Funding was provided by the NIH K08 NS064094 and UCSF REAC grants. The authors would like to acknowledge Nikita Derguin, Zinalda Vexler, and Joel Faustino for their assistance in the development of this technique.

All animal research was approved by the University of California, San Francisco Committee on Animal Research and was performed in accordance with the Guide for the Care and Use of Laboratory Animals (US Department of Health and Human Services, Publication No. 85-23, 1985). Animals were closely monitored by veterinarians of the UCSF Institutional Animal Care and Use Committee (IACUC), accredited by AAALAC. One female Sprague-Dawley rat with an 8 day-old litter (10 pups per litter) was obtained. The mother and her pups were given food and water ad libitum and housed in a temperature- and light-controlled animal care facility with daily enrichment, per IACUC protocol, until the pups were 10 days old. All surgical instruments used in this procedure were autoclaved to ensure sterility. Sterility of instrument tips is maintained throughout the surgery.
1. Middle Cerebral Artery Occlusion
<ol>
	<li>Weigh the pup and ensure that it is the proper weight (19-21 g). Anesthetize the pup in 3% isoflurane in 100% O2 and ensure that there is no response to a foot pinch. Maintain the body surface temperature between 35.5 &#176;C and 37 &#176;C with the use of a heating pad under the surgical stage.</li>
	<li>Secure the animal in a supine position with tape across the shoulder regions. Using sterile cotton swabs, swab the anterior cervical area with povidone-iodine solution followed by a swab of 70% ethanol in double-distilled water, alternating between each solution for four swabs total.</li>
	<li>Locally infiltrate 0.25% bupivacaine into the planned incision site. Using a stereoscope, make a midline 5 to 7 mm anterior cervical incision to expose the common carotid artery (CCA). Place 2-4 retractors to keep the cavity open and the artery exposed.</li>
	<li>Locate the internal carotid artery (ICA), occipital artery (OA), and external carotid artery (ECA). Groom the arteries to get a clear view. Be careful not to disturb the vagus nerve. 
	NOTE: For sham-operated pups, the inicision is left open and the arteries are exposed, after which the incision is sutured closed. The total anesthesia time is equivalent to that of the occlusion surgery.</li>
	<li>Cut 1.5 cm of 6-0 silk braided suture thread. Unbraid the suture, pulling out single strands. Make sure that the single strands are neat and not frayed. 
	NOTE: If necessary, smoothe the frayed ends by dipping the single strand in sterile water and grooming the strand with forcep tips.</li>
	<li>While holding the suture strand with 45-degree forceps, move the forceps in a sweeping arc motion to go under the ICA so that the forcep tips emerge between the ICA and OA. 
	NOTE: If the dissection is done well, this step will be relatively easy. If the ICA and OA are touching, be careful not to rupture the arteries when using the forceps to isolate the ICA. If bleeding occurs, apply pressure to the artery with the forceps until the bleeding stops. Absorb the blood with a sterile swab.</li>
	<li>Grab the end of the suture strand that is held by the forceps and pull it so that the end is easy to access. Release the strand from the forceps and back the forceps out from under the ICA, reversing the motion in step 1.5 (Figure 1A).</li>
	<li>Tie a temporary ligature around the ICA at the base, closest to where it separates from the CCA. 
	NOTE: It is important to tie the knot so that the end of the strand that will be pulled to remove the knot is long enough (greater than 1 mm, less than 3 mm) to easily grasp with forceps, while ensuring that there is an appropriate amount of suture strand on the other side of the knot for retraction.</li>
	<li>Carefully retract the ICA laterally and use a clip to secure the strand to excess skin near the axilla region on the side opposite the incision. Ensure that this retracting strand is taut enough to stop blood flow before proceeding to the next step, to minimize the risk of uncontrolled bleeding. Observe that the artery is flat and pale. 
	NOTE: Do not over-retract, as it can cause a partial or full tear of the ICA. The retraction can be adjusted just before making the arteriotomy by pulling on the strand on the side opposite to the clip.</li>
	<li>Use 45&#176; forceps to grasp another unbraided suture strand and loop it under and around the ICA, as in step 1.5. Position this strand lateral to the retraction strand (Figure 1B). 
	NOTE: This step can also be done prior to retraction. If unsure about the quality of retraction, this knot can be very loosely tied before the next step.</li>
	<li>Cut a 0.2 mm arteriotomy midway between the tied and untied ligatures, erring closer to the tied ligature. 
	NOTE: Blood remaining in the tied-off artery may empty through the arteriotomy but should not exceed 5 &#181;L. If bleeding persists, carefully pull the retraction strand to increase retraction, with care to avoid damaging the artery from excessive tension.</li>
	<li>Using a metric ruler, measure the occluding suture and cut the suture with an extra allowance of 2-3 mm for the removal of the occluder during reperfusion. Hold the occluder with 45-degree forceps and use straight forceps to create a bend at the appropriate length to reach the MCA, marking a stopping point for advancement. 
	NOTE: A 10 mm occlusion length from the silicone tip end to the bend is used for P10 Sprague Dawley or Long Evans rat pups in this weight range.</li>
	<li>Using 45&#176; forceps, feed the silicone-coated nylon occlusion suture into the arteriotomy and advance the suture to the bend that marks the pre-determined distance to the MCA (Figure 1C). Ensure that advancement feels smooth; immediately stop advancement if resistance is felt. During advancement, aim the suture in a direction that is parallel to the CC/ECA, towards the head. 
	NOTE: If the suture is advanced dorsally, toward the animal&#39;s spine, it may run into the pterygopalatine artery (PTA). If resistance is felt after 3-5 mm of advancement, the suture has hit the PTA junction. Back the occluder out of the artery until the silicone head is near the arteriotomy before adjusting the advancement direction. It is not necessary to completely remove the occluder from the arteriotomy.</li>
	<li>Secure the occluder by tying a temporary ligature, using the strand from step 1.10 (Figure 1D).</li>
	<li>Remove the retractor clip. Trim the strands of both of the temporary ligatures so that the strand of the knot that is pulled to remove the knot is easy to grasp with straight forceps and is longer than the strand that is pulled to tighten the knot. 
	NOTE: The strands must be short enough so that they do not tangle in the cavity after closure.</li>
	<li>Remove the retractors and close the cavity using 6-0 braided silk to create three to four interrupted sutures.</li>
	<li>Remove the pup from anesthesia and place it on a heating pad in room air. Monitor the pup until it has regained sufficient consciousness to maintain sternal recumbency and ensure that it has fully recovered before returning it to the dam. Ensure that the pup maintains a body surface temperature between 35.5 &#176;C and 37 &#176;C.</li>
	<li>During occlusion, diffusion-weighted magnetic resonance imaging (DW-MRI) under isoflurane anesthesia can be used to verify the appropriate induction of injury. Figure 2 demonstrates ongoing ischemic injury detected by DW-MRI during tMCAO9. 
	NOTE: Diffusion-weighted spin echo planar imaging is performed 10-15 min prior to reperfusion. The entire brain is imaged with serial 2-mm-thick coronal sections using the following pulse sequence settings: TR/TE=5,000/60 ms, 4 averages, field of view = 35 mm, data matrix = 128x128, diffusion gradient duration = 20 ms, separation = 29.7 ms, amplitude = 70mT/m, and b-factor = 1,045 s/mm2. Animals exhibiting a lack of cortical involvement or atypical ischemic injury, such as in the brainstem, are excluded.</li>
</ol>
2. Reperfusion
NOTE: Occlusion is performed for 3 h to cause a moderate-to-severe amount of injury involving the striatum and cortex.
<ol>
	<li>At approximately 2 h and 50 min following occlusion of the MCA, anesthetize the pup in 3% isoflurane in 100% O2. Maintain a body surface temperature between 35.5 &#176;C and 37 &#176;C with the use of a heating pad under the surgical stage.</li>
	<li>Remove the interrupted sutures from step 1.16 and locate the junction, which is marked by the two ligatures and the tail end of the occluding suture. 
	NOTE: Sham-operated animals are induced and remain under anesthesia for an equivalent time period to the occluded animals. The incision is once again opened and sutured closed. For sham-operated animals, proceed to step 2.11.</li>
	<li>Carefully untie the most lateral knot previously tied in step 1.14 by pulling the longer strand. 
	NOTE: If there is resistance in untying the knot, stop to ensure that the correct strand of the knot is being pulled. If resistance continues, increase magnification for a better view. Be cautious when untying, as it is possible to damage the artery during this step.</li>
	<li>Remove the suture strand from the cavity.</li>
	<li>At exactly 3 h following MCA occlusion, slowly back the occluding suture out of the artery. There will be no resistance. 
	NOTE: In most cases, there will be no bleeding. If a small amount of bleeding occurs, apply pressure to the artery at the arteriotomy site.</li>
	<li>Use forceps to apply pressure to the arteriotomy, as the next step restores ICA blood flow for reperfusion.</li>
	<li>Carefully untie the medial knot using the same method as in step 2.3.</li>
	<li>Remove the suture strand from the body and apply a hemostatic agent to the arteriotomy to stop bleeding.</li>
	<li>Ensure that the bleeding has stopped. 
	NOTE: Return of the artery&#39;s original shape and red color confirms that ICA blood flow has been restored.</li>
	<li>Remove the retractors and close the cavity with three to four interrupted sutures of 6-0 braided silk.</li>
	<li>Remove the pup from anesthesia and place it on a heating pad in room air. Monitor it until it has regained sufficient consciousness to maintain sternal recumbency and ensure full recovery before returning it to the dam. Ensure that the pup maintains a body surface temperature between 35.5 &#176;C and 37 &#176;C.</li>
	<li>Inspect the pups daily for 5 days. Recprd their weights and inspect the incision sites closely for appropriate healing. Remove the sutures after 7-14 days. Post-tMCAO injury can be imaged with MRI, if desired9. 
	NOTE: Animals may lose up to 1 g of weight on the first day but will typically regain the weight without intervention and be within a similar weight range to controls by days 4-5. Rarely, a pup may lose excessive weight or have difficulty feeding, requiring oral gavage feeds for 2-3 days.</li>
	<li>At P21, injury can be reliably assessed with sensorimotor behavioral tests, such as rotarod or cylinder rearing. Cognitive testing can be performed as early as 4-6 weeks of age using assessments such as novel object recognition or the Morris water maze13.</li>
	<li>Euthanize the rats by intraperitoneal injection of Euthasol (50 mg/kg) for brain harvest11,12.</li>
	<li>Histological analysis to assess injury volume or response to interventions can be performed with cresyl violet or H&#38;E staining (Figure 3)10.</li>
</ol>

<ol>
	<li>Grunt, S., 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=Incidence+and+outcomes+of+symptomatic+neonatal+arterial+ischemic+stroke.">Incidence and outcomes of symptomatic neonatal arterial ischemic stroke.</a> Pediatrics. 135 (5), 1220-1228 (2015).</li><li>Rice, J. E., Vannucci 3rd, R. C., Brierley, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+immaturity+on+hypoxic-ischemic+brain+damage+in+the+rat.">The influence of immaturity on hypoxic-ischemic brain damage in the rat.</a> Ann Neurol. 9 (2), 131-141 (1981).</li><li>Koizumi, J., Yoshida, Y., Nakazawa, T., Ooneda, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+studies+of+ischemic+brain+edema.+I:+a+new+experimental+model+of+cerebral+embolism+in+rats+in+which+recirculation+can+be+introduced+in+the+ischemic+area.">Experimental studies of ischemic brain edema. I: a new experimental model of cerebral embolism in rats in which recirculation can be introduced in the ischemic area.</a> Jpn J Stroke. 8 (8), (1986).</li><li>Longa, E. Z., Weinstein, P. R., Carlson, S., Cummins, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+middle+cerebral+artery+occlusion+without+craniectomy+in+rats.">Reversible middle cerebral artery occlusion without craniectomy in rats.</a> Stroke. 20 (1), 84-91 (1989).</li><li>Labat-gest, V., Tomasi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photothrombotic+ischemia:+a+minimally+invasive+and+reproducible+photochemical+cortical+lesion+model+for+mouse+stroke+studies.">Photothrombotic ischemia: a minimally invasive and reproducible photochemical cortical lesion model for mouse stroke studies.</a> J Vis Exp. (76), (2013).</li><li>Renolleau, S., Aggoun-Zouaoui, D., Ben-Ari, Y., Charriaut-Marlangue, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+of+transient+unilateral+focal+ischemia+with+reperfusion+in+the+P7+neonatal+rat:+morphological+changes+indicative+of+apoptosis.">A model of transient unilateral focal ischemia with reperfusion in the P7 neonatal rat: morphological changes indicative of apoptosis.</a> Stroke. 29 (7), 1454-1460 (1998).</li><li>Perlman, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intervention+strategies+for+neonatal+hypoxic-ischemic+cerebral+injury.">Intervention strategies for neonatal hypoxic-ischemic cerebral injury.</a> Clin Ther. 28 (9), 1353-1365 (2006).</li><li>Derugin, N., Ferriero, D. M., Vexler, Z. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+reversible+focal+cerebral+ischemia:+a+new+model.">Neonatal reversible focal cerebral ischemia: a new model.</a> Neurosci Res. 32 (4), 349-353 (1998).</li><li>Gonzalez, F. F., 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=Erythropoietin+increases+neurogenesis+and+oligodendrogliosis+of+subventricular+zone+precursor+cells+after+neonatal+stroke.">Erythropoietin increases neurogenesis and oligodendrogliosis of subventricular zone precursor cells after neonatal stroke.</a> Stroke. 44 (3), 753-758 (2013).</li><li>Larpthaveesarp, A., Georgevits, M., Ferriero, D. M., Gonzalez, F. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+erythropoietin+therapy+improves+histological+and+behavioral+outcomes+after+transient+neonatal+stroke.">Delayed erythropoietin therapy improves histological and behavioral outcomes after transient neonatal stroke.</a> Neurobiol Dis. , (2016).</li><li>Rutherford, M. A., Ramenghi, L. A., Cowan, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+stroke.">Neonatal stroke.</a> Arch Dis Child Fetal Neonatal Ed. 97 (5), 377-384 (2012).</li><li>van der Aa, N. E., Benders, M. J., Groenendaal, F., de Vries, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+stroke:+a+review+of+the+current+evidence+on+epidemiology,+pathogenesis,+diagnostics+and+therapeutic+options.">Neonatal stroke: a review of the current evidence on epidemiology, pathogenesis, diagnostics and therapeutic options.</a> Acta Paediatr. 103 (4), 356-364 (2014).</li><li>Gonzalez, F. F., 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=Erythropoietin+sustains+cognitive+function+and+brain+volume+after+neonatal+stroke.">Erythropoietin sustains cognitive function and brain volume after neonatal stroke.</a> Dev Neurosci. 31 (5), 403-411 (2009).</li><li>Dudink, J., 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=Evolution+of+unilateral+perinatal+arterial+ischemic+stroke+on+conventional+and+diffusion-weighted+MR+imaging.">Evolution of unilateral perinatal arterial ischemic stroke on conventional and diffusion-weighted MR imaging.</a> AJNR Am J Neuroradiol. 30 (5), 998-1004 (2009).</li><li>Derugin, N., 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=Magnetic+resonance+imaging+as+a+surrogate+measure+for+histological+sub-chronic+endpoint+in+a+neonatal+rat+stroke+model.">Magnetic resonance imaging as a surrogate measure for histological sub-chronic endpoint in a neonatal rat stroke model.</a> Brain Res. 1066 (1-2), 46-56 (2005).</li><li>Dzietko, M., Wendland, M., Derugin, N., Ferriero, D. M., Vexler, Z. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+resonance+imaging+(MRI)+as+a+translational+tool+for+the+study+of+neonatal+stroke.">Magnetic resonance imaging (MRI) as a translational tool for the study of neonatal stroke.</a> J Child Neurol. 26 (9), 1145-1153 (2011).</li><li>Mancuso, A., Derugin, N., Hara, K., Sharp, F. R., Weinstein, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mild+hypothermia+decreases+the+incidence+of+transient+ADC+reduction+detected+with+diffusion+MRI+and+expression+of+c-fos+and+hsp70+mRNA+during+acute+focal+ischemia+in+rats.">Mild hypothermia decreases the incidence of transient ADC reduction detected with diffusion MRI and expression of c-fos and hsp70 mRNA during acute focal ischemia in rats.</a> Brain Res. 887 (1), 34-45 (2000).</li><li>Kasdorf, E., Hyperthermia Perlman, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation,+and+Perinatal+Brain+Injury.">Inflammation, and Perinatal Brain Injury.</a> Pediatric Neurology. 49 (1), 8-14 (2013).</li><li>Bouley, J., Fisher, M., Henninger, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+between+coated+vs.+uncoated+suture+middle+cerebral+artery+occlusion+in+the+rat+as+assessed+by+perfusion/diffusion+weighted+imaging.">Comparison between coated vs. uncoated suture middle cerebral artery occlusion in the rat as assessed by perfusion/diffusion weighted imaging.</a> Neurosci Lett. 412 (3), 185-190 (2007).</li><li>Shimamura, N., Matchett, G., Tsubokawa, T., Ohkuma, H., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+silicon-coated+nylon+suture+to+plain+nylon+suture+in+the+rat+middle+cerebral+artery+occlusion+model.">Comparison of silicon-coated nylon suture to plain nylon suture in the rat middle cerebral artery occlusion model.</a> J Neurosci Methods. 156 (1-2), 161-165 (2006).</li><li>Kirton, A., deVeber, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paediatric+stroke:+pressing+issues+and+promising+directions.">Paediatric stroke: pressing issues and promising directions.</a> Lancet Neurol. 14 (1), 92-102 (2015).</li><li>Nelson, K. 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The authors have nothing to disclose.

Critical steps within the protocol
First, it is important to maintain normothermia from the initiation of anesthesia until full recovery, as there are known effects of both hypothermia17 and hyperthermia18 on the progression of brain injury in both immature and mature animals. Second, while securing the animal and retracting the incision, optimal positioning to monitor breathing and to ensure that the trachea is free of compression is essential. Third, avoid squeezing or stretching the vagus nerve, as this may cause changes in heart rate with vagal stimulation. Fourth, because retraction of the ICA is necessary to control bleeding during the arteriotomy, attention must be paid to the degree of tension during retraction to avoid damaging the artery. If the artery does tear from retraction, or if there is a poor arteriotomy incision, the animal should be excluded from analysis due to the risk of hemorrhage and poor reperfusion.
Modifications and troubleshooting
Using MRI as a guide, the suture length may be optimized to ensure that the silicone tip properly occludes the MCA to create the focal ischemia. If MRI is not available, pups may be euthanized before reperfusion for dissection to visualize the placement of the suture. Adjust the suture length as needed. The pup weight highly correlates with the occluding suture length requirements. The occlusion time can be modified to adjust the degree of injury severity.
In addition, suture shape and length are critical. For P10 Sprague-Dawley and Long Evans rats weighing 19-21 g, 10 mm is the optimal length of insertion in our experience. Further insertion of the occluding suture may result in perforation of the MCA. Furthermore, the consistency in the shape of the occluding filament in each surgery will result in an increased consistency of injury pattern19,20. For this reason, we recommended using professionally-manufactured sutures for this specific purpose. It is also important to note that the injury pattern may differ between practitioners due to seemingly minute differences in technique.
Limitations of the technique
Performing this technique in a small, developing rodent requires significant experience. If performed correctly, the surgeon is able to cause a very consistent injury pattern across animals of different sizes and attain a survival rate greater than 95%. Furthermore, proper surgical tools are essential. Surgical instruments must be well maintained to ensure that all instrument tips approximate properly.
Significance of this technique with respect to existing or alternative methods
While hypoxia-ischemia, or the Rice-Vannucci model2, is most commonly used to study hypoxic-ischemic injury in the developing brain, it is important to note that this model of tMCAO is distinct from HI in that there is transient focal ischemia without global hypoxia, followed by a reperfusion phase when the obstruction is removed and blood flow is restored. This causes a more consistent and reproducible injury and is more clinically translational by causing an injury pattern similar to that seen in full-term neonatal stroke. This enables the study of focal injury patterns and compensatory responses in uninjured tissue.
Future applications after mastering this technique
This model is similar to the most common cause of stroke in human neonates, a transient occlusive thrombus that occurs during the perinatal period11,21. The etiology is not entirely clear and is most likely multifactorial, but it is presumed in most cases to result from emboli passing from the placenta11. In addition, many newborns with presumed perinatal stroke often present with later seizure activity or subtle focal neurological exam abnormalities22. This makes the use of a consistent, translational injury model to identify mechanisms of injury progression and possible therapeutic strategies crucial.

Stroke during the neonatal period is a significant cause of death and disability, occurring in as many as 1 in 2,300 live births1. This leads to altered central nervous system development and increased long-term morbidity, including increased incidence of epilepsy, cerebral palsy, mental retardation, and other types of motor or cognitive dysfunction. The lifelong effects of early stroke make translational animal models essential for examining the mechanisms of injury and repair in this population, including strategies to protect the injured brain or to enhance repair.
Different ischemia models have been used to study brain injury in adult animals, and while the Rice-Vannucci (Modified Levine)2 procedure is commonly used to study hypoxic-ischemic injury in the developing brain, focal ischemia-reperfusion is a distinct mechanism of injury causing focal injury, with an injured core and penumbra and uninjured remote tissue. The Koizumi3 and Longa4 models were developed in adult rats to achieve transient middle cerebral artery occlusion via the common carotid artery (CCA) and external carotid artery (ECA), respectively. In both models, permanent ligation and cauterization of artery branches are important to minimize bleeding and to streamline the surgical procedure, which also causes adverse effects on the animal&#39;s ability to feed and to gain weight following injury. Furthermore, there are distinct injury mechanisms in the immature brain and specific patterns of injury seen as a result.
More recently, photothrombotic stroke (Rose-Bengal method)5 and permanent MCA ligation6 have been used to study neonatal and adult stroke. Both photothrombotic stroke and MCA ligation create permanent changes in cerebral blood flow that result in a lack of reperfusion. Reperfusion is a critical component of the development and progression of focal injury, with increased excitotoxicity, free radical formation, and nitric oxide production leading to delayed cell death that involves signaling cascades that are distinct from the ischemic phase7. Hypoxia-ischemia involves permanent unilateral carotid ligation followed by global hypoxia, which also differs from the cause of hypoxic-ischemic injury in humans and does not cause a consistent focal injury pattern, making study of the injured core and penumbra more challenging.
We have previously described a non-hemorrhagic ischemia-reperfusion stroke model in the immature rat using transient middle cerebral artery occlusion (MCAO)8,9,10. This is a less invasive method that accesses and occludes the MCA through the internal carotid artery without permanent ligation or cauterization. This provides a model of injury similar to the most common cause of stroke in the perinatal period11,12. This ischemia-reperfusion model of injury results in damage to the ipsilateral striatum and parieto-temporal cortex. This model of tMCAO also allows control over the severity of injury by varying the duration of occlusion. Examination of signaling pathways and histological changes in the injured core and penumbra and in the uninjured ipsilateral and contralateral tissue can further elucidate the mechanisms of injury and repair in the immature brain. This study will demonstrate this important injury model for the developing brain.

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	<tbody>
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			<td>Henry Schein</td>
			<td>50033</td>
			<td>anesthetic, at 3%&#160;</td>
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			<td>Trinocular Surgioscope</td>
			<td>World Precision Instruments</td>
			<td>PSMT5N</td>
			<td></td>
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			<td>Heating pad</td>
			<td>Sunbeam</td>
			<td>000731-500-000</td>
			<td>low to medium setting</td>
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			<td>IR Thermometer</td>
			<td>Extech Instruments</td>
			<td>72-5270</td>
			<td></td>
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			<td>Retraction kit for small animals&#160;</td>
			<td>Fine Science Tools</td>
			<td>18200-20</td>
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			<td>CermaCut Scissors</td>
			<td>Fine Science Tools</td>
			<td>14958-09</td>
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			<td>Dumont #5SF Forceps</td>
			<td>Fine Science Tools</td>
			<td>112522-00</td>
			<td>2x</td>
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			<td>Dumont #5/45 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-35</td>
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			<td>B-2 Micro Clamp</td>
			<td>Fine Science Tools</td>
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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.

The severity of injury caused by tMCAO is highly dependent upon both the occlusion time and the experience of the surgeon. A 90-min occlusion often produces a mild to moderate injury pattern, while 3 h produces a moderate-to-severe injury. Severity of injury may be assessed through a variety of methods, including MRI, histology, or short- or long-term behavioral analyses. Figure 2 demonstrates an example of the DW-MRI performed 75 min into a 90-min occlusion, confirming ischemic injury involving the ipsilateral hemisphere. Diffusion weighted imaging demonstrates increased diffusion in the ipsilateral striatum and the majority of the ipsilateral cortex, without contralateral changes, during the acute ischemic phase. This correlates to a moderate level of long-term injury involving both the cortex and the deep gray matter.
Occlusion of the MCA results in cell death that begins in the striatum for less severe injury and develops worsening cortical and hippocampal injury for longer occlusion times and more severe injury. During optimization of the surgical technique, such as determining the suture insertion length, MRI is highly recommended, as it allows for the confirmation of proper suture placement and visualization of edema and injury progression during occlusion14,15,16. If MRI is not available, H&#38;E or cresyl violet staining are simple and reliable histological methods to determine injury morphology and can be used at both early and late time points after the tMCAO. Figure 3 demonstrates a moderate-to-severe injury pattern on histopathological examination following a 3 h occlusion, demonstrating cyst formation and volume in the ipsilateral striatum and cortex.
Unbiased stereological quantification of cresyl-violet-stained sections to calculate the ratio of ipsilateral to contralateral hemispheric volume confirms ipsilateral ischemic-reperfusion injury. Specifically, after anesthetizing the animal with Euthasol, the brain was harvested by transcardiac perfusion in 0.1 M PBS with 4% paraformaldehyde. Following overnight postfixation and equilibration in 30% sucrose, the entire brain was sectioned at 50 &#181;m intervals, and every twelfth section was selected, mounted, and stained with cresyl violet10.
Even with mild injury, locomotor changes, such as circling and hemiparesis, are noted during the occlusion period. With more severe injury, these changes will persist after reperfusion. Additional behavioral testing can be used to assess the severity of injury, including rotarod or cylinder rearing testing for sensorimotor function and the Morris water maze for cognitive function13.
<img alt="Figure 1" src="/files/ftp_upload/54830/54830fig1.jpg" /> 
Figure 1: Live Surgical Images of the tMCAO Procedure. (A) The first suture strand is looped around the ICA, as detailed in step 1.6. (B) The first temporary ligature is tied and the ICA is retracted. The second suture strand is looped around the ICA, lateral to the first suture strand, as detailed in step 1.9. (C) The silicone coated occlusion suture is fed into the arteriotomy site, as detailed in step 1.12. (D) The second temporary ligature is tied to secure the occluder in place, as detailed in step 1.14. Scale bar = 1 mm.
<img alt="Figure 2" src="/files/ftp_upload/54830/54830fig2.jpg" /> 
Figure 2: MRI During Occlusion Demonstrates the Appropriate Unilateral Injury. Anterior to posterior, coronal image slices of the DW-MRI, performed during a 90 min occlusion, demonstrate increased diffusion involving the ipsilateral hemisphere (arrows), which is consistent with ongoing ischemic injury in the acute phase. Reprinted with permission from Stroke11. <a href="http://cloudflare.jove.com/files/ftp_upload/54830/54830fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54830/54830fig3.jpg" /> 
Figure 3: Unilateral Injury Involving the Striatum and Cortex at 4 weeks following tMCAO. Posterior to anterior, cresyl-violet-stained coronal brain sections (each 50 &#181;m) harvested from P38 animals demonstrate fairly severe injury (the arrows show ipsilateral cyst formation and reduced cortical and striatal volume) following a 3 h tMCAO at P10. The round hole on the left side represents a contralateral hemisphere identifier. Scale bar = 5 mm. Reprinted with permission from Neurobiology of Disease12. <a href="http://cloudflare.jove.com/files/ftp_upload/54830/54830fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats at JoVE.com

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

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 ...

transient-middle-cerebral-artery-occlusion-model-neonatal-stroke-p10

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10.5K Views.  University of California, San Francisco. 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.

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

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 ...

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

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

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

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

Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in 20091, 2. Several models of cerebral ischemia have been developed to mimic the human condition of stroke. It has been suggested that up to 80% of all strokes result from ischemic damage in the middle cerebral artery (MCA) area3. In the early 1990s, endothelin-1 (ET-1) 4 was used to induce ischemia by applying it directly adjacent to the surface of the MCA after craniotomy. Later, this model was modified 5 by using a stereotaxic injection of ET-1 adjacent to the MCA to produce focal cerebral ischemia. The main advantages of this model include the ability to perform the procedure quickly, the ability to control artery constriction by altering the dose of ET-1 delivered, no need to manipulate the extracranial vessels supplying blood to the brain as well as gradual reperfusion rates that more closely mimics the reperfusion in humans5-7. On the other hand, the ET-1 model has disadvantages that include the need for a craniotomy, as well as higher variability in stroke volume8. This variability can be reduced with the use of laser Doppler flowmetry (LDF) to verify cerebral ischemia during ET-1 infusion. Factors that affect stroke variability include precision of infusion and the batch of the ET-1 used6. Another important consideration is that although reperfusion is a common occurrence in human stroke, the duration of occlusion for ET-1 induced MCAO may not closely mimic that of human stroke where many patients have partial reperfusion over a period of hours to days following occlusion9, 10. This protocol will describe in detail the ET-1 induced MCAO model for ischemic stroke in rats. It will also draw attention to special considerations and potential drawbacks throughout the procedure.

Several animal models of cerebral ischemia have been developed to simulate the human condition of stroke. This protocol describes the endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAO) model for ischemic stroke in rats. In addition, important considerations, advantages, and shortcomings of this model are discussed.

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in ...

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

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

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

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

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 ...

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

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2 (i.e. room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...