outline the infarcted area at 10X objective. Change the objective to 100X to perform neutrophil quantification (do not forget to select the 100X from the objective drop down menu).</li>
		<li>Click on the menu &#8220;Probes&#8221; and select the &#8220;Optical Fractionator&#8221;. Set the &#8220;XY Placement of counting Frames&#8221; as 230 for both X and Y grid size. Set &#8220;Grid Rotation&#8221; as 0. Set the &#8220;Block Advance&#8221; as 30 &#181;m. Click OK.</li>
		<li>Select the &#8220;neutrophil&#8221; button from the Marker Bar. Mark stained neutrophils in the infarcted area. Once finish, deactivate the Optical Fractionator probe by clicking on &#8220;Probes&#8221; and &#8220;Optical Fractionator&#8221;. Deactivate the neutrophil button from the marker bar.</li>
		<li>Repeat the same procedure for each section (4.2.4-4.2.9).</li>
		<li>Export the results of the quantification to a spreadsheet file.
		<ol>
			<li>Click &#8220;Probes&#8221; in the Stereo Investigator Menu. Select &#8220;Display Probe Run List&#8221; to open the &#8220;Previous Stereological Runs&#8220; dialog.</li>
			<li>Select all the sections by clicking over them. Press the &#8220;View Results&#8221; Button to open &#8220;the Optical Fractionator Results&#8221; where the estimated number of neutrophils is displayed.</li>
			<li>Export the results to a spreadsheet file by clicking the &#8220;Copy all Results to Clipboard&#8221; and paste to a spreadsheet file.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
5. Brain Dissociation and Flow Cytometry Analysis
NOTE: Myeloid subpopulation characterization by flow cytometry on fresh brain tissue can be used as an alternative to the previous neutrophil characterization performed on fixed and immunostained brain sections.
<ol>
	<li>Brain dissociation and single cell suspension preparation
	<ol>
		<li>Make 10 ml/animal of a RPMI (Roswell Park Memorial Institute medium)-Percoll solution containing 8 ml of RPMI, 1 ml of 30% Percoll and 1 ml of a 10x PBS (phosphate buffered saline).</li>
		<li>Remove mouse brain after MCAO as described in the step 2.3.</li>
		<li>Under a microscope, dissect out core and peri-infarct areas of the ipsilateral cortex from brain ischemic mouse (or a similar region for sham and na&#239;ve animals) from the brain. Weight brain tissue and place it into 5 ml of ice-cold RPMI-Percoll (the weight step can be performed for normalization between experimental groups).</li>
		<li>Dissociate the tissue in a single cell suspension using a Potter-Elvehjem-type tissue grinder with Teflon pestles.</li>
		<li>Transfer cell suspensions to a 50 ml ultracentrifuge tube and add 5 more ml of RPMI-Percoll solution to the samples.</li>
		<li>Centrifuge the cell suspensions at 7,800 x g for 30 min at 25 &#186;C. After centrifugation, ensure a white-colored layer corresponding to myelin appears at the top of the solution.</li>
		<li>Remove the myelin layer carefully and filter the whole cell suspensions, including the pelleted cells, through 40-&#181;m nylon mesh strainer.</li>
		<li>Add 10 ml of RPMI on the strainer to ensure that all the cells are filtered and place the solution in a 50 ml tube. Add 30 ml of RPMI and centrifuge the 50 ml of cell suspension at 600 x g for 10 min at RT.</li>
		<li>Discard the supernatant and resuspend the leukocyte pellet with 1 ml of PBS. Add 4 ml of lysis buffer (150 mM NH4Cl, 10 mM NaHCO3 and 0.1 mM EDTA) and incubate the cells for 2-3 min at RT to lyse erythrocytes from the cell suspension. Centrifuge the solution at 600 x g for 10 min at 4 &#186;C.</li>
	</ol>
	</li>
	<li>Cell staining and flow cytometry
	<ol>
		<li>Prepare a labeling cocktail for flow cytometry, containing 200 &#956;l of PBS-BSA (1%) 1:100 Fc-Block, 1:40 anti-CD45-PercP rat, 1:40 anti-Ly6G-APC rat, 1:40 anti CD11b-FITC rat and 1:40 anti-Ly-6C-PE rat per sample.</li>
		<li>Resuspend the cells in the labeling cocktail and incubate the samples for 45 min in ice.</li>
		<li>Wash the labeling cocktail with 1 ml of cold PBS and centrifuge the cells at 600 x g for 10 min at 4 &#186;C. Resuspend the pellet with 100 &#181;l of FACS Flow and acquire the entire cell suspension by flow cytometry.</li>
		<li>Use a flow cytometry analysis software to characterize and quantify cell populations.
		<ol>
			<li>Main leukocyte subsets corresponding to the labeling cocktail prepared in this protocol can be characterized as follows: CD45loCD11b+: resident microglial cells; CD45hiCD11b+Ly-6G+: neutrophils; CD45hiCD11b+Ly-6G-Ly-6Chi: pro-inflammatory monocytes.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+two+pathophysiologies+of+focal+brain+ischemia:+implications+for+translational+stroke+research.">The two pathophysiologies of focal brain ischemia: implications for translational stroke research.</a> J Cereb Blood Flow Metab. 32, 1310-1316 (2012).</li><li>Garc&#237;a-Y&#233;benes, I., 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=Iron+overload,+measured+as+serum+ferritin,+increases+brain+damage+induced+by+focal+ischemia+and+early+reperfusion.">Iron overload, measured as serum ferritin, increases brain damage induced by focal ischemia and early reperfusion.</a> Neurochem Int. 61, 1364-1369 (2012).</li><li>Sobrado, M., 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=Synthesis+of+lipoxin+A4+by+5-lipoxygenase+mediates+PPARgamma-dependent,+neuroprotective+effects+of+rosiglitazone+in+experimental+stroke.">Synthesis of lipoxin A4 by 5-lipoxygenase mediates PPARgamma-dependent, neuroprotective effects of rosiglitazone in experimental stroke.</a> J Neurosci. 29, 3875-3884 (2009).</li><li>Cuartero, M. I., 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=N2+Neutrophils,+Novel+Players+in+Brain+Inflammation+After+Stroke:+Modulation+by+the+PPAR&#947;+Agonist+Rosiglitazone.">N2 Neutrophils, Novel Players in Brain Inflammation After Stroke: Modulation by the PPAR&#947; Agonist Rosiglitazone.</a> Stroke. 44, 3498-3508 (2013).</li><li>Ford, A. L., Foulcher, E., Goodsall, A. L., Sedgwick, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+digestion+with+dispase+substantially+reduces+lymphocyte+and+macrophage+cell-surface+antigen+expression.">Tissue digestion with dispase substantially reduces lymphocyte and macrophage cell-surface antigen expression.</a> Journal of Immunological Methods. 194, 71-75 (1996).</li><li>Pfenninger, C. V., 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=CD133+is+not+present+on+neurogenic+astrocytes+in+the+adult+subventricular+zone,+but+on+embryonic+neural+stem+cells,+ependymal+cells,+and+glioblastoma+cells.">CD133 is not present on neurogenic astrocytes in the adult subventricular zone, but on embryonic neural stem cells, ependymal cells, and glioblastoma cells.</a> Cancer Research. 67, 5727-5736 (2007).</li><li>Tham, C. S., Lin, F. F., Rao, T. S., Yu, N., Webb, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+activation+state+and+lysophospholipid+acid+receptor+expression.">Microglial activation state and lysophospholipid acid receptor expression.</a> International Journal of Developmental Neuroscience : the Official Journal of the International Society for Developmental Neuroscience. 21, 431-443 (2003).</li><li>Nikodemova, M., Watters, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+isolation+of+live+microglia+with+preserved+phenotypes+from+adult+mouse+brain.">Efficient isolation of live microglia with preserved phenotypes from adult mouse brain.</a> Journal of Neuroinflammation. 9, 147 (2012).</li></ol>

The authors declare that they have no competing financial interests.

The cerebral ischemia model introduced here gives highly reproducible infarct volumes determined 24-48 hr and 7 days after MCA ligation by different approaches8,15,17. This MCAO model has a low mortality rate (less than 1%) compared to others, minimizing the number of animals used in studies. A critical step to obtain this low rate of mortality is to maintain proper aseptic conditions to avoid infections which could impair survival after stroke induction. This MCAO model can not only be used as a permanent MCAO model, which is considered a clinical relevant model for translational research18, but also as a transitory model by transient ligation of the CCA and MCA with an slipknot and posterior reperfusion at the desired time19. This method has been successfully used in mice and rats17,20. All this evidence indicates that MCAO by ligation is a high versatile model of cerebral ischemia with multiple applications. A critical step of this technique is that it requires invasive surgery under a stereomicroscope; the craniotomy should be performed very carefully as to not to damage the zygomatic bone as well as the MCA. However, the use of sham animals (which are subjected to the surgical procedure but CCA and MCA ligation is not performed) provides a useful tool to discriminate surgical procedure-dependent effects. The extent of brain injury following this technique can be quantified by several methods. Our protocol of brain sectioning, Nissl staining and subsequent estimation of the volume by Cavalieri allows for an accurate quantification of the damaged region and minimizes the number of mice used in this type of studies since serial brain sections can also be used for different immunohistochemical analysis. For a better performance of this methodology, it is critical to choose the appropriate parameters used in the stereology software (Table 1) which will be necessary for estimating the volume of the different regions by the formula: V = 1/ssf*af*t*&#931;Pi (Ssf is the slice sampling fraction, t is the mean thickness of the sections, af is the area of the grid spacing and &#8721;P the number of points hitting the structure).
The distal MCAO by ligation can be useful to characterize the infiltrated leukocyte and immune cell subpopulations8,13 that participate in the inflammatory process following brain injury1-4. Here, we propose two different methodologies for brain immune cell characterization, a precise stereological approach and a flow cytometric analysis for better characterizing multiple leukocytes subpopulations.
Taking advantage of serial brain sectioning, the quantification of the total number of neutrophils can be achieved by the optical fractionator method16, which estimates the total number of cells from the number of cells sampled with a Systematic Randomly Sampled (SRS) set of unbiased virtual counting spaces covering the entire region of interest, in our case the infarct region, with uniform distance between unbiased virtual counting spaces in directions X, Y and Z. This method provides an accurate tool to estimate total neutrophil numbers in the ischemic brain at different times after ligation. Although it has not been shown in this study, this protocol can also be used for estimation of the different neutrophil subpopulations after ischemia21 and for an accurate quantification of any other cell population found in the ischemic brain like other infiltrated leukocytes (monocytes/macrophages) and also for estimating surviving neurons or even for neurogenesis quantification after stroke. The most important step for an accurate estimation in the desired area is the selection of the appropriate parameters, as the ones shown in the Table 3 for neutrophil quantification in the ischemic area. These parameters will be used for the stereology software to calculate the total positive cell number (N) by using the equation N = &#931;Q- x 1/ssf x 1/asf x 1/tsf (&#931;Q- is the total number of cells counted with the fractionator, ssf is the section sampling fraction, asf is the sampling fraction area, and tsf is the sampling fraction thickness)5. Although this methodology is slower than other quantification techniques (for example, analysis of neutrophil markers by mg of tissue, densitometry of representative images or number of neutrophils per field), it has the advantage to be an unbiased and a solid technique which provides a precise quantification of cell numbers.
The brain leukocyte isolation approach allows for a simultaneous identification and quantification of several immune cell subtypes without the need to bias the system by in vivo staining or genetic manipulations. Subsequent cell sorting from the characterized myeloid populations or their immunomagnetic separation can be used for multiple downstream applications, such as further studies on gene or protein expression. The accurate characterization of neutrophils, monocytes and microglia obtained with this method provides high specificity with respect to existing methods such as the immunohistochemical studies, an advantage that allows allocating specific functions to the different myeloid cells that mediate brain innate immune response. In addition, it can be further extended to characterize other brain populations with the appropriate label, like blood born macrophages (CD11b+CD45hiCD68+), and it can be applied for the study of other CNS pathologies or injuries. Therefore this technique provides an essential tool to explore the heterogeneity of the inflammatory response in the brain. A main limitation of this technique resides on the preparation of the leukocyte suspensions from fresh brain tissue, which can alter the activation state of the cells or their antigen alteration. Although this technique allows a more detailed qualitative characterization compared to immunohistochemical studies, it provides a less accurate quantification based on cell isolation. Despite this, the efficiency of our cell isolation protocol is similar to other published methodologies9 and it can be efficiently used to detect differences in the number of brain immune cells between control and MCAO groups or even between MCAO groups subjected to different treatments8.
Critical steps of this protocol are the tissue dissection, the tissue disruption procedure, and the myelin removal. Regarding the tissue collection, a normalization step can be included (by weighing the tissue collected) to avoid variability due to different dissection performances. In addition, normalization between different MCAO groups can also occur through infarct volume (previously determined by Magnetic Resonance). Another way to solve this problem is to use the whole ipsilateral hemisphere of both ischemic and control groups, or even use the contralateral hemisphere of the ischemic mouse as a control to minimize the number of animals used. While this approach avoids differences between each dissection, it has a main disadvantage; a dilution factor is added by increasing the total number of cells but not the number of myeloid cells which are exclusively located in the core and peri-infarct areas of the ipsilateral cortex. Regarding tissue disruption, this protocol illustrates the steps for the mechanical disruption of brain cells, avoiding enzymatic treatments and preventing surface antigen alteration9,22, an essential issue for further qualitative and quantitative analysis of the inflammatory cell subpopulations. In addition to the preparation of the cell suspension of interest, myelin removal from brain samples is a highly recommended step to avoid interference with downstream applications, such as immunomagnetic cell separation or flow cytometry23,24. This can be accomplished using different methods, such sucrose or Percoll gradients, or anti-myelin beads. Here, and based on previous studies that compare different methods for brain cell suspension isolation, mechanical disruption in combination with Percoll usage to remove myelin is used to improve cell yields and viability25.

Morphological, phenotypic and gene expression alterations of microglia, as well as extravasation and activation of haematogenous macrophages and neutrophils are believed to play a pivotal role in the pathophysiological cascade following brain injury1-4. This protocol provides two approaches to estimate the number of different myeloid cell subsets of the ischemic brain and to perform their phenotypical characterization. In addition, it also provides a description of an experimental model of cerebral ischemia in mice, which consists of the transient or permanent ligation of both distal Middle Cerebral Artery (MCA) and the common carotid artery (CCA), including how to evaluate the infarct by the accurate Cavalieri method using a stereological software.
The first approach to characterize myeloid cell subsets of the ischemic brain is a stereological method based on the optical fractionator approach5. This is the most commonly used stereological probe in life sciences and provides a high degree of precision with low coefficients of error5-7. This is the best choice for cell quantification when the tissue is cut into sections, as it avoids the bias on cell estimation because of the fragmentation of the tissue into sections. This method is a very powerful way to characterize numbers, dynamics and phenotypic changes of infiltrated neutrophil subpopulations of the ischemic tissue8.
The second characterization approach is based on a modified protocol from Campanella and collaborators9 that provides a simple way to isolate brain leukocyte suspensions and to characterize them by flow cytometry. In contrast to conventional immunohistochemical techniques, flow cytometry characterization allows differentiating between microglia (CD45loCD11b+) and infiltrated myeloid cells (CD45hiCD11b+) based on their different expression levels of CD45 9-13. In addition, pro-inflammatory monocytes (CD45hiCD11b+Ly-6G-Ly-6Chi), neutrophils (CD45hiCD11b+Ly-6G+) and other leukocytes subsets of the ischemic tissue can be distinguished. This approach provides a reliable and rapid assay to assess neuroinflammation in experimental models of brain ischemia. However, tissue processing can influence the activation state and numbers of the different cell populations found in the ischemic tissue providing a semi-quantitative characterization.

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			<td height="21" style="height:21px;width:373px;">Photonic Led F1</td>
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			<td height="21" style="height:21px;width:373px;">Temp Controller&#160;</td>
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			<td height="21" style="height:21px;width:373px;">Volvere GX</td>
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			<td height="21" style="height:21px;width:373px;">Microscope</td>
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			<td height="21" style="height:21px;width:373px;">Stainless Steel Burrs</td>
			<td style="width:215px;">FST</td>
			<td style="width:215px;">19007-14</td>
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			<td height="21" style="height:21px;width:373px;">Forceps Dumont #5/45&#160;</td>
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			<td height="21" style="height:21px;width:373px;">Forceps Dumont #5SF</td>
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			<td style="width:215px;">11252-30</td>
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			<td height="21" style="height:21px;width:373px;">Suture 6/0</td>
			<td style="width:215px;">LorcaMarin</td>
			<td style="width:215px;">55108</td>
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			<td height="21" style="height:21px;width:373px;">Suture 9/0</td>
			<td style="width:215px;">LorcaMarin</td>
			<td style="width:215px;">61966</td>
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			<td height="21" style="height:21px;width:373px;">Nikon Eclipse&#160;</td>
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			<td style="width:215px;">TE300</td>
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			<td height="21" style="height:21px;width:373px;">Isoflurane</td>
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			<td height="21" style="height:21px;width:373px;">Sodium Pentobarbital</td>
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			<td style="width:215px;">570681</td>
			<td>Chemical</td>
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			<td height="21" style="height:21px;width:373px;">Freezing microtome</td>
			<td style="width:215px;">Leica Microsystems GmbH</td>
			<td style="width:215px;">SM2000R</td>
			<td>Equipment</td>
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			<td height="21" style="height:21px;width:373px;">Superfrost slides</td>
			<td style="width:215px;">Thermo Scientific&#160;</td>
			<td style="width:215px;">2014-07</td>
			<td>Lab material</td>
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			<td height="21" style="height:21px;width:373px;">Cresyl violet acetate</td>
			<td style="width:215px;">Sigma</td>
			<td style="width:215px;">C5042</td>
			<td>Chemical</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Microscope&#160;</td>
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			<td style="width:215px;">Nikon Eclipse TE300</td>
			<td>Equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">XYZ motorized computer stage and controller</td>
			<td style="width:215px;">Ludl electronics Products</td>
			<td style="width:215px;"></td>
			<td>Equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">Stereo Investigator System</td>
			<td style="width:215px;">Microbrightfield</td>
			<td style="width:215px;">Version 7.003 software</td>
			<td>Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">5 ml Tissue Grinder, Potter-Elv with teflon pestle</td>
			<td style="width:215px;">Thomas Scientific</td>
			<td>0913X70</td>
			<td>Lab Material</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polypropylene 50mL Oak Ridge Centrifuge Tube</td>
			<td style="width:215px;">Nalgene</td>
			<td>3119-0050&#160;</td>
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			<td height="21" style="height:21px;width:373px;">Percoll</td>
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			<td style="width:215px;">p1644-100</td>
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			<td height="21" style="height:21px;width:373px;">RPMI 1640</td>
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			<td height="21" style="height:21px;width:373px;">Beckman Ultracentrifugue</td>
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			<td style="width:215px;"></td>
			<td>Equipment</td>
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			<td height="21" style="height:21px;width:373px;">Beckman Coulter ultracentrifuge rotor 45 Ti</td>
			<td style="width:215px;">Beckman Coulter</td>
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			<td>Equipment</td>
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			<td height="21" style="height:21px;width:373px;">BD Sterile Cell Strainer, 40 Micron</td>
			<td style="width:215px;">BD</td>
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			<td height="21" style="height:21px;width:373px;">Bovine Serum Albumin</td>
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			<td style="width:215px;">A3733-500G</td>
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			<td height="21" style="height:21px;width:373px;">FcR Blocking Reagent, mouse</td>
			<td style="width:215px;">Miltenyi</td>
			<td style="width:215px;">130-092-575</td>
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			<td>Antibody</td>
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			<td height="21" style="height:21px;width:373px;">PerCP/Cy5.5 anti-mouse Ly-6C</td>
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			<td style="width:215px;">128012</td>
			<td>Antibody</td>
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			<td height="21" style="height:21px;width:373px;">BD FACS Flow</td>
			<td style="width:215px;">BD</td>
			<td style="width:215px;">342003</td>
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			<td height="21" style="height:21px;width:373px;">BD FACSCalibur; 4-color</td>
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			<td style="width:215px;">342975</td>
			<td>Equipment</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:373px;">BD Cell Quest Pro Software</td>
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			<td style="width:215px;"></td>
			<td>Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:373px;">FlowJo software</td>
			<td style="width:215px;">Treestar inc.</td>
			<td style="width:215px;"></td>
			<td>Software</td>
		</tr>
	</tbody>
</table>

stereological and flow cytometry characterization of leukocyte subpopulations in models of transient or permanent cerebral ischemia

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after stroke. These myeloid cell subpopulations can display different phenotypes and functions and need to be distinguished and characterized to study their regulation and contribution to tissue damage. This protocol provides two different methodologies for brain immune cell characterization: a precise stereological approach and a flow cytometric analysis. The stereological approach is based on the optical fractionator method, which calculates the total number of cells in an area of interest (infarcted brain) estimated by a systematic random sampling. The second characterization approach provides a simple way to isolate brain leukocyte suspensions and to characterize them by flow cytometry, allowing for the characterization of microglia, infiltrated monocytes and neutrophils of the ischemic tissue. In addition, it also details a cerebral ischemia model in mice that exclusively affects brain cortex, generating highly reproducible infarcts with a low rate of mortality, and the procedure for histological brain processing to characterize infarct volume by the Cavalieri method.

The cerebral ischemia model shown here generates infarcts seen exclusively in the cortex without affecting the striatal tissue since the lenticulostriatal branches of the MCA that irrigate the striatum are not occluded (Figure 1). By Nissl staining, the damaged area can be identified as a hypochromic cortical area (Figure 2). This model is characterized by highly reproducible infarct volumes at 24 hr after MCAO (%IH 15.89 &#177; 0.28) estimated by Cavalieri method in Nissl-stained sections (Figure 2). Estimation of the infarcted volume by the Cavalieri method is an accurate approach with a low error that is reflected in the coefficient of error (CE) of Gundersen in the contralesional and ipsilesional hemispheres as well as in the infarcted area (Table 2). Damaged tissue volume can be expressed in mm3 but also as % IH using the formula in the section 3.2.13. In addition, the estimation of the total volume of the ipsilesional and contralesional regions allows for the calculation of edema index to correct the infarcted volume and to avoid an overestimation of the damaged tissue (Table 2).
Given the serial sectioning processing of the brain tissue, this can be taken advantage of to perform an accurate estimation of the total number of cell subpopulations, like infiltrated neutrophils, in the ischemic area, using the Optical Fractionator approach (Figure 3 and Table 4) with parameters shown in Table 3. This protocol estimates a total number of neutrophils (Ly6G-positive cells) in the infarcted area of 23,328 &#177; 3,623 at 24 hr and 82,856 &#177; 8,143 at 48 hr in mice after pMCAO (Figure 3D). In agreement with previous studies, neutrophil infiltration is directly correlated with the infarct size (Figure 3E). Estimation of the number of neutrophils by the Optical Fractionator method is an accurate approach with a low error that is reflected by the CE of Gundersen (Table 4).
The leukocyte isolation and flow cytometry characterization protocol allows the isolation of 47,922 &#177; 23,174 myeloid cells from the cortex of the ipsilesional hemisphere of ischemic mice. This comprises 10-30% of the total events found in the cell suspension. The vast majority of captured events using this protocol present a low FSC parameter, associated to cellular debris (Figure 4). CD11b staining shows that CD11b+ cells have a higher FSC value (Figure 4), suggesting that cell debris is not labeled with this marker and, as previously indicated, that it can be excluded from further analysis by setting the FSC threshold at 2009. The variable amount of cell debris obtained with this method suggests that differences on sample processing (timing, tissue conservation, sample temperature, efficient myelin removal, etc.) can account for it. In addition, the use of cell strainers is also needed to avoid the presence of cellular clamps in the samples; this step needs to be done prior to cell staining. Using the gating strategy shown in the Figure 5 which is based on CD11b and CD45 expression, we can discriminate between resident and infiltrated myeloid cells in the ischemic tissue. This CD11b+ population increases in the ischemic hemisphere when compared with the naive and with the sham group, in which these cells are mostly associated to a low expression of CD45 indicating that microglia is proliferating after ischemia (Figure 4, Figure 5 and Table 5). This difference is likely due to cell infiltration from the periphery, as evidenced by the appearance of a CD11b+CD45hi cell subpopulation in the ischemic brain (Figure 4, Figure 5 and Table 5) which is low number in na&#239;ve and in sham brains. The contribution of infiltrates to the CD11b+ cell population in brain ischemia is a very dynamic process4. In the MCAO model by ligature, it can vary from 30% to 60% of the total CD11b+ cells depending on the size of the lesion and of the time when the characterization has been done. Neutrophils, characterized as Ly-6G+ cells, are the most numerous infiltrated cell population found at 24 hr after MCAO in the ischemic mouse brain using this model of cerebral ischemia, as they comprise the 70-80% of the CD11b+CD45hi. The rest of the cells are mostly a subpopulation of CD11b+CD45hiLy-6G-Ly-6Chi pro-inflammatory monocytes. In this model, this population will increase in number in the ischemic brain areas from 24 to 48 hr after MCAO.
<img alt="Figure 1" src="/files/ftp_upload/52031/52031fig1.jpg" /> 
Figure 1: Surgical procedure for the MCA ligation. (A) After retraction of the temporal muscle, a small craniotomy is performed in the mouse skull. MCA is ligated by a knot or a slipknot using a 9/0 suture. (B) Representative images of the cortical lesion generated by the MCAO model. <a href="https://www.jove.com/files/ftp_upload/52031/52031fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52031/52031fig2.jpg" /> 
Figure 2: Quantification of infarct volume by Cavalieri. (A) Representative images of Nissl-stained brain sections 24 hr after permanent MCA ligation. The high magnification shows the hipocromatic area after Nissl staining which identifies the damaged area (core) after MCAO. The Peri-Infarct (P.I) region is also shown. (B) Quantification of the infarct volume represented as % Infarcted Hemisphere (%IH) by using the Cavalieri method in serial Nissl-stained sections, 24 hr after MCAO (n = 50 mice).
<img alt="Figure 3" src="/files/ftp_upload/52031/52031fig3.jpg" /> 
Figure 3: Stereological quantification of neutrophils in the infarcted area 24 and 48 hr after ligation, using the Optical Fractionator method. (A) Representative image of infiltrated neutrophils (Ly6G-positive cells) in the ischemic area at 24 hr after MCAO. Delimitation shows the infarcted area. (B, C) Examples of an optical dissector for neutrophil quantification by the optical fractionator. (D) Quantification of total neutrophils in the infarcted region at 24 and 48 hr (n = 4 mice). (E) Correlation of the number of neutrophils with the infarct size at 24 and 48 hr after MCA ligation. <a href="https://www.jove.com/files/ftp_upload/52031/52031fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52031/52031fig4.jpg" /> 
Figure 4: Representative dot-plot scatter analysis of brain leukocytes obtained from na&#239;ve, sham and ischemic (24 hr after MCAO) hemispheres on the basis of physical parameters (SSC and FSC). A population of events that express CD11b (red) was identified in all groups. This population was gated and characterized according to the expression of CD45. Cells expressing low levels of CD45 (green) were present in the ischemic and non-ischemic brain cortex and corresponded to microglial cells. In contrast, cells expressing high levels of CD45 (yellow) were only found in the ischemic hemisphere and in lesser extent in sham group. Further analysis of the CD11b+CD45hi population indicates that neutrophils (Ly-6G+ cells, blue) and pro-inflammatory monocytes (Ly-6Chi cells, orange) are the main cell subpopulations found in brain infiltrates 24 hr after stroke.<a href="https://www.jove.com/files/ftp_upload/52031/52031fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52031/52031fig5.jpg" /> 
Figure 5: Gating strategies to differentiate resident from infiltrated myeloid cells in the ischemic tissue. CD11b+ cell were first gated according to isotype fluorescence intensity (A). A representative dot plot of cell suspensions of the ischemic brain is shown in the up-right corner of the panel. In addition, typical values for the total number of events and for the number of CD11b+ events acquired using this technique is shown (no. of cells/ ischemic brain hemisphere; (A) CD45 Fluorescence intensity analysis of the CD11b+ cells is shown in panel B. A representative dot plot analysis of CD45 and CD11b expression of the gated CD11b+ subpopulation is shown in the up-right corner of the panel. In addition, the typical number of CD45lo CD45hi cells acquired per ischemic brain hemisphere using this technique is shown (B).
<table border="0" cellpadding="0" cellspacing="0">
	<colgroup>
		<col span="2" />
	</colgroup>
	<tbody>
		<tr>
			<td colspan="2">Parameters used for Cavalieri method</td>
		</tr>
		<tr>
			<td>Section Thickness (t)</td>
			<td>30 &#181;m</td>
		</tr>
		<tr>
			<td>Objective</td>
			<td>10X</td>
		</tr>
		<tr>
			<td>Slice sampling fraction (ssf)</td>
			<td>1/20</td>
		</tr>
		<tr>
			<td>Distance between sections</td>
			<td>600 &#181;m</td>
		</tr>
		<tr>
			<td>Grid Spacing&#160;</td>
			<td>100 &#181;m</td>
		</tr>
	</tbody>
</table>
Table 1: Parameters used for the stereological quantification of the infarcted tissue.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Results</td>
			<td>Contralesional</td>
			<td>Ipsilesional</td>
			<td>Infarct</td>
		</tr>
		<tr>
			<td>Area (&#181;m&#178;)</td>
			<td>151,460,000</td>
			<td>155,060,000</td>
			<td>22,600,000</td>
		</tr>
		<tr>
			<td>Volume (&#181;m&#179;)</td>
			<td>90,876,000,000</td>
			<td>93,036,000,000</td>
			<td>13,560,000,000</td>
		</tr>
		<tr>
			<td>Volume Corrected for&#160; 
			Over Projection (&#181;m&#179;)</td>
			<td>89,957,100,000</td>
			<td>92,046,300,000</td>
			<td>13,416,600,000</td>
		</tr>
		<tr>
			<td>Coefficient of Error (Gundersen), 
			m=0</td>
			<td>0.068</td>
			<td>0.077</td>
			<td>0.067</td>
		</tr>
		<tr>
			<td>Coefficient of Error (Gundersen), 
			m=1</td>
			<td>0.015</td>
			<td>0.017</td>
			<td>0.015</td>
		</tr>
		<tr>
			<td>Coefficient of Error (Gundersen), 
			alpha(q)</td>
			<td>0.068</td>
			<td>0.077</td>
			<td>0.067</td>
		</tr>
		<tr>
			<td colspan="2">% Infarcted Hemisphere</td>
			<td colspan="2">14.90</td>
		</tr>
		<tr>
			<td colspan="2">Brain Oedema (Ips Vol/Cont Vol)</td>
			<td colspan="2">1.02</td>
		</tr>
	</tbody>
</table>
Table 2: Representative examples of contralesional, ipsilesional and infarct volumes by the Cavalieri method using the Stereo Investigator Software.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td colspan="2">Parameters used for The Optical fractionator</td>
		</tr>
		<tr>
			<td>Section Thickness (tsf)</td>
			<td>30 &#181;m</td>
		</tr>
		<tr>
			<td>Objective</td>
			<td>100X</td>
		</tr>
		<tr>
			<td>Slice sampling fraction (ssf)</td>
			<td>1/10</td>
		</tr>
		<tr>
			<td>Counting Frame Height</td>
			<td>40 &#181;m</td>
		</tr>
		<tr>
			<td>Counting Frame Width</td>
			<td>40 &#181;m</td>
		</tr>
		<tr>
			<td>X grid size</td>
			<td>230 &#181;m</td>
		</tr>
		<tr>
			<td>X grid size</td>
			<td>230 &#181;m</td>
		</tr>
		<tr>
			<td>Safe Guard</td>
			<td>2 &#181;m</td>
		</tr>
		<tr>
			<td>Optical Disector Height</td>
			<td>14 &#181;m</td>
		</tr>
	</tbody>
</table>
Table 3: Parameters used for the stereological quantification of infiltrated neutrophils after brain ischemia with the optical fractionator probe by using the Stereo Investigator Software.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td colspan="2">Estimation of Neutrophils by the Optical Fractionator</td>
		</tr>
		<tr>
			<td>Number Of Sampling Sites</td>
			<td>430</td>
		</tr>
		<tr>
			<td>Shape Factor</td>
			<td>6.24</td>
		</tr>
		<tr>
			<td>Total markers Counted</td>
			<td>166</td>
		</tr>
		<tr>
			<td>Estimated Neutrophils by Optical Fractionator</td>
			<td>117,608.03</td>
		</tr>
		<tr>
			<td>Coefficient of Error (Gundersen), m=0</td>
			<td>0.22</td>
		</tr>
		<tr>
			<td>Coefficient of Error (Gundersen), m=1</td>
			<td>0.09</td>
		</tr>
	</tbody>
</table>
Table 4: Representative example of the estimated infiltrated neutrophils after brain ischemia with the Optical Fractionator probe by using the Stereo Investigator Software.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td>Cd11b+</td>
			<td>Neutrophils</td>
			<td>Monocytes</td>
			<td>Microglia</td>
		</tr>
		<tr>
			<td>Naive</td>
			<td>25,863 &#177; 4,575.8</td>
			<td>473 &#177; 75.8</td>
			<td>525 &#177; 191.4</td>
			<td>19,012 &#177; 1,523</td>
		</tr>
		<tr>
			<td>Sham 24 hr</td>
			<td>24,563 &#177; 5,263</td>
			<td>873 &#177; 192.5</td>
			<td>1,124 &#177; 391.5</td>
			<td>23,734 &#177; 2,910</td>
		</tr>
		<tr>
			<td>pMCAO 24 hr</td>
			<td>47,922 &#177; 23,174</td>
			<td>4,874 &#177; 748.7&#160;</td>
			<td>4,826 &#177; 1,345&#160;</td>
			<td>35,395 &#177; 10,833&#160;</td>
		</tr>
	</tbody>
</table>
Table 5: Representative results of the estimated myeloid cells after brain ischemia with the Flow cytometry approach.

Watch this Scientific Journal Video about Stereological and Flow Cytometry Characterization of Leukocyte Subpopulations in Models of Transient or Permanent Cerebral Ischemia at JoVE.com

Stereological and Flow Cytometry Characterization of Leukocyte Subpopulations in Models of Transient or Permanent Cerebral Ischemia

Brain myeloid cells characterization following stroke can be performed by stereology using the optical fractionator method, or by a flow cytometric analysis of brain leukocytes suspensions. Both are useful techniques to perform an accurate phenotypical distinction of the main myeloid cell subsets found in the ischemic brain.

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after ...

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15.5K Views.  Universidad Complutense de Madrid y Instituto de Investigación Hospital 12 de Octubre, Madrid. Brain myeloid cells characterization following stroke can be performed by stereology using the optical fractionator method, or by a flow cytometric analysis of brain leukocytes suspensions. Both are useful techniques to perform an accurate phenotypical distinction of the main myeloid cell subsets found in the ischemic brain.

Video: Stereological and Flow Cytometry Characterization of Leukocyte Subpopulations in Models of Transient or Permanent Cerebral Ischemia

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 ...

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury (IRI) of the Rat Cremaster Muscle

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, intestinal ischemia as well as following transplant and cardiovascular surgery.1 Reperfusion of previously ischemic tissue, while essential for the prevention of irreversible tissue injury, elicits excessive inflammation of the affected tissue. Adjacent to the production of reactive oxygen species, activation of the complement system and increased microvascular permeability, the activation of leukocytes is one of the principle actors in the pathological cascade of inflammatory tissue damage during reperfusion.2, 3 Leukocyte activation is a multistep process consisting of rolling, firm adhesion and transmigration and is mediated by a complex interaction between adhesion molecules in response to chemoattractants such as complement factors, chemokines, or platelet-activating factor.4
While leukocyte rolling in postcapillary venules is predominantly mediated by the interaction of selectins5 with their counter ligands, firm adhesion of leukocytes to the endothelium is selectin-controlled via binding to intercellular adhesion molecules (ICAM) and vascular cellular adhesion molecules (VCAM).6, 7 
Gold standard for the in vivo observation of leukocyte-endothelial interaction is the technique of intravital microscopy, first described in 1968.8
Though various models of IRI (ischemia-reperfusion injury) have been described for various organs, 9-12 only few are suitable for direct visualization of leukocyte recruitment in the microvascular bed on a high level of image quality.8 
We here promote the digital intravital epifluorescence microscopy of the postcapillary venule in the cremasteric microcirculation of the rat 13 as a convenient method to qualitatively and quantitatively analyze leukocyte recruitment for IRI-research in striated muscle tissue and provide a detailed manual for accomplishing the technique. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly appreciate, and safely perform the method.
In a step by step protocol we depict how to get started with respiration controlled anesthesia under sufficient monitoring to keep the animal firmly anesthetized for longer periods of time. We then describe the cremasteric preparation as a thin flat sheet for outstanding optical resolution and provide a protocol for leukocyte imaging in IRI that has been well established in our laboratories.

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury IRI of the Rat Cremaster Muscle

Digital intravital epifluorescence microscopy of postcapillary venules in the cremasteric microcirculation is a convenient method to gain insights into leukocyte-endothelial interaction in vivo in ischemia-reperfusion injury (IRI) of striated muscle tissue. We here provide a detailed protocol to safely perform the technique and discuss its applications and limitations.

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, ...

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow (ischemia) 1. Middle cerebral artery (MCA) delivers a great majority of blood to the lateral surface of the cortex 2, is the most common site of human stroke 3, and ischemia within its territory can result in extensive dysfunction or death 1,4,5. Survivors of ischemic stroke often suffer loss or disruption of motor capabilities, sensory deficits, and infarct. In an effort to capture these key characteristics of stroke, and thereby develop effective treatment, a great deal of emphasis is placed upon animal models of ischemia in MCA.
Here we present a method of permanently occluding a cortical surface blood vessel. We will present this method using an example of a relevant vessel occlusion that models the most common type, location, and outcome of human stroke, permanent middle cerebral artery occlusion (pMCAO). In this model, we surgically expose MCA in the adult rat and subsequently occlude via double ligature and transection of the vessel. This pMCAO blocks the proximal cortical branch of MCA, causing ischemia in all of MCA cortical territory, a large portion of the cortex. This method of occlusion can also be used to occlude more distal portions of cortical vessels in order to achieve more focal ischemia targeting a smaller region of cortex. The primary disadvantages of pMCAO are that the surgical procedure is somewhat invasive as a small craniotomy is required to access MCA, though this results in minimal tissue damage. The primary advantages of this model, however, are: the site of occlusion is well defined, the degree of blood flow reduction is consistent, functional and neurological impairment occurs rapidly, infarct size is consistent, and the high rate of survival allows for long-term chronic assessment.

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

We describe a highly reproducible method for the permanent occlusion of a rodent major cerebral blood vessel. This technique can be accomplished with very little peripheral damage, minimal blood loss, a high rate of long-term survival, and consistent infarct volume commensurate with the human clinical population.

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

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 ...

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 ...

Simultaneous PET/MRI Imaging During Mouse Cerebral Hypoxia-ischemia

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics disturbance in affected cells. Diffusion weighted magnetic resonance imaging (MRI) identifies regions that are damaged, potentially irreversibly, by hypoxia-ischemia. Alterations in glucose utilization in the affected tissue may be detectable by positron emission tomography (PET) imaging of 2-deoxy-2-(18F)fluoro-&#7429;-glucose ([18F]FDG) uptake. Due to the rapid and variable nature of injury in this animal model, acquisition of both modes of data must be performed simultaneously in order to meaningfully correlate PET and MRI data. In addition, inter-animal variability in the hypoxic-ischemic injury due to vascular differences limits the ability to analyze multi-modal data and observe changes to a group-wise approach if data is not acquired simultaneously in individual subjects. The method presented here allows one to acquire both diffusion-weighted MRI and [18F]FDG uptake data in the same animal before, during, and after the hypoxic challenge in order to interrogate immediate physiological changes.

The method presented here uses simultaneous positron emission tomography and magnetic resonance imaging. In the cerebral hypoxia-ischemia model, dynamic changes in diffusion and glucose metabolism occur during and after injury. The evolving and irreproducible damage in this model necessitates simultaneous acquisition if meaningful multi-modal imaging data are to be acquired.

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics ...

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA thrombolysis) rarely produces benefits in mechanical occlusion-based experimental models, which dominate preclinical stroke research. This split between the bench and bedside suggests the need to employ tPA-responsive models in preclinical stroke research. To this end, a simple and tPA-reactive thrombotic stroke model is invented and described here. This model consists of transient occlusion of the unilateral common carotid artery and delivery of 7.5% oxygen through a face mask in adult mice for 30 min, while maintaining the animal rectal temperature at 37.5 &#177; 0.5 &#176;C. Although reversible ligation of the unilateral carotid artery or hypoxia each suppressed cerebral blood flow only transiently, the combination of both insults caused lasting reperfusion deficits, fibrin and platelet deposition, and large infarct in the middle cerebral artery-supplied territory. Importantly, tail-vein injection of recombinant tPA at 0.5, 1, or 4 hr post-tHI (10 mg/kg) provided time-dependent reduction of the mortality rate and infarct size. This new stroke model is simple and can be standardized across laboratories to compare experimental results. Further, it induces thrombosis without craniectomy or introducing pre-formed emboli. Given these unique merits, the tHI model is a useful addition to the repertoire of preclinical stroke research.

Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients.

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA ...

Performing Permanent Distal Middle Cerebral with Common Carotid Artery Occlusion in Aged Rats to Study Cortical Ischemia with Sustained Disability

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, obesity and diabetes. Accordingly, when evaluating therapies for stroke in animals, it is important to select a model with excellent face validity. Ischemic stroke accounts for 80% of all strokes, and the majority of these occur in the territory of the middle cerebral artery (MCA), often inducing infarcts that affect the sensorimotor cortex, causing persistent plegia or paresis on the contralateral side of the body. We demonstrate in this video a method for producing ischemic stroke in elderly rats, which causes sustained sensorimotor disability and substantial cortical infarcts. Specifically, we induce permanent distal middle cerebral artery occlusion (MCAO) in elderly female rats by using diathermy forceps to occlude a short segment of this artery. The carotid artery on the ipsilateral side to the lesion was then permanently occluded and the contralateral carotid artery was transiently occluded for 60 min. We measure the infarct size using structural T2-weighted magnetic resonance imaging (MRI) at 24 hr and 8 weeks after stroke. In this study, the mean infarct volume was 4.5% &#177; 2.0% (standard deviation) of the ipsilateral hemisphere at 24 hr (corrected for brain swelling using Gerriet&#8217;s equation, n = 5). This model is feasible and clinically relevant as it permits the induction of sustained sensorimotor deficits, which is important for the elucidation of pathophysiological mechanisms and novel treatments.

Here we present a protocol to produce permanent distal middle cerebral artery occlusion in elderly female rats with simultaneous occlusion of the carotid arteries to generate large cortical infarcts and sustained deficits. We show confirmation of the lesion size using structural MRI at 24 hr and 8 weeks after stroke.

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, ...