Special thanks to the Gidday lab for their work in developing the RHP protocol, as well as the Neuro-Models Facility (UTSW) for their assistance in the tMCAo surgeries. This work was supported by grants from the American Heart Association (AMS), The Haggerty Center for Brain Injury and Repair (UTSW; AMS), and The Spastic Paralysis Research Foundation of the Illinois-Eastern Iowa District of Kiwanis International (JMG).

NOTE: This protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at UT Southwestern Medical Center which abides by the National Institutes for Health (NIH) policy for experimental animal usage.
1. Repetitive Hypoxic Preconditioning
<ol>
	<li>Custom design four flowmeters on gas regulators and attach to standard 15 L induction chambers with PVC tubes to allow compressed gas from the oxygen (O2) tanks to flow into the chambers via an inlet port. See Equipment and Materials for more details about custom design.</li>
	<li>Divide mice into 2 groups: Repetitive Hypoxic Preconditioning (RHP) Group, which receive exposures to 8% and 11% O2, and the Control Group, which receive exposures to 21% O2 (room air) concurrently. See Table 1 for the variations in frequency, intensity (8 and 11% O2 or 21% O2), and duration (2 or 4 hr) of RHP exposures.</li>
	<li>Remove the top filter lid of each cage and place the cage, with food and water bottles intact, in the chambers connected to their respective O2 tanks. Close and secure the lid of the chamber.
	<ol>
		<li>Open the main gas valve for the tanks and set the initial flow for each induction chamber to 2 L per min (LPM) for the first 5 mins of exposure. Reduce the flow rate to 1 LPM for the remainder of the exposure.</li>
		<li>At the end of the exposure, reduce the flow to to 0 LPM and close the gas valve for the tanks.</li>
		<li>Open the chamber lids and replace the filter top lid on each cage. Place the cages in standard housing until the next hypoxic exposure.</li>
	</ol>
	</li>
	<li>Spray down each induction chamber with NPD (Steris) or an equivalent disinfectant/deodorizer after each use.</li>
	<li>Expose both 21% and RHP mice at the same time of day over the course of two weeks as described in Table 1.</li>
</ol>
2. Transient Middle Cerebral Artery Occlusion (tMCAo)
<ol>
	<li>See Discussion for more details on the timing of stroke following final RHP exposure.</li>
	<li>Prepare an aseptic surgery workplace. Clean surrounding workplace with 70% Ethanol or an equivalent disinfectant and autoclave all surgical tools.
	<ol>
		<li>Set up the Laser Doppler Flowmetry (LDF) instrument to measure relative changes in cerebral blood flow (CBF). Turn on the heating pad to 37 &#176;C. Turn on the incubator to 34 &#176;C.</li>
	</ol>
	</li>
	<li>Anesthetize mice with a brief exposure to a mix of 4% isoflurane/70% NO2/30% O2 in a small induction chamber. Confirm proper anesthesia by lightly pinching the paw. If the mouse withdraws its paw, return the mouse to the induction chamber and continue isoflurane exposure.</li>
	<li>Remove mice from the anesthesia induction chamber and quickly insert the mouse&#8217;s nose in the nose cone. Open the gas flow to the nose cone, closing off flow to the anesthesia induction chamber.
	<ol>
		<li>Without changing the 70% NO2/30% O2 gas mixture, fix isoflurane at 1.8% as the maintenance dose for the remainder of the procedure. Breathing should remain slow and regular throughout the procedure but if breathing becomes rapid and shallow, increase the isoflurane dose. Maintenance dose may vary between equipment manufacture and animal used in the experiment.</li>
	</ol>
	</li>
	<li>Using a microshaver, shave the hair on the temporal region between the corner of the eye and the ear as well as ventral midline of the neck. Remove excess fur and apply ocular lubricant with a sterile cotton swab to keep the corneas from drying out during the procedure. Wipe down incision area with alcohol pads and swab providone-iodine with a sterile cotton swab to maintain aseptic conditions.</li>
	<li>Administer analgesics according to rodent surgical guidelines.</li>
	<li>Make an incision through the temporal skin between the eye and the ear. Expose the temporalis muscle. Using surgical micro-scissors, cut the temporal muscle ligament at the temporal ridge within the area of white muscle striation.
	<ol>
		<li>Gently push the muscle bulk laterally with forceps to visualize the middle cerebral artery (MCA) through the skull. After incision of the temporal muscle, the area may fill with blood. Gently use a cotton swab to staunch any potential bleeding.</li>
		<li>Target the the LDF probe tip to the MCA area. Record the vessel chosen as this area varies between mice.</li>
		<li>Hold the LDF in place and flush with the skull until a stable red blood cell flux is read and record this value as the baseline CBF. Ideal baseline CBF on a Laser Doppler Flowmetry are &#62;600 flux, but this will vary with manufacturer. If baseline CBF is &#60;400 flux, the flow recording is most likely from a nearby vein, or an incomplete placement of the probe over the target vessel.</li>
	</ol>
	</li>
	<li>After baseline CBF is established, reposition the mouse so that the neck is exposed. Support its head and keep the mouse under steady anesthesia from the nosecone.</li>
	<li>Make a ventral midline incision from just below the mandible to the clavicle.
	<ol>
		<li>Using forceps, blunt dissect all superficial fascia to expose the left common carotid artery (CCA). Separate the CCA from connective tissue and the vagus nerve.</li>
		<li>Permanently ligate the CCA with a 6.0 silk suture. Place the ligating suture as proximal as possible to allow enough room for occluding filament placement.</li>
		<li>Loop and loosely tie a second silk 6.0 suture around the CCA distal to the occluding suture. Be careful not to occlude the artery as the occluding filament will subsequently be threaded through the carotid.</li>
		<li>Use an 8 x 2 mm light micro serrafine areterial clamp to clamp the CCA distal to the loosely-tied silk suture. Gently lift the CCA with forceps and make a small longitudinal incision, as proximal to the ligating suture as possible with 3 mm vannas.</li>
		<li>Thread a 12 mm silicon-coated 6.0 gauge nylon occluding filament through the incision to enter the artery lumen, and then advance it a few mm. Loosely tighten the second loose silk suture around the tip of the occluding filament to ensure blood flow does not push the filament out of the CCA and remove the arterial clamp.</li>
		<li>At the first bifurcation of the CCA into the internal carotid artery (ICA) and the external carotid artery (ECA), thread the occluding filament into the right branch of the first bifurcation to enter the ICA.Advance the occluding filament 9 to 10.5 mm past the second silk suture into the left internal carotid artery (ICA).</li>
		<li>Shortly after entering the ICA, advance the occluding filament into the left branch at the second bifurcation between the ICA and the pterogopalantine artery (PPA). Visualization of the PPA is unlikely so proceed until you feel a mild resistance with full placement of the occluding filament. Lifting the ICA with forceps may help the filament to thread more easily into the left branch of the second birfurcation. Tighten the second silk suture.</li>
		<li>Turn the mouse so the incision over the MCA is visible. With the LDF equipment, confirm that the CBF is blocked through LDF readings. A successful occlusion is a &#62;80% reduction from baseline CBF.</li>
		<li>Completely tighten and double-knot the second silk suture around the occluding filament when the proper position is achieved. If necessary, slightly push in or pull out the occluding filament to achieve CBF criteria for a successful occlusion (e.g. &#62;80% reduction from baseline CBF).</li>
		<li>Close the neck and head opening with 6.0 nylon sutures.</li>
	</ol>
	</li>
	<li>Place mice in the 34 &#176;C incubator for the duration of the occlusion. Recommended length of occlusion is 60 min, but this varies for age, strain-dependent differences in cerebrovascular anatomy,25 and the extent of injury desired (mild, moderate, severe). Make sure that animals regain consciousness within mins of coming off anesthesia.</li>
	<li>Re-anesthetize the animals with isoflurane, as described in step 2.3, 5 min before the end of the pre-defined occlusion period, open the scalp incision and confirm that the MCA perfusion is still reduced using transcranial LDF readings. If CBF is not sufficiently reduced (e.g. &#60;20% baseline CBF), the MCA has reperfused at some point during the occlusion and the mouse should be excluded from further experimentation.</li>
	<li>Open the midline neck incision and loosely tie a third silk suture around the CCA, distal to the second silk suture but proximal to the CCA bifurcation to ensure that the external carotid artery (ECA) will remain viable after the filament is removed.
	<ol>
		<li>Cut or untie the knot that holds the occluding filament (i.e., second silk suture) and withdraw the occluding filament slowly. Once removed, quickly close the third silk suture around the CCA to minimize backflow of blood from the ICA. Double-knot this suture and close the incision with 6.0 nylon sutures.</li>
		<li>Reposition the mouse to quantify the level of CBF after 5 min of reperfusion. Successful reperfusion is generally defined as a CBF of &#62;50% of baseline CBF, but, as with occlusion flow, investigators can establish their own criterion. If animals exhibit a CBF below 50% of their baseline, it is likely the MCA is &#8216;permanently&#8217; occluded, and thus represents another study exclusion criterion.</li>
		<li>Close both incisions with 6.0 nylon sutures. Provide saline, anesthetic (Lidocaine), and antibiotics according to rodent guidelines. However some small doses of antibiotics (3 mg/kg of minocycline) have been found to be neuroprotective following stroke.26</li>
	</ol>
	</li>
	<li>After regaining consciousness in the heated incubator following surgery, place mice in a clean, sterile cage. Provide moistened food or nutritional hydration dietary gel supplements and a petri dish of water as the animals will have restricted mobility following stroke.&#160;Monitor animals closely during recovery for excessive post-surgical pain and death.</li>
</ol>
3. Evans Blue (EB) Injection
<ol>
	<li>Inject EB 22 hr after reperfusion and should circulate in the bloodstream for 2 hr prior to sacrifice and TTC staining.</li>
	<li>Make an EB solution for injection (2% EB in saline) and gently mix at room temperature. Filter solution through filter paper or push through a 0.2 &#181;m filter attached to a small syringe to remove undissolved EB and store at room temperature.</li>
	<li>Prepare the amount of EB needed for injection (4 ml/kg of body weight) Draw the desired amount of dye into a 0.3 cc insulin syringe with a 29 gauge needle and ensure that the solution is at room temperature
	<ol>
		<li>Restrain mouse using flat bottom restrainer. Hold the tail so that the lateral vein is uppermost. Lateral veins are located on either side of the centerline of the tail. Hold the tip of the tail to keep the mouse steady for injection.</li>
		<li>Insert the needle into the vein approximately 3.5 mm, being careful not to perforate the vein. Confirm that the needle is in the vein by drawing back on the syringe and looking for traces of blood.</li>
		<li>Inject all of the dye over the course of 1 min. If the solution is going into the vein there should be minimal resistance as pressure is applied to the syringe. Confirm successful systemic venous administration of EB by an immediate color change in the tail, limbs, and eyes of the mouse.</li>
		<li>Remove the needle from the tail and gently apply pressure using clean gauze in order to stop bleeding.</li>
	</ol>
	</li>
	<li>Begin timing when the mouse&#8217;s skin turns blue. Allow the EB to circulate for 2 hr to penetrate the weakened BBB.</li>
</ol>
4. 2,3,5- Triphenyltetrazolium Chloride (TTC) Staining
<ol>
	<li>Perfusion and TTC staining should occur 24 hr after reperfusion.</li>
	<li>Prepare a 2% TTC solution prior to the designated time of sacrifice. Add 10 g of TTC powder to 500 ml of 0.01 M phosphate buffered saline (PBS), pH 7.4. Heat solution to 37 &#176;C in water bath to facilitate solubilizing of the TTC. CAUTION: TTC powder and solution are a skin, lung, and eye irritant. Wear appropriate personal protective equipment when handling these materials.
	<ol>
		<li>Once the powder has completely dissolved, immediately transfer to a bottle, cover in foil, and store at 4 &#176;C. TTC and tissue stained with TTC are light sensitive.</li>
	</ol>
	</li>
	<li>At 24 hr after tMCAo and 2 hr after EB administration, sacrifice the animal with an isoflurane overdose in a small induction chamber. Perfusion should begin immediately following sacricice in order to minimize autolysis which begins in the absence of oxygen following death.</li>
	<li>Quickly secure the animal on a Styrofoam platform with forearms pinned through the paws. Cut a lateral incision through the abdominal wall from the midline just below the ribcage. Carefully cut through the diaphragm to expose the heart.
	<ol>
		<li>Start the perfusion pump at 5 ml/min flow rate with a 60 cc syringe filled with ice cold 0.01 M PBS and connected to a 27 guage winged infusion needle. Place the tip of the needle about 0.5 cm into the left ventricle of the heart and cut the right atrium. Progressively diluted venous blood should flow out of the atrium during the perfusion until venous blood appears colorless. Transcardially perfuse 30 ml 0.01 M phosphate buffered saline (PBS) through the heart.</li>
	</ol>
	</li>
	<li>Add 5 ml of TTC solution into transparent 20 scintillation bottles.</li>
	<li>Immediately following perfusion, decapitate the animals and dissect out the brains using small scissors and a spatula if necessary. Examine the brains to exclude animals which underwent subarachnoid hemorrhage at the Circle of Willis, secondary to suture placement. Check that the hemisphere contralateral to the occluded MCA appears pale, without noticeable EB leakage or edema.</li>
	<li>Pour PBS into an acrylic brain matrix designed to make 1.0 mm thick coronal sections of a mouse brain. Place the brain, dorsal side up, into the matrix and immediately pour PBS over the brain. Keep the brain moist.
	<ol>
		<li>Remove the olfactory bulbs by inserting a stainless steel 0.21 mm thick blade into the second slot from the rostral side of the matrix.</li>
		<li>Remove the cerebellum by inserting a blade in the fourth slot from the caudal side of the matrix.</li>
		<li>Insert a blade into the middle slot of the remaining slots in the matrix. Insert the remaining blades, evenly bisecting the remaining tissue to ensure the most even distribution of tissue during the slicing.</li>
		<li>Once all blades have been inserted, add 1 to 2 drops of PBS to the brain to moisten it.</li>
		<li>Remove the blades one at a time from the matrix beginning with the rostral region. Keep the first 7 slices for TTC analysis after tMCAo. Use a small spatula to carefully transfer the slices from the blade to the TTC-filled vial.</li>
	</ol>
	</li>
	<li>After all sections are in the vial, cap it and place in a warm water bath until the sections turn pink. Gently turn the bottle in the bath if necessary to avoid section overlap, which could lead to uneven staining. Then dispose of the TTC and pour a 4% paraformaldehyde solution into the vial to cover brain sections to terminate the TTC chemical reaction.</li>
	<li>Immediately arrange the sections on a clean 1&#34; x 3&#8221; glass slide and orient the sections from rostral to caudal.
	<ol>
		<li>When the sections are arranged on the slide, scan the slide using a standard scanner. Set the resolution at a minimum of 600 dpi for image analysis. Be sure to include the name of the animal and a metric ruler in the scanned image.</li>
		<li>Flip over the slide and scan the back side to ensure all data is collected.</li>
	</ol>
	</li>
</ol>
5. Infarct Volume Quantification
<ol>
	<li>Quantify the infarct volume by using a standard image analysis software (e.g., ImageJ).</li>
	<li>Scan images at a high resolution (e.g. 600 dpi) for adequate analysis. Crop images. Standardize the scale for all images using the metric ruler included in the scanned image.</li>
	<li>Calculate the total volume of the contralateral hemisphere using the following formula. Repeat this formula to calculate the total volume of the ipsilateral hemisphere. 
	Sum of total contralateral hemisphere of each slice x slice thickness</li>
	<li>Calculate the indirect infarct volume. Control for ipsilateral edema from stroke by using the healthy, contralateral hemisphere as a control.27 Use the following formula to calculate the indirect infarct volume: 
	Total volume of contralateral hemisphere - 
	(Total volume of ipsilateral hemisphere &#8211;Average volume of 3 measurements of infarct)</li>
</ol>
6. Blood-brain Barrier (BBB) Integrity Quantification
<ol>
	<li>To prepare for EB quantification, first weigh 2.5 inch weigh boats. Record the weight and label two weigh boats for each animal: one for the ipsilateral hemisphere and one for the contralateral hemisphere.</li>
	<li>After scanning the TTC-stained sections, bisect each section with a disposable razor blade into ipsilateral and contralateral hemispheres. Place the ipsilateral hemispheres from all 7 sections in a weigh boat and record the weight. Repeat for contralateral hemisphere.</li>
	<li>Immediately transfer the weight boats to an oven set for 56 &#176;C for 48 hr.</li>
	<li>Weigh the dried sections. Transfer both hemispheres into separate 1.5 ml microcentrifuge tubes.
	<ol>
		<li>Calculate the amount of formamide needed for each hemisphere (8 ml/g of dry tissue), and add to their respective microcentrifuge tubes. Formamide is light sensitive and cover all formamide-treated samples in foil from this point onward.</li>
		<li>Transfer the microcentrifuge tubes to an incubator set to 56 &#176;C for another 48 hr.</li>
	</ol>
	</li>
	<li>After the 48 hr, pipette out the blue supernatant into another set of labeled microcentrifuge tubes. Push tissue to the bottom of the tube to maximize the volume of extracted supernatant. Keep the tubes of supernatant and dispose of all tissue.</li>
	<li>Prepare exponential serial dilutions of EB in formamide for the standard curve. Include 1 blank (formamide only) and then 10 exponential solutions from 0.125 ug/ml through 64 ug/ml of EB in formamide.
	<ol>
		<li>Pipette 300 &#181;l of the dilutions made for the standard curve into a 96-well plate. Pipette 300 &#181;l of the supernatant into corresponding wells.</li>
		<li>Measure the absorbance on a spectrophotometer at 620 nm.</li>
		<li>Compare the standard curve of the EB dilutions with the absorbance of the supernatant samples. The optical density is directly proportional to the integrity of the BBB.</li>
		<li>Assume the optical density of the contralateral hemisphere as background and use the formula (ipsilateral-contralateral)/contralateral to determine fold change. For further details about statistical analysis see Martin et al., 2010.18</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

A single exposure to systemic hypoxia (i.e., 2 hr of 11% O2) in mice &#8220;transiently&#8221; protects the brain from tMCAo,29 meaning the epigenetic response to the hypoxic preconditioning challenge is short-lasting, and the baseline phenotype is restored within days. Repetitive presentations of the hypoxic preconditioning stimulus dramatically extend the duration of the neuroprotective phenotype.6 Many studies have shown that the frequency, magnitude, and duration of the repet...

As the leading cause of adult disability and the fourth leading cause of death, stroke is one of the most debilitating disease states facing the adult population of the United States.1 Animal models of stroke allow for experimental investigation of new methods of reducing ischemic injury and improving post-stroke recovery. One novel avenue for such translational research is preconditioning. Preconditioning is the intentional use of a non-damaging stimulus to reduce damage from a subsequent, and more severe, injury.2 Hypoxic preconditioning has been shown to produce pleiotropic changes in the brain that provide protection against stroke in both in vivo and in vitro studies.3 However, a single exposure to hypoxia only offers short-term neuroprotection, inducing less than 72 hr of tolerance against ischemia in adult mice.4 Even after four weeks of 14 hr daily exposures to hypobaric hypoxia, Lin et al. found that neuroprotection was only sustained for one week.5 Repetitive hypoxic preconditioning (RHP) is characterized by stochastic variations in frequency, duration, and intensity of hypoxic exposures. In contrast to a single preconditioning challenge, RHP induces a cerebroprotective phenotype that lasts up to eight weeks in mice.6 RHP reduced infarct volumes, blood-brain barrier (BBB) disruption, vascular inflammation, and leukocyte diapedesis for weeks after the final hypoxic exposure. RHP specifically reduced inflammation in the ischemic brain by reducing T cell, monocyte, and macrophage populations, while maintaining B cell populations in the ischemic hemisphere.7 In fact, RHP induced an immunosuppressive phenotype in mice prior to any CNS injury, including stroke. RHP-treated B cells isolated from RHP-treated healthy mice exhibited a unique anti-inflammatory phenotype, with a downregulation of both antigen presentation and antibody production. The overall reduction in pro-inflammatory adaptive immune mechanisms makes RHP an excellent methodology to induce endogenous immunosuppression for not only CNS-specific inflammatory diseases, but also systemic injury or disease models that include a pro-inflammatory pathology.
RHP reduces both infarct volume and BBB disruption following a transient middle cerebral artery occlusion (tMCAo). Animal models of stroke, such as the commonly used tMCAo, dramatically improve the understanding of the pathophysiology of stroke, as well as the design of more effective neurotherapeutics. First developed by Koizumi et al., in 1986,8 the tMCAo procedure is a widely used method of inducing stroke in rodents and one of the preferred methods for investigating inflammation following reperfusion. As the methods for tMCAo evolve, the more recent use of silicone-coated filaments further reduce the risk of subarachnoid hemorrhage compared to other models9,10 and improve reliability, though unfortunately tMCAo often produces a wide variation in infarct volumes.11-13 Most of these studies delineate infarction regions in coronal brain sections by staining with 2,3,5- triphenyltetrazolium chloride (TTC), considered a gold standard for infarct quantification because it is a simple and inexpensive way to produce vivid, replicable results. TTC serves as a substrate of dehydrogenases present in mitochondria. When brain slices are exposed to the TTC solution, TTC is selectively taken into living cells where its non-soluble reduction product, formazan, precipitates to a deep red color in viable mitochondria. Because of mitochondrial dysfunction in the ischemic tissue, this tissue remains white, allowing for differentiation of damaged and healthy tissue.14
RHP also reduces BBB disruption in the ischemic hemisphere.6 Therefore, the dual quantification of BBB integrity within the same brains as TTC-based infarct volume determinations15 would provide useful information about the full efficacy of endogenous protection, and potential causal relations between BBB disruption and infarction in untreated and treated animals. The influx of peripheral blood through a disrupted BBB, secondary to stroke, increases leukocyte populations, pro-inflammatory cytokines, oxidative stress, vasogenic edema, and hemorrhagic transformation in the ischemic hemisphere, ultimately increasing the rates of infection and mortality in patients with ischemic stroke.16,17 A common method of measuring BBB disruption in animal models is through quantification of Evans blue (EB) dye leak into the brain.15,18-21 EB selectively binds to serum albumin, a globular protein (MW=65 kDa) that does not cross the BBB in uninjured animals.22 Following ischemic stroke, EB infiltrates the brain, and fluoresces at 620 nm, allowing for measurement of optical density within the perfused injured parenchyma.22 The optical density is directly proportional to the permeability of the BBB when EB has been washed out of the post-mortem cortical vasculature by transcardiac perfusion. With the immediate processing of TTC-stained brains in animals with EB administration, both the infarct volume and BBB disruption can be effectively quantified. It should be noted, however, that neuronal injury and BBB disruption are not concomitant processes in the post-stroke brain,23,24 so the selection of time of sacrifice is an important consideration.
The protocol that follows details the RHP method, the tMCAo method for inducing a temporary arterial occlusion that models middle cerebral artery occlusions in human patients, and the dual histological methods for determining neural and vascular stroke injury endpoints. TTC measures cell death and cumulative tissue damage, allowing for the quantification of an overall infarct volume, while EB provides for the hemispheric quantification of BBB damage.

<table border="0" cellpadding="0" cellspacing="0" width="830">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:249px;">Material/ Equipment</td>
			<td style="width:171px;">Company</td>
			<td style="width:127px;">Catalog Number</td>
			<td style="width:283px;">Comments/Description</td>
		</tr>
		<tr height="220">
			<td height="220" style="height:220px;width:249px;">Flowmeters, regulators</td>
			<td style="width:171px;">VetEquip, Inc</td>
			<td style="width:127px;">Specialty order</td>
			<td style="width:283px;">Four flowmeters are attached to 6.0 mm flexible PVC tubing which connects to the inlet port on each induction chamber with a plastic female connector. These flowmeters are bolted to a 6.5&#34; x 1&#34; x 1&#34; metal bar. This metal bar is bolted to a MI-246-P pressure gauge with a DISS outlet. This pressure gauge and flowmeter equipment can be attached to each new gas cylinder with a wrench.</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:249px;">21% O2 tank</td>
			<td style="width:171px;">AirGas</td>
			<td style="width:127px;">OX USP200</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:249px;">11% O2 tank</td>
			<td style="width:171px;">AirGas</td>
			<td style="width:127px;">Specialty order</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:249px;">8% O2 tank&#160;</td>
			<td style="width:171px;">AirGas</td>
			<td style="width:127px;">Specialty order</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">15L induction chambers</td>
			<td style="width:171px;">VetEquip</td>
			<td style="width:127px;">941454</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:249px;">Moor Laber Dopper Flow&#160;</td>
			<td style="width:171px;">Moor Instruments&#160;</td>
			<td style="width:127px;">moorVMS-LDF1-HP</td>
			<td style="width:283px;">0.8mm diameter probe&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">High Intensity Illuminator&#160;</td>
			<td style="width:171px;">Nikon</td>
			<td style="width:127px;">NI-150</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:249px;">Zoom Stereo Microscope&#160;</td>
			<td style="width:171px;">NIkon</td>
			<td style="width:127px;">SMZ800</td>
			<td style="width:283px;">Other surgical microscopes may be used.&#160;</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:249px;">Kent Scientific Right Temperature CODA</td>
			<td style="width:171px;">Kent Scientific Corporation</td>
			<td style="width:127px;">Discontinued</td>
			<td style="width:283px;">Recommended replacement is PhysioSuite with RightTemp Temperature Monitoring and Homeothermic Control (Kent Scientific, #PS-RT).</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:249px;">Hovabator Incubator</td>
			<td style="width:171px;">Stromberg&#39;s</td>
			<td style="width:127px;">2362-E</td>
			<td style="width:283px;">Our model is the 2362N. 2362E is a later model and includes an electronic thermostat.&#160;</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:249px;">V010 Anesthesia system&#160;</td>
			<td style="width:171px;">VetEquip</td>
			<td style="width:127px;">901807</td>
			<td style="width:283px;">Includes: ten foot high-pressure oxygen hose, frame, flowmeter, oxygen flush assembly, vaporizer, breathing circuit, chamber, nosecones, waste gas evacuation tubing and two VapoGuard filters</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">250 mL isoflurane&#160;</td>
			<td style="width:171px;">Butler Schein</td>
			<td style="width:127px;">NDC-11695</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:249px;">D-6 Vet Trim Animal Cordless Trimmer&#160;</td>
			<td style="width:171px;">Andis&#160;</td>
			<td style="width:127px;">#23905</td>
			<td style="width:283px;">Replacement blades are available from Andis (#23995)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">Betadine&#160;</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:127px;">19-898-867&#160;</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:249px;">Q-tips</td>
			<td style="width:171px;">Multiple sellers&#160;</td>
			<td style="width:127px;">Catalog number not available&#160;</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">Gauze Pads</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:127px;">67622</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">Surgical drape</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:127px;">GM300&#160;</td>
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			<td height="40" style="height:40px;width:249px;">Silk Sutures&#160;</td>
			<td style="width:171px;">Look/Div Surgical Specialties</td>
			<td style="width:127px;">SP115</td>
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			<td height="40" style="height:40px;width:249px;">Nylon Sutures</td>
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			<td height="20" style="height:20px;width:249px;">Durmont #5 forceps (2)&#160;</td>
			<td style="width:171px;">Fine Science Tools&#160;</td>
			<td style="width:127px;">11251-35</td>
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			<td height="40" style="height:40px;width:249px;">3mm Vannas</td>
			<td style="width:171px;">Kent Scientific Corporation</td>
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			<td height="20" style="height:20px;width:249px;">Hartman Hemostats&#160;</td>
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			<td style="width:171px;">Washington University</td>
			<td style="width:127px;">Specialty order</td>
			<td style="width:283px;">Filaments are silicone coated at Washington Univeristy and provided to UTSW facilities for a fee.&#160;</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:249px;">Evans Blue</td>
			<td style="width:171px;">Sigma Aldritch</td>
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			<td style="width:283px;">150 mm, circles, Grade 1&#160;</td>
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			<td style="width:283px;">Scintillation tubes with TTC may be manually held under running warm water as an alternative to the water bath.</td>
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			<td style="width:127px;">Catalog number not available&#160;</td>
			<td style="width:283px;">Reusable cryostat blades are an inexpensive alternative.</td>
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			<td height="40" style="height:40px;width:249px;">4% paraformaldehyde</td>
			<td style="width:171px;">Santa Cruz Biotechnology&#160;</td>
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			<td height="20" style="height:20px;width:249px;">Superfrost microscope slides&#160;</td>
			<td style="width:171px;">Fisher Scientific</td>
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			<td height="40" style="height:40px;width:249px;">HP Scanjet G4050</td>
			<td style="width:171px;">Multiple sellers&#160;</td>
			<td style="width:127px;">Catalog number not available&#160;</td>
			<td style="width:283px;">Other commercial scanners are suitable for this step in the protocol.</td>
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			<td height="40" style="height:40px;width:249px;">ImageJ&#160;</td>
			<td style="width:171px;">National Institute of Health</td>
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			<td style="width:171px;">Mettler Toledo&#160;</td>
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			<td height="20" style="height:20px;width:249px;">Precision Compact Oven&#160;&#160;</td>
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			<td height="40" style="height:40px;width:249px;">1.7 mL microcentrifuge tubes (Eppendorfs)</td>
			<td style="width:171px;">Denville Scientific&#160;</td>
			<td style="width:127px;">C2170</td>
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			<td height="20" style="height:20px;width:249px;">Formamide</td>
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			<td style="width:127px;">BP228-100</td>
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			<td height="20" style="height:20px;width:249px;">96-well plates</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:127px;">07-200-9</td>
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			<td height="40" style="height:40px;width:249px;">Epoch Microplate Spectrophotometer&#160;</td>
			<td style="width:171px;">BioTek&#160;</td>
			<td style="width:127px;">Catalog number not available&#160;</td>
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</table>

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 study included 25 male Swiss Webster mice that were 10 weeks of age at the start of randomization into RHP (n = 10) or 21% O2 (n = 15) groups. Two weeks after the final RHP exposure, surgical procedures were performed, with groups blinded and counterbalanced between days. Following tMCAo, 1 mouse died during post-operative recovery and 1 mouse was excluded from the study because it did not meet the reperfusion CBF criterion. Both excluded mice were from the 21% O2 group. In accordance with ARRI...

Watch this Scientific Journal Video about Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice at JoVE.com

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

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 ...

quantification-neurovascular-protection-following-repetitive-hypoxic

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13.7K visualizações.  University of Texas Southwestern Medical Center. 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.

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

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 ...

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 ...

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

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

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

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

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 ...

Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ischemic in origin and occur in the territory of the middle cerebral artery (MCA). Currently the only Food and Drug Administration (FDA) approved drug for the treatment of this devastating disease is tissue plasminogen activator (tPA). However, tPA has a small therapeutic window for administration (3 - 6 hr), and is only effective in 4% of the patients who actually receive it. Current research focuses on understanding the pathophysiology of stroke in order to find potential therapeutic targets. Thus, reliable models are crucial, and the MCA occlusion (MCAo) model (also termed the intraluminal filament or suture model) is deemed to be the most clinically relevant surgical model of ischemic stroke, and is fairly non-invasive and easily reproducible. Typically the MCAo model is used with rodents, especially with mice due to all the genetic variations available for this species. Here we describe (and present in the video) how to successfully perform the MCAo model (with reperfusion) in mice to generate reliable and reproducible data.

In order to understand the pathophysiology of stroke, it is important to use reliable models. This paper will describe one of the most frequently used stroke models in mice, termed the middle cerebral artery occlusion (MCAo) model (also termed the intraluminal filament or suture model) with reperfusion.

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ...

Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature and mature brain. Stroke occurs commonly in the perinatal period in humans, and transient ischemia-reperfusion is the most common form of stroke in neonates. The reperfusion phase is a critical component of injury progression, which occurs over a period of days to weeks, and of the endogenous response to injury. This postnatal day 10 (p10) rat model of transient middle cerebral artery occlusion (tMCAO) creates a unilateral, non-hemorrhagic focal ischemia-reperfusion injury that can be utilized to study the mechanisms of focal injury and repair in the full-term-equivalent brain. The injury pattern that is produced by tMCAO is consistent and highly reproducible and can be confirmed with MRI or histological analyses. The severity of injury can be manipulated through changes in occlusion time and other methods that will be discussed.

Neonatal stroke is a significant cause of early brain injury requiring a translational model with consistent focal injury patterns and high reproducibility in order to enable study. This study describes the detailed surgical procedure for creating a non-hemorrhagic, unilateral focal ischemia-reperfusion injury in full-term-equivalent rodents.

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature ...

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

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

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

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