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 repetitive stimulus train are critical determinants of this response. For example, simply repeating the same intensity and duration of hypoxia (2 hr of 11% oxygen) 3 times per week (M-W-F) over 2 weeks did not extend the therapeutic window for neurovascular protection from tMCAo,6 but was sufficient to induce long-term ischemic tolerance in the retina.30 Interestingly, although 5 exposures to systemic hypoxia (5 hr of 8% O2) spaced 6 days apart protected against tMCAo induced 3 days after the last hypoxic exposure, neuroprotection was lost if the 5 hypoxic exposures were placed 3 days apart.31 The reason for this difference remains unclear, but may have been due to the duration of the severe hypoxia relative to this protocol and/or insufficient recovery time between hypoxic challenges of this severity.
In short, titration of the repetitive hypoxic challenge across the domains of frequency, magnitude, and duration, seems critical to the development of tolerance while not inducing injury, not to mention tissue-specific and perhaps species-specific effects. For example, 3 of 9 of the RHP exposures involve exposure to 8% O2 for 4 hr, though 6 hr of 8% O2 induces hippocampal neuronal death.32 Therefore, there must be a strict adherence to the RHP protocol with regard to duration and severity of hypoxic exposures to induce endogenous protection. Although the potential effect of circadian rhythm on preconditioning-induced protection needs further exploration,33 performing RHP exposures at the same time of day for a particular cohort reduces the risk of any potential confounding effect. In terms of dose-response and efficacy, the most robust RHP-mediated protection from focal stroke occurred when the tMCAo was induced 2 weeks following the final hypoxic exposure,3 at a time when RHP-treated healthy mice exhibit an immunosuppressive phenotype (in the absence of ischemic injury).7 This 2 week time point may not coincide with the period of maximal protection against other acute CNS injuries, or in other organ systems. Both the RHP protocol, and the temporal features of the epigenetic response, will undoubtedly need optimization for each new translational use.
One of the greatest limitations of the tMCAo procedure is the heterogeneous distribution of infarct volumes, as shown by this representative data set. The collateral circulation provided by the Circle of Willis, leptomeningeal anastomoses, and dorsal collateral junctions can lead to inconsistent infarct volumes.34 Variations between the Circle of Willis in different strains of mice, as well as the presence and patency of posterior communicating arteries,25,35 can further reduce consistency of infarct volumes within groups. Robust sample sizes and replication are often necessary to produce conclusive results and should be included in the design of the experiment after powering accordingly. Other methods of stroke induction, particularly distal focal ischemic methods such as occlusion of the primary MCA branch via a burr hole in the lateral skull, or photothrombosis of distal branches of the MCA, often produce less variation in stroke volume. Photothrombotic strokes are limited, however, as they produce simultaneous extracellular and intracellular edema, a phenomenon not seen in ischemic strokes in humans.36 Conversely, tMCAos are more directly applicable to the clinical setting as over 60% of all strokes in humans occur due to obstruction of the middle cerebral artery.37 Finally, the anesthesia used during tMCAo, isoflurane, has been shown to induce neuroprotection.38 In order to account for this neuroprotection, isoflurane levels should be consistent between surgeries and between treated and untreated experimental groups, and maintained at &#60;3% to reduce its potential neuroprotective effects.39
Time is also critical in the TTC staining process. Although there is great variation in the stroke literature about the time in which TTC staining can be performed after ischemia, varying from 4 hr to 7 days post-stroke,18,40 TTC staining should occur at 24 to 48 hr after stroke for the most consistent and clearly delineated infarct volumes. TTC infarct volumes have been found to stabilize 24 hr after stroke but after 48 hr, an influx of macrophages into the ischemic brain makes defining the infarct volume difficult.14,40 Furthermore, the staining of brain tissue with TTC should immediately follow sacrifice. Delaying staining will decrease the quality of the stain because of increased mitochondrial death resulting from animal death, not cerebral infarction. A similar increase in mitochondrial death will occur if the brain is not kept moist during the insertion of the blades. While this protocol clearly illustrates the benefit of infarct analysis with TTC staining, other immunohistological stains can be used to quantify the infarct volume following tMCAo. Cresyl violet or fluoro-jade staining have less rigid time requirements for staining after sacrifice, but these classic stains require very thin sections obtained by cryostat or microtome, and thus require more time for integrative summation and analysis.14 Furthermore, these stains cannot be used in conjunction with EB quantification of BBB disruption, as developed by others,15 eliminating the possibility of direct comparison between infarct volume and BBB integrity in a given brain. It should be noted that RHP-treated animals typically exhibited pink as opposed to pure white volumes.6 In order to avoid washout of infarct in RHP-treated mice, we use a shorter staining period for TTC that results in more pink healthy tissue as opposed to dark red. Evans Blue processing may also darken the infarct area so pure white infarcts are unlikely with the dual quantification staining presented in this protocol.
In order to compare EB quantification within and between groups, all animals must undergo equivalent circulation times for EB. Others have injected EB immediately following reperfusion and allowed EB to circulate for up to 72 hr,41 or at 4 hr post-tMCAo to circulate for 4 hr,15 as alternative approaches. The amount of EB that crosses the disrupted BBB and enters brain tissue represents the integral accumulation of albumin-bound dye from time of injection to time of sacrifice. Thus, the method described in this paper reveals the status of the BBB at precisely the time between 22 and 24 hr of reperfusion, and may miss earlier or later openings or closings of the barrier. Conversely, the method employed by Wang et al. reveals the status of the BBB for 72 hr following stroke, including all changes in BBB status during that 3 day post-stroke time.42 Neither protocol is better or worse; although there remains a risk of &#8220;missing&#8221; a transient opening of the BBB by using shorter circulation times, it allows investigators to identify specific temporal features of BBB pathophysiology and to pair it with other methods, as shown here. Others have combined TTC and EB by injecting EB intracardially 1 min after transcardial perfusion.18 However, this method produced inconsistent results and is a far more complicated procedure than the protocol described above.
One of the most promising future directions for RHP is the translational application of this preconditioning protocol to other disease states. Intermittent hypoxic exposures have been found to be protective in pro-inflammatory CNS conditions other than ischemia. In a rat model of Alzheimer&#8217;s, 2 weeks of 4 hr daily exposures to hypobaric hypoxia reduced the impairment of memory retention and loss of cortical neurons after intracerebral injections of beta-amyloid.43 Intermittent hypoxia (2 weeks of 15 hr daily exposures to hypobaric hypoxia) was also found to be protective in rats following L-buthionine-[S,R]-sulfoximine (L-BSO) infusion, a model of Parkinson&#8217;s which impairs striatal dopaminergic transmission.44 However, these models of Alzheimer&#8217;s and Parkinson&#8217;s only investigated the short-term effects of hypoxic preconditioning.43,44 Investigating the long-term neuroprotection induced by RHP may be a fruitful future direction for research. Furthermore, the neuroprotection induced by RHP may extend to improved BBB integrity in mouse models of Alzheimer&#8217;s, which could be analyzed with the EB.45 The dual TTC and EB analysis would be even more useful in Parkinson&#8217;s model as it induces striatal lesions46 and BBB disruption,47 which can be quantified with TTC48 and EB,49 respectively. Overall RHP is a simple, powerful form of preconditioning with great translational potential as it uniquely induces long-term, anti-inflammatory, and neuroprotective effects. The dual quantification of TTC and EB could also be easily applied to other disease states that involve mitochondrial disruption, cell death, and BBB leakage.

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><tbody><tr><td>Flowmeters, regulators</td><td>VetEquip, Inc</td><td>Specialty order</td><td>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&quot; x 1&quot; x 1&quot; 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><td>21% O&lt;sub&gt;2&lt;/sub&gt; tank</td><td>AirGas</td><td>OX USP200</td><td></td></tr><tr><td>11% O&lt;sub&gt;2 &lt;/sub&gt;tank</td><td>AirGas</td><td>Specialty order</td><td></td></tr><tr><td>8% O&lt;sub&gt;2&lt;/sub&gt; tank&amp;nbsp;</td><td>AirGas</td><td>Specialty order</td><td></td></tr><tr><td>15 L induction chambers</td><td>VetEquip</td><td>941454</td><td></td></tr><tr><td>Moor Laber Dopper Flow&amp;nbsp;</td><td>Moor Instruments&amp;nbsp;</td><td>moorVMS-LDF1-HP</td><td>0.8 mm diameter probe&amp;nbsp;</td></tr><tr><td>High Intensity Illuminator&amp;nbsp;</td><td>Nikon</td><td>NI-150</td><td></td></tr><tr><td>Zoom Stereo Microscope&amp;nbsp;</td><td>NIkon</td><td>SMZ800</td><td>Other surgical microscopes may be used.&amp;nbsp;</td></tr><tr><td>Kent Scientific Right Temperature CODA</td><td>Kent Scientific Corporation</td><td>Discontinued</td><td>Recommended replacement is PhysioSuite with RightTemp Temperature Monitoring and Homeothermic Control (Kent Scientific, #PS-RT).</td></tr><tr><td>Hovabator Incubator</td><td>Stromberg&#039;s</td><td>2362-E</td><td>Our model is the 2362N. 2362E is a later model and includes an electronic thermostat.&amp;nbsp;</td></tr><tr><td>V010 Anesthesia system&amp;nbsp;</td><td>VetEquip</td><td>901807</td><td>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><td>250 ml isoflurane&amp;nbsp;</td><td>Butler Schein</td><td>NDC-11695</td><td></td></tr><tr><td>D-6 Vet Trim Animal Cordless Trimmer&amp;nbsp;</td><td>Andis&amp;nbsp;</td><td>#23905</td><td>Replacement blades are available from Andis (#23995)</td></tr><tr><td>Betadine&amp;nbsp;</td><td>Fisher Scientific</td><td>19-898-867&amp;nbsp;</td><td></td></tr><tr><td>Q-tips</td><td>Multiple sellers&amp;nbsp;</td><td>Catalog number not available&amp;nbsp;</td><td></td></tr><tr><td>Gauze Pads</td><td>Fisher Scientific</td><td>67622</td><td></td></tr><tr><td>Surgical drape</td><td>Fisher Scientific</td><td>GM300&amp;nbsp;</td><td></td></tr><tr><td>Silk Sutures&amp;nbsp;</td><td>Look/Div Surgical Specialties</td><td>SP115</td><td></td></tr><tr><td>Nylon Sutures</td><td>Look/Div Surgical Specialties</td><td>SP185</td><td></td></tr><tr><td>Durmont #5 forceps (2)&amp;nbsp;</td><td>Fine Science Tools&amp;nbsp;</td><td>11251-35</td><td>Angled 45&amp;deg;</td></tr><tr><td>Surgical Scissors</td><td>Fine Science Tools&amp;nbsp;</td><td>14028-10</td><td></td></tr><tr><td>3 mm Vannas</td><td>Kent Scientific Corporation</td><td>INS600177</td><td>Straight blade</td></tr><tr><td>Hartman Hemostats&amp;nbsp;</td><td>Fine Scientific Tools</td><td>13002-10</td><td></td></tr><tr><td>Occluding filaments</td><td>Washington University</td><td>Specialty order</td><td>Filaments are silicone coated at Washington Univeristy and provided to UTSW facilities for a fee.&amp;nbsp;</td></tr><tr><td>Evans Blue</td><td>Sigma Aldritch</td><td>E2129-10G</td><td></td></tr><tr><td>Filter Paper&amp;nbsp;</td><td>Sigma Aldritch</td><td>WHA1001150</td><td>150 mm, circles, Grade 1&amp;nbsp;</td></tr><tr><td>Weigh Boats</td><td>Fisher Scientific</td><td>02-202-101</td><td>2.5&quot; diameter</td></tr><tr><td>0.9% Sodium Chloride Injection USP&amp;nbsp;</td><td>Baxter Pharmaceutics&amp;nbsp;</td><td>2B1321</td><td></td></tr><tr><td>0.3 cc insulin syringe with 29 gauge needle</td><td>Becton Dickinson Labware</td><td>309301</td><td></td></tr><tr><td>Flat bottom restrainer&amp;nbsp;</td><td>Braintree Scientific&amp;nbsp;</td><td>FB&amp;nbsp;M</td><td>2.0&quot; diameter</td></tr><tr><td>TTC</td><td>Sigma</td><td>T8877</td><td></td></tr><tr><td>10x PBS, pH 7.4</td><td>Fisher Scientific</td><td>BP399-20</td><td></td></tr><tr><td>Water Bath</td><td>Multiple sellers&amp;nbsp;</td><td>Catalog number not available&amp;nbsp;</td><td>Scintillation tubes with TTC may be manually held under running warm water as an alternative to the water bath.</td></tr><tr><td>Styrofoam board</td><td>Multiple sellers&amp;nbsp;</td><td>Catalog number not available&amp;nbsp;</td><td></td></tr><tr><td>Large Syringe Kit</td><td>PumpSystems Inc</td><td>P-SYRKIT-LG</td><td></td></tr><tr><td>Perfusion Pump</td><td>PumpSystems Inc</td><td>NE-300&amp;nbsp;</td><td></td></tr><tr><td>60 cc syringe</td><td>Fisher Scientific</td><td>NC9203256</td><td></td></tr><tr><td>27 gauge winged infusion set</td><td>Kawasumi Laboratories, Inc</td><td>D3K1-25G 1</td><td></td></tr><tr><td>20 ml scintillation vial</td><td>Fisher Scientific</td><td>50-367-126</td><td></td></tr><tr><td>Stainless steel spatula</td><td>Fisher Scientific</td><td>14-373-25A</td><td></td></tr><tr><td>Alto acrylic 1.0 mm mouse brain, coronal</td><td>CellPoint Scientific&amp;nbsp;</td><td>Catalog number not available&amp;nbsp;</td><td></td></tr><tr><td>0.21 mm stainless steel blades, 25 pk</td><td>CellPoint Scientific&amp;nbsp;</td><td>Catalog number not available&amp;nbsp;</td><td>Reusable cryostat blades are an inexpensive alternative.</td></tr><tr><td>4% paraformaldehyde</td><td>Santa Cruz Biotechnology&amp;nbsp;</td><td>SC-281692</td><td></td></tr><tr><td>Superfrost microscope slides&amp;nbsp;</td><td>Fisher Scientific</td><td>12-550-15</td><td></td></tr><tr><td>HP Scanjet G4050</td><td>Multiple sellers&amp;nbsp;</td><td>Catalog number not available&amp;nbsp;</td><td>Other commercial scanners are suitable for this step in the protocol.</td></tr><tr><td>ImageJ&amp;nbsp;</td><td>National Institute of Health</td><td>Catalog number not available&amp;nbsp;</td><td></td></tr><tr><td>Analytical Balance</td><td>Mettler Toledo&amp;nbsp;</td><td>XSE 205U</td><td></td></tr><tr><td>Precision Compact Oven&amp;nbsp;&amp;nbsp;</td><td>Thermo Scientific&amp;nbsp;</td><td>PR305225M</td><td></td></tr><tr><td>1.7 ml microcentrifuge tubes (Eppendorfs)</td><td>Denville Scientific&amp;nbsp;</td><td>C2170</td><td></td></tr><tr><td>Formamide</td><td>Fisher Scientific</td><td>BP228-100</td><td></td></tr><tr><td>96-well plates</td><td>Fisher Scientific</td><td>07-200-9</td><td></td></tr><tr><td>Epoch Microplate Spectrophotometer&amp;nbsp;</td><td>BioTek&amp;nbsp;</td><td>Catalog number not available&amp;nbsp;</td><td></td></tr></tbody></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 ARRIVE guidelines,28 Table 2 shows surgical parameters. Indirect infarct volumes, hemispheric swelling (i.e., edema), and Evans blue (EB) extravasations are shown in Figure 2. All data were analyzed with unpaired t-tests (mean, standard deviation shown), and outliers detected with a False Discovery Rate less than 1% (Graphpad Prism), which removed 4 outliers (2 from 21%, 2 from RHP) from the EB data set.
In this cohort, RHP reduced infarct volumes by almost half (33.9 &#177; 23.0 mm3) compared to 21% O2-treated mice (62.6 &#177; 42.6 mm3), but this difference was non-significant (p = 0.10). However, an a priori Power analysis predicted a required n = 10 per group, which we did not achieve with the RHP-treated mice after outliers were removed. This should be considered in designing future experiments using RHP. There was no effect of RHP on hemispheric swelling, a metric derived from the TTC volume analysis that could be equated with edema.6 RHP-treated mice exhibited a trend (p = 0.05) in the reduction of BBB disruption within the ischemic hemisphere normalized to the contralateral hemisphere. Figure 2D shows a range of values for EB leak for both treatment groups.
<img alt="Figure 1" src="/files/ftp_upload/52675/52675fig1.jpg" /> 
Figure 1: Custom-designed RHP chambers. Upper panel depicts the custom-built flowmeters for individual monitoring of air flow in up to four chambers. Air impermeable tubing is shown leaving the flowmeter and attaching to the inlet port of the 15 L induction chamber in the lower panel. Outlet port remains open during RHP to allow for air circulation.
<img alt="Figure 2" src="/files/ftp_upload/52675/52675fig2.jpg" /> 
Figure 2: RHP reduces infarct volumes and BBB disruption. RHP (A) reduces infarct volumes by 46% compared to control mice, but has (B) no affect on hemispheric swelling derived from the TTC data. (C) In the same animals, RHP reduces BBB disruption (p = 0.05) as defined by Evans Blue leak normalized to the contralateral hemisphere. (D) Representative TTC stains from both RHP-treated and 21% O2-exposed control mice with corresponding infarct volumes and Evans Blue leak shown below each sample. Values shown are mean +/- standard deviation.

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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

Nanyang Technological University

Research

JoVE Journal

Medicine

13.7K Views.  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.

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

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

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

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

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

2-Vessel Occlusion/Hypotension: A Rat Model of Global Brain Ischemia

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ischemia produces damage to specific brain regions shown to be highly sensitive to ischemia 1. Hippocampal neurons have higher sensitivity to ischemic insults compared to other cell populations, and specifically, the CA1 region of the hippocampus is particularly vulnerable to ischemia/reperfusion 2.
The design of therapeutic interventions, or study of mechanisms involved in cerebral damage, requires a model that produces damage similar to the clinical condition and in a reproducible manner. Bilateral carotid vessel occlusion with hypotension (2VOH) is a model that produces reversible forebrain ischemia, emulating the cerebral events that can occur during cardiac arrest and resuscitation. We describe a model modified from Smith et al. (1984) 2, as first presented in its current form in Sanderson, et al. (2008) 3, which produces reproducible injury to selectively vulnerable brain regions 3-6. The reliability of this model is dictated by precise control of systemic blood pressure during applied hypotension, the duration of ischemia, close temperature control, a specific anesthesia regimen, and diligent post-operative care. An 8-minute ischemic insult produces cell death of CA1 hippocampal neurons that progresses over the course of 6 to 24 hr of reperfusion, while less vulnerable brain regions are spared. This progressive cell death is easily quantified after 7-14 days of reperfusion, as a near complete loss of CA1 neurons is evident at this time.
In addition to this brain injury model, we present a method for CA1 damage quantification using a simple, yet thorough, methodology. Importantly, quantification can be accomplished using a simple camera-mounted microscope, and a free ImageJ (NIH) software plugin, obviating the need for cost-prohibitive stereology software programs and a motorized microscopic stage for damage assessment.

Bilateral carotid occlusion coupled with systemic hypotension produces global brain ischemia in the rat, resulting in damage to the hippocampus with reproducible severity. Animal subjects are impaired with predictable patterns of brain damage, they recover expediently, and mortality rates are comparatively low.

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ...

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

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

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

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

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

Neuroscience

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 ...

Induction of Ischemic Stroke and Ischemia-reperfusion in Mice Using the Middle Artery Occlusion Technique and Visualization of Infarct Area

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of stroke on tissue injury and to evaluate the effectiveness of therapeutic interventions, several experimental models in a variety of species were developed. They include complete global cerebral ischemia, incomplete global ischemia, focal cerebral ischemia, and multifocal cerebral ischemia. The model described in this protocol is based on the middle cerebral artery occlusion (MCAO) and is related to the focal ischemia category. This technique produces consistent focal ischemia in a strictly defined region of the hemisphere and is less invasive than other methods. The procedure described is performed on mice, given the availability of several genetic variants and the high number of tests standardized for mice to aid in the behavioral and neurodeficit evaluation.

We describe a mouse model of stroke induced by the occlusion of the middle cerebral artery using a silicone coated suture. The protocol can be applied to induce permanent occlusion or a temporary ischemia, followed by reperfusion.

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of ...

Experimental Models to Study the Neuroprotection of Acidic Postconditioning Against Cerebral Ischemia

Stroke is one of the leading causes of mortality and disability worldwide, with limited therapeutic approaches. As an endogenous strategy for neuroprotection, postconditioning treatments have proven to be promising therapies against cerebral ischemia. However, complicated procedures and potential safety issues limit their clinical application. To overcome these disadvantages, we have developed acidic postconditioning (APC) as a therapy for experimental focal cerebral ischemia. APC refers to the mild acidosis treatment by inhaling CO2 during reperfusion following ischemia. Here we present two models to execute APC in vitro and in vivo, respectively. The oxygen-glucose deprivation (OGD) treatment of mice and the corticostriatal occlusion and middle cerebral artery occlusion (MCAO) of mice were employed to mimic cerebral ischemia. APC can be simply achieved by transferring brain slices to acidic buffer bubbled with 20% CO2, or by mice inhaling 20% CO2. APC showed significant protective effects against cerebral ischemia, as reflected by tissue viability and brain infarct volume.

Acidic postconditioning protects against cerebral ischemia. Here we present two models to execute APC. They are achieved respectively by transferring corticostriatal slices to acidic buffer after oxygen-glucose deprivation in vitro and by inhaling 20% CO2 after middle cerebral artery occlusion in vivo.

Stroke is one of the leading causes of mortality and disability worldwide, with limited therapeutic approaches. As an endogenous strategy for ...

Assessing Neuroprotective Effects of Glycyrrhizae Radix et Rhizoma Extract Using a Transient Middle Cerebral Artery Occlusion Mouse Model

Ischemia followed by reperfusion of cerebral blood flow after a stroke leads to the death of nerve cells and loss of brain tissue. The most commonly used animal model for studying stroke is the middle cerebral artery occlusion (MCAO) model. Previous research studies have reported different infarct sizes even when the same experimental animal species was used under similar MCAO conditions. Therefore, we developed an improved experimental method to address this discrepancy. Mice were subjected to MCAO using a filament as the occlusion material to mimic human stroke conditions and filament thickness was optimized to establish more reproducible infarction volume. Mice treated with a methanol extract of Glycyrrhizae Radix et Rhizome (GRex) following stroke induction showed a significantly decreased total infarction volume and increased number of surviving cells relative to the untreated control group. This modified experimental protocol successfully and reproducibly demonstrated the beneficial effect of GRex on ischemic stroke.

In this study, we modify an existing experimental method to obtain more reproducible results, by establishing a middle cerebral artery occlusion (MCAO) mouse model. Oral administration of Glycyrrhizae Radix et Rhizome (GR) methanol extract (GRex), following stroke induction, significantly decreased total infarction volume relative to the untreated control group.

Ischemia followed by reperfusion of cerebral blood flow after a stroke leads to the death of nerve cells and loss of brain tissue. The most commonly used ...

Two-vessel Occlusion Mouse Model of Cerebral Ischemia-reperfusion

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse model is useful for investigating the pathophysiology of cerebral ischemia-reperfusion and determining potential therapeutic strategies for patients with stroke. Variations in the anatomy of the circle of Willis of C57BL/6 mice affects their infarct volume after cerebral-ischemia-induced injury. Studies have indicated that distal MCA occlusion (MCAO) can overcome this problem and result in a stable infarct size. In this study, we establish a two-vessel occlusion mouse model of cerebral ischemia-reperfusion through the interruption of the blood flow to the right MCA. We distally ligate the right MCA and right common carotid artery (CCA) and restore blood flow after a certain period of ischemia. This ischemia-reperfusion injury induces an infarct of stable size and a behavioral deficit. Peripheral immune cells infiltrate the ischemic brain within the 24 h infiltration period. Additionally, the neuronal loss in the cortical area is less for a longer reperfusion duration. Therefore, this two-vessel occlusion model is suitable for investigating the immune response and neuronal recovery during the reperfusion period after cerebral ischemia.

A mouse model of cerebral ischemia-reperfusion is established to investigate the pathophysiology of stroke. We distally ligate the right middle cerebral artery and right common carotid artery and restore blood flow after 10 or 40 min of ischemia.

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse ...

A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. To investigate pathological mechanisms of retinal ischemia, relevant experimental models need to be developed. Anatomically, a main retinal blood supplying vessel is the ophthalmic artery (OpA) and OpA originates from the internal carotid artery of the common carotid artery (CCA). Thus, disruption of CCA could effectively cause retinal ischemia. Here, we established a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion (tBCCAO) to tie the right CCA with 6-0 silk sutures and to occlude the left CCA transiently for 2 seconds via a clamp, and showed that tBCCAO could induce acute retinal ischemia leading to retinal dysfunction. The current method reduces reliance on surgical instruments by only using surgical needles and a clamp, shortens occlusion time to minimize unexpected animal death, which is often seen in mouse models of middle cerebral artery occlusion, and maintains reproducibility of common retinal ischemic findings. The model can be utilized to investigate the pathophysiology of ischemic retinopathies in mice and further can be used for in vivo drug screening.

Here, we describe a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion using simple sutures and a clamp. This model can be useful for understanding the pathological mechanisms of retinal ischemia caused by cardiovascular abnormalities.

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. ...

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

Transient Middle Cerebral Artery Occlusion Model of Stroke

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

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

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

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

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

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