This study was supported by the NIH grant NS074559 (to C. K.). We thank all collaborators who contributed to our research articles that the present methodology report is based upon.

This protocol is approved by the Institutional Animal Care and Use Committee (IACUC) of Emory University and follows the National Institutes of Health Guideline for Care and Use of Laboratory Animals.
1. Setup
<ol>
	<li>Prepare the surgical bed on warming pad connected with heat pump at 37 &#176;C for at least 15 min before the surgery. Place a neck roll using the barrel of 3 ml syringe on the surgical bed. Prepare the anesthesia gas with 2% isoflurane in medical air.</li>
	<li>Prepare autoclaved forceps, scissors, micro needle holders, hemostat, cotton swabs and sutures. Prepare tissue glue and eye ointment.</li>
	<li>Set up the hypoxia system and temperature controllers with heating lamp and rectal probe. Prepare hypoxia gas with 2% isoflurane in 7.5% O2 balanced by 92.5% N2.</li>
	<li>One hour before surgery, mice are analgesized by subcutaneous injection of a slow-release Meloxicam (4.0 mg/kg).</li>
</ol>
2. Transient Cerebral Hypoxia-ischemia (Figure 1B)
<ol>
	<li>Anesthetize 10-13 week old male C57BL/6 mice weighing 22 to 30 g in the anesthesia induction chamber with 3% isoflurane until the animal is unresponsive to foot squeeze, and then remove the hair on right neck using an electronic shaver.</li>
	<li>Place mice on the surgical bed connected with 2% isoflurane in medical air at flow rate of 2 L/min. Secure forelimbs stretched out along neck roll at sides using medical tape.</li>
	<li>Clean the surgical site for incision with betadine followed by alcohol and then cotton swabs.</li>
	<li>Under dissecting microscope, make a 0.5 cm right-cervical incision using a straight forceps and a micro scissors about 0.2 cm lateral from midline skin.</li>
	<li>Use a pair of fine serrated forceps to pull apart the fascia and tissue to expose the right common carotid artery (RCCA). Carefully separate the RCCA from the vagal nerve using a pair of fine smooth forceps.</li>
	<li>Live knot two precut 5-0 silk suture (releasable) on the RCCA, and then sew up the skin using 4-0 Nylon monofilament suture (Figure 1C).</li>
	<li>Apply eye ointment on both eyes to prevent dryness.</li>
	<li>Quickly transfer the mice to hypoxia system and put nose and mouth in face-mask with 2% isoflurane in 7.5% O2 at flow rate of &#160; 0.5-1 L/min for 30 min.
	<ol>
		<li>During hypoxia, use temperature controllers with heating lamps to control the rectal temperature at 37.5 &#177; 0.5 &#176;C. Monitor the respiratory rate at 80-120 breaths/min. The maintenance of the body temperature above 37 &#176;C during the hypoxia is important to create consistent brain infarction. Low respiratory rate usually happens after 20 min hypoxia. Remove the face-mask and allow normal air supply if the respiratory rate drops below to 40.&#160;This&#160;takes 1-2 min and does not count&#160;into the 30 min hypoxia duration.</li>
	</ol>
	</li>
	<li>After hypoxia, transfer mice to a surgical bed and release the two sutures from RCCA. Close the wound using tissue glue, and then return mice to the cage. Exclude the animals if both of two live knots are unexpectedly released after hypoxia.</li>
	<li>Monitor the mice for 5-10 min to recover from hypoxia and anesthesia. Place the wetted food in the cage and return it to animal care facility. 
	Note: Animals showing mild to severe circling behavior at 24 hr after tHI are correlated with brain infraction. Most animals with seizure symptoms die before the&#160;24 hr timepoint&#160;after tHI.</li>
</ol>
3. Laser Speckle Contrast Imaging
Note: Although this is not an essential procedure of the tHI model, a two-dimensional laser speckle contrast imaging system16 can be used to characterize the changes of cerebral blood flow (CBF) during or after transient hypoxia-ischemia. To document the alterations of CBF under tHI, record immediately after the step 2.6. Alternatively, to compare CBF recovery after tHI insult, these procedures can be performed following the step 2.10.
<ol>
	<li>Place an anesthetized mouse&#160;in the prone position and perform a 1 cm-long midline incision on the scalp with the skull exposed but unopened.</li>
	<li>Monitor CBF in both cerebral hemispheres under a blood flow imager according to manufacturer&#8217;s protocol and start recording the cerebral blood flow immediately after the CCAO surgery (step 2.6). Continue for 50 min.</li>
	<li>Show CBF image with arbitrary units in a 16-color palette and analyze in real-time the selected regions using the MoorFLPI software following the manufacturer&#8217;s instructions (Figure 2).</li>
	<li>After recording the CBF image, close the scalp with tissue glue and return the animal to the cage.</li>
</ol>
4. tPA Administration
<ol>
	<li>Inject animals at the tail vein with the solvent or 10 mg/kg recombinant tPA (220-300 &#956;l&#160;of 1 mg/m tPA) at 0.5, 1, or 4 hr&#160;after tCCAo plus hypoxia (Figure 4).</li>
</ol>
5. Brain Damage Detection with Several Different Options
Note: To collect brain samples, euthanize the mice at 1, 4 or 24 hr after tHI.

<ol>
	<li>Perform quantification of infarct volume by in&#160;vivo 2,3,5-Triphenyltetrazolium chloride (TTC) method at 24 hrs after tHI insult as previous described.17 
	<ol>
		<li>Intraperitoneally inject animals with 1.4 M mannitol solution (~0.1 ml/g body weight) 30 min before transcardial perfusion. Transcardial perfuse mice with PBS followed by 10 ml&#160;of 2% TTC.</li>
		<li>Remove the brain of animals with surgical instruments after 10 min and place into 4% paraformaldehyde for fixation overnight and section into 1 mm thickness with a vibratome.</li>
		<li>Snap a series of four sectioned brain slides by digital microscope and quantify the infarct volume as the ratio of the infarcted area (white area in the right side) to the area of the undamaged, contralateral hemisphere using the ImageJ software.</li>
	</ol>
	</li>
	<li>Alternatively, perform thrombosis formation by immunofluorescence at 1 hr&#160;after tHI insult.
	<ol>
		<li>Freeze the fixed brain in O.C.T. compound and section the brains at 12 &#956;m thickness using a cryostat.</li>
		<li>Incubate the brain slide with rabbit anti-fibrinogen antibody (1:100) following by goat anti-rabbit Alexa Fluro 488 dye (1:200) to observe the fluorescence on a fluorescent microscope.</li>
	</ol>
	</li>
	<li>Alternatively, perform vessel obstruction by tail vein injection of 100 &#181;l&#160;2% Evans blue dye at 4 hr after tHI insult.
	<ol>
		<li>Euthanize the mice and quickly cut head to remove brains into 4% paraformaldehyde after Evans blue injection. Note: It takes 5-10 min&#160;for Evans blue circulation with blue color of both fore- and hind limbs.</li>
		<li>Section fixed brains at 100 &#956;m thickness using a sliding microtome and observe the fluorescence using a 680 nm emission filter on a fluorescent microscope.</li>
	</ol>
	</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+two+pathophysiologies+of+focal+brain+ischemia:+implications+for+translational+stroke+research.">The two pathophysiologies of focal brain ischemia: implications for translational stroke research.</a> J. Cereb. Blood Flow Metab. 32 (7), 1310-1316 (2012).</li><li>Macrae, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+stroke+research--advantages+and+disadvantages+of+the+most+common+rodent+models+of+focal+ischaemia.">Preclinical stroke research--advantages and disadvantages of the most common rodent models of focal ischaemia.</a> Br. J. Pharmacol. 164 (4), 1062-1078 (2011).</li><li>Niessen, F., Hilger, T., Hoehn, M., Hossmann, K. 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Pharm. 157 (7), 1154-1156 (2009).</li><li>Dirnagl, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+concerted+appeal+for+international+cooperation+in+preclinical+stroke+research.">A concerted appeal for international cooperation in preclinical stroke research.</a> Stroke. 44 (6), 1754-1760 (2013).</li><li>Khatri, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revascularization+end+points+in+stroke+interventional+trials:+recanalization+versus+reperfusion+in+IMS-I.">Revascularization end points in stroke interventional trials: recanalization versus reperfusion in IMS-I.</a> Stroke. 36 (11), 2400-2403 (2005).</li><li>Rosenberg, R. D., Aird, W. 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Stroke is a major health issue of growing significance for any society with an aging population. Globally, stroke is the second-leading cause of death with an estimated 5.9 million fatal events in 2010, equivalent to 11.1% of all deaths18. Stroke is also the third-leading cause of disability-adjusted life years (DALYs) lost globally in 2010, rising from the fifth position in 199019. These epidemiological data highlight the need of more effective therapies of acute (ischemic) stroke. However, despite...

Thrombolysis and recanalization is the most effective therapy of acute ischemic stroke in clinical practice1. Yet, the majority of preclinical neuroprotection research was performed in a transient mechanic obstruction model (intraluminal suture middle cerebral artery occlusion) that produces rapid recovery of cerebral blood flow upon removal of the vascular occlusion and shows little to no benefits by tPA thrombolysis. It has been suggested that the dubious choice of stroke models contributed, at least in part, to the difficulty in translating neuroprotective therapy to patients2,3. Hence, there is an increasing call for employing tPA-responsive thromboembolic stroke models in preclinical research, but such models also have technical problems (see Discussion)4-7. Here we describe a new thrombotic stroke model based on unilateral transient hypoxic-ischemic (tHI) insult and its responses to intravenous tPA therapy8.
The tHI stroke model was developed based on the Levine procedure (permanent ligation of the unilateral common carotid artery followed by exposure to transient hypoxia in a chamber) that was invented for experiments with adult rats in 19609. The original Levine procedure faded into obscurity because it only produced variable brain damage, but the same insult caused consistent neuropathology in rodent pups when it was re-introduced by Robert Vannucci and his colleagues as a model of neonatal hypoxic-ischemic encephalopathy (HIE) in 198110. In recent years, some investigators re-adapted the Levine-Vannucci model to adult mice by adjusting the temperature in the hypoxic chamber11. It is plausible that the inconsistent brain lesions in the original Levine procedure may arise from fluctuating body temperatures of adult rodents in the hypoxic chamber. To test this hypothesis, we modified the Levine procedure by administering hypoxic gas through a facemask, while maintaining the rodent core temperature at 37 &#176;C&#160;on the surgical table12. As expected, stringent body temperature control greatly increased the reproducibility of HI-induced brain pathology. The HI insult also triggers coagulation, autophagy, and gray- and white-matter injury13. Other investigators have also used the HI model to investigate post-stroke inflammatory responses14.
A unique feature of the HI stroke model is that it closely follows the Virchow&#8217;s triad of thrombus formation, including the stasis of blood flow, endothelial injury (e.g. due to HI-induced oxidative stress), and hypercoagulability (HI-induced platelet activation) (Figure 1A)15. As such, the HI model may capture some pathophysiological mechanisms relevant to real-world ischemic stroke. With this idea in mind, we further refined the HI model with reversible ligation of the unilateral common carotid artery (therefore to create a transient HI insult), and tested its responses to tPA thrombolysis with or without Edaravone. Edaravone is a free radical scavenger already approved in Japan to treat ischemic stroke within 24 hr of onset9. Our experiments showed that as brief as 30 min transient HI triggers thrombotic infarction, and that combined tPA-Edaravone treatment confers synergistic benefits8. Here we describe detailed surgical procedures and methodological considerations of the tHI model, which can be used to optimize reperfusion treatments of acute ischemic stroke.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>adult male mice</td>
			<td>Charles River</td>
			<td>C57BL/6&#160;</td>
			<td>10~13 weeks old (22~30 g)</td>
		</tr>
		<tr>
			<td>Mobile Laboratory Animal Anesthesia System</td>
			<td>VetEquip</td>
			<td>901807</td>
			<td>anesthesia</td>
		</tr>
		<tr>
			<td>Medical air (Compressed) air tank</td>
			<td>Airgas</td>
			<td>UN1002</td>
			<td>anesthesia</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Piramal Healthcare</td>
			<td>NDC 66794-013-25</td>
			<td>anesthesia</td>
		</tr>
		<tr>
			<td>Multi-Station Lab Animal AnesthesiaSystem</td>
			<td>Surgivet</td>
			<td>V703501</td>
			<td>hypoxia system</td>
		</tr>
		<tr>
			<td>7.5% O2 balanced by 92.5% N2 tank</td>
			<td>Airgas</td>
			<td>UN1956</td>
			<td>hypoxia system</td>
		</tr>
		<tr>
			<td>Temperature Controller with heating lamp&#160;</td>
			<td>Cole Parmer&#160;</td>
			<td>EW-89000-10</td>
			<td>temperature controllers</td>
		</tr>
		<tr>
			<td>Rectal probe</td>
			<td>Cole Parmer&#160;</td>
			<td>NCI-00141PG</td>
			<td>temperature controllers</td>
		</tr>
		<tr>
			<td>Dissecting microscope&#160;</td>
			<td>Olympus&#160;</td>
			<td>SZ40</td>
			<td>surgical setup</td>
		</tr>
		<tr>
			<td>Heat pump with warming pad</td>
			<td>Gaymar&#160;</td>
			<td>TP700</td>
			<td>surgical setup</td>
		</tr>
		<tr>
			<td>Fine curved forceps (serrated)</td>
			<td>FST</td>
			<td>11370-31</td>
			<td>surgical instrument</td>
		</tr>
		<tr>
			<td>Fine curved forceps (smooth)</td>
			<td>FST</td>
			<td>11373-12</td>
			<td>surgical instrument</td>
		</tr>
		<tr>
			<td>micro scissors</td>
			<td>FST</td>
			<td>15000-03</td>
			<td>surgical instrument</td>
		</tr>
		<tr>
			<td>micro needle holders</td>
			<td>FST</td>
			<td>12060-01</td>
			<td>surgical instrument</td>
		</tr>
		<tr>
			<td>Halsted-Mosquito hemostats</td>
			<td>FST</td>
			<td>13008-12</td>
			<td>surgical instrument</td>
		</tr>
		<tr>
			<td>5-0 silk suture&#160;</td>
			<td>Harvard Apparatus</td>
			<td>624143</td>
			<td>surgical supplies</td>
		</tr>
		<tr>
			<td>4-0 Nylon monofilament suture</td>
			<td>LOOK</td>
			<td>766B</td>
			<td>surgical supplies</td>
		</tr>
		<tr>
			<td>Tissue glue</td>
			<td>Abbott Laboratories</td>
			<td>NC9855218</td>
			<td>surgical supplies</td>
		</tr>
		<tr>
			<td>Puralube Vet ointment</td>
			<td>Fisher</td>
			<td>NC0138063&#160;</td>
			<td>eye dryness prevention&#160;</td>
		</tr>
		<tr>
			<td>MoorFLPI-2 blood flow imager</td>
			<td>Moor</td>
			<td>780-nm laser source</td>
			<td>Laser Speckle Contrast Imaging</td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td>Sigma</td>
			<td>M4125</td>
			<td>in-vivo TTC</td>
		</tr>
		<tr>
			<td>2,3,5-triphenyltetrazolium chloride (TTC)&#160;</td>
			<td>Sigma</td>
			<td>T8877</td>
			<td>in-vivo TTC</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Stoelting</td>
			<td>51425</td>
			<td>brain section for in-vivo TTC&#160;</td>
		</tr>
		<tr>
			<td>Digital microscope</td>
			<td>Dino-Lite</td>
			<td>AM2111</td>
			<td>whole-braina imaging</td>
		</tr>
		<tr>
			<td>O.C.T compound</td>
			<td>Sakura Finetek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>goat anti-rabbit Alexa Fluro 488</td>
			<td>Invitrogen</td>
			<td>A11008</td>
			<td>Immunohistochemistry</td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Vibratome</td>
			<td>ultrapro 5000</td>
			<td>brain section for IHC</td>
		</tr>
		<tr>
			<td>Evans blue</td>
			<td>Sigma</td>
			<td>E2129</td>
			<td>Detecting vascular perfusion</td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Electron Microscopy Sciences</td>
			<td>5000</td>
			<td>brain section for histology</td>
		</tr>
		<tr>
			<td>Avertin (2, 2, 2-Tribromoethanol)</td>
			<td>Sigma</td>
			<td>T48402</td>
			<td>euthanasia</td>
		</tr>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Olympus</td>
			<td>DP73</td>
			<td></td>
		</tr>
	</tbody>
</table>

a thrombotic stroke model based on transient cerebral hypoxia-ischemia

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

Two-dimensional laser speckle contrast imaging (LSCI)16 was used to compare the alterations of cerebral blood flow (CBF) by 30 min transient unilateral carotid occlusion (tCCAO), 30 min exposure to hypoxia (7.5% oxygen), and 30 min unilateral carotid ligation under hypoxia (tHI). This experiment revealed that tCCAO under normoxia suppressed the CBF on the carotid ligated hemisphere to ~50% of the baseline value, which quickly recovered to above 85% after release of the carotid occlusion (R in Figure 2A...

Watch this Scientific Journal Video about A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia at JoVE.com

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

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

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

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14.6K Views.  The Emory University School of Medicine. Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients. 