Name: a model for encephalomyosynangiosis treatment after middle cerebral artery occlusion-induced stroke in mice
Uploaded: 2022-06-22T15:00:00.000+00:00
Duration: 6 min 54 s
Description: Watch this Scientific Journal Video about A Model for Encephalomyosynangiosis Treatment after Middle Cerebral Artery Occlusion-Induced Stroke in Mice at JoVE.com

This work was supported by Research Excellence Program-UConn Health (to Ketan R Bulsara and Rajkumar Verma) and UConn Health start-up (to Rajkumar Verma).

All experiments were approved by the Institutional Animal Care and Use Committee of UConn Health and conducted in accordance with US guidelines. The following protocol should work in any species or strain of rodent. Here, 8- to 12-week-old, age- and weight-matched C57BL/6 wild-type male mice were used. Mice were fed standard chow diet and water ad libitum. Standard housing conditions were maintained at 72.3 &#176;F&#160;and 30%-70% relative humidity with a 12 h light/dark cycle.
1. Pre-surgery preparation
<ol>
	<li>Sterilize all instruments by autoclaving prior to surgery. Sanitize the operating surface with 70% ethanol and warm the operating surface to 37 &#176;C with an electric heating pad.</li>
	<li>Use an induction chamber to anesthetize the mouse with 4%-5% isoflurane for induction. Deliver 1.5%-2.0% isoflurane via nose cone for maintenance until the end of surgery. Ensure prior to surgery that the mouse is properly anesthetized by assessing the lack of a response to a firm hind foot pinch and loss of the postural reaction and righting reflex.</li>
	<li>Place the mouse on its left side on the operating surface and apply eye ointment to protect both eyes.</li>
	<li>Shave hair over the surgical field (i.e., right lateral cranium between eye and ear) with&#160;electric clippers. Clean the surgical field&#160;in concentric circles outward from the middle of the surgical site,&#160;with 70% ethanol followed by povidone solution, and repeat these steps 2x. 
	NOTE:&#160;Due to the surgery site being close to the eye, removing 150% of the area surrounding a surgical site may not be possible to avoid irritation or accidental injury to the eye.</li>
	<li>Administer a single dose of 0.25% bupivacaine (up to 8 mg/kg body weight) by subcutaneous injection as pre-operative analgesia at the site of surgery.</li>
	<li>Set up a surgical microscope at 4x magnification. The microscope is used for all surgical steps.</li>
</ol>
2. Surgery procedure
NOTE: The surgery steps are presented in Figure 1. For this protocol, three mice were allocated to sham group, three mice for EMS alone, 12 mice for MCAo, and 23 mice for MCAo + EMS group.
<ol>
	<li>MCAo surgery 
	NOTE: MCAo is a well-characterized model of ischemic stroke in rodents, as described by us and others12,13,14. The surgery steps are described in brief here. Focal transient cerebral ischemia was induced by a 60 min right MCAo under isoflurane anesthesia followed by reperfusion for 7 or 21 days.
	<ol>
		<li>Make a midline ventral neck incision followed by unilateral right MCAo by advancing a 10-11 mm long 6.0 silicone rubber-coated monofilament from the internal carotid artery bifurcation via an external carotid artery stump. In sham mice, perform identical surgeries except for the advancement of the suture into the internal carotid artery.</li>
		<li>Measure rectal temperatures using a temperature control system, maintaining the temperature at ~37 &#176;C during surgery with an automatic heating pad.</li>
		<li>Use laser Doppler flowmetry to measure cerebral blood flow prior to suture insertion by placing the Doppler probe against the lateral skull (corresponding to the MCA territory) and recording the value8. To confirm occlusion reduction to 15% of baseline cerebral blood flow, use the same procedure after the suture is advanced. To confirm reperfusion, use the same procedure after the suture is removed.</li>
		<li>Feed all animals with wet mash until sacrifice and/or 1 week after surgery to ensure adequate nutrition for chronic endpoints, as animals have rearing deficits after stroke.</li>
	</ol>
	</li>
	<li>EMS surgery
	<ol>
		<li>After 60 min of MCAo, randomize mice into MCAo-only or MCAo + EMS groups. Perform EMS 4 h after MCAo (MCAo + EMS group) or sham surgery for select experiments (EMS-only group). Change into a new pair of sterile surgical gloves before the surgery. 
		NOTE: The mice recovered from anesthesia after 60 minutes of&#160;MCAo and were re-anesthetized before the EMS surgery.</li>
		<li>For groups that receive EMS (MCAo + EMS or EMS-only groups), make a 10-15 mm skin incision with scissors, extending from 1-2 mm rostral to the right ear to 1-2 mm caudal to the right eye. 
		NOTE: Sterile scissors were used to prevent accidental damage to the temporalis muscles underneath.</li>
		<li>Retract skin flaps using clamps and visually identify the temporalis muscle and the skull.</li>
		<li>Bluntly dissect the temporalis muscle away from the skull using scissors with a spreading technique. Perform a 2-3 mm myotomy directed ventrally along the caudal border of the muscle to facilitate ventral reflection.</li>
		<li>Perform a craniotomy ~5 mm in diameter at the skull underneath the reflected temporalis muscle using a micro drill.</li>
		<li>Remove the dura mater with tweezers to expose the pial surface of the brain. Use extreme caution to avoid accidental injury to the brain.</li>
		<li>Suture the dorsal border of the temporalis muscle to the subcutaneous tissue of the dorsal skin flap with 6-0 monocryl filaments, making it flush to the exposed cerebral cortex.</li>
		<li>Close the skin incision with 6-0 monofilament suture. Place the mouse back into its cage and monitor until recovery from anesthesia. Return the mouse to its housing facility.</li>
	</ol>
	</li>
</ol>
3. Post-operative considerations
<ol>
	<li>Monitor the mice for illness and the surgical site for infection daily. Give subcutaneous normal saline (1% volume by body weight) daily to support hydration.</li>
	<li>Monitor for severe dehydration (loss of body weight &#62;20%) until 7 days after surgery. Administer an additional bolus of subcutaneous normal saline 1% volume by body weight if &#62;20% weight loss.</li>
	<li>Proceed with injections, physiologic monitoring, and other testing without special considerations. 
	NOTE: In this procedure, the use of opioids or non-steroidal anti-inflammatory drugs (NSAIDs) to treat post-surgery was avoided due to the known effects of these agents on stroke outcome or infarct size in consultation with in-house institutional animal care and use committee15,16,17,18. However, the use of post-operative analgesia is highly encouraged for EMS surgery with other models. Please consult the Institutional Animal Care and Use Committee (IACUC) for this.</li>
</ol>

<ol>
	<li>Stroke, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Last+updated+10/22/20.">Last updated 10/22/20.</a> , (2020).</li><li>Cipolla, M. J., McCall, A. L., Lessov, N., Porter, 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=Reperfusion+decreases+myogenic+reactivity+and+alters+middle+cerebral+artery+function+after+focal+cerebral+ischemia+in+rats.">Reperfusion decreases myogenic reactivity and alters middle cerebral artery function after focal cerebral ischemia in rats.</a> Stroke. 28 (1), 176-180 (1997).</li><li>Arai, K., 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=Cellular+mechanisms+of+neurovascular+damage+and+repair+after+stroke.">Cellular mechanisms of neurovascular damage and repair after stroke.</a> Journal of Child Neurology. 26 (9), 1193-1198 (2011).</li><li>Ergul, A., Alhusban, A., Fagan, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis:+a+harmonized+target+for+recovery+after+stroke.">Angiogenesis: a harmonized target for recovery after stroke.</a> Stroke. 43 (8), 2270-2274 (2012).</li><li>Imai, H., 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=The+importance+of+encephalo-myo-synangiosis+in+surgical+revascularization+strategies+for+moyamoya+disease+in+children+and+adults.">The importance of encephalo-myo-synangiosis in surgical revascularization strategies for moyamoya disease in children and adults.</a> World Neurosurgery. 83 (5), 691-699 (2015).</li><li>Ravindran, K., Wellons, J. C., Dewan, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+outcomes+for+pediatric+moyamoya:+a+systematic+review+and+meta-analysis.">Surgical outcomes for pediatric moyamoya: a systematic review and meta-analysis.</a> Journal of Neurosurgery: Pediatrics. 24 (6), 663-672 (2019).</li><li>Kim, H. 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=The+neovascularization+effect+of+bone+marrow+stromal+cells+in+temporal+muscle+after+encephalomyosynangiosis+in+chronic+cerebral+ischemic+rats.">The neovascularization effect of bone marrow stromal cells in temporal muscle after encephalomyosynangiosis in chronic cerebral ischemic rats.</a> Journal of Korean Neurosurgical Society. 44 (4), 249-255 (2008).</li><li>Srivastava, 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=Neuroprotective+and+neuro-rehabilitative+effects+of+acute+purinergic+receptor+P2X4+(P2X4R)+blockade+after+ischemic+stroke.">Neuroprotective and neuro-rehabilitative effects of acute purinergic receptor P2X4 (P2X4R) blockade after ischemic stroke.</a> Experimental Neurology. , 329 (2020).</li><li>Cao, R., 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=VEGFR1-mediated+pericyte+ablation+links+VEGF+and+PlGF+to+cancer-associated+retinopathy.">VEGFR1-mediated pericyte ablation links VEGF and PlGF to cancer-associated retinopathy.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (2), 856-861 (2010).</li><li>Hedlund, E., Hosaka, K., Zhong, Z., Cao, R., Cao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malignant+cell-derived+PlGF+promotes+normalization+and+remodeling+of+the+tumor+vasculature.">Malignant cell-derived PlGF promotes normalization and remodeling of the tumor vasculature.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (41), 17505-17510 (2009).</li><li>Cao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+angiogenesis+for+ischemic+disorders:+what+is+missing+for+clinical+benefits.">Therapeutic angiogenesis for ischemic disorders: what is missing for clinical benefits.</a> Discovery Medicine. 9 (46), 179-184 (2010).</li><li>Verma, R., 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=Inhibition+of+miR-141-3p+ameliorates+the+negative+effects+of+poststroke+social+isolation+in+aged+mice.">Inhibition of miR-141-3p ameliorates the negative effects of poststroke social isolation in aged mice.</a> Stroke. 49 (7), 1701-1707 (2018).</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>Engel, O., Kolodziej, S., Dirnagl, U., Prinz, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+stroke+in+mice-middle+cerebral+artery+occlusion+with+the+filament+model.">Modeling stroke in mice-middle cerebral artery occlusion with the filament model.</a> Journal of Visualized Experiments. 47 (47), 2423 (2011).</li><li>P&#233;trault, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neither+nefopam+nor+acetaminophen+can+be+used+as+postoperative+analgesics+in+a+rat+model+of+ischemic+stroke.">Neither nefopam nor acetaminophen can be used as postoperative analgesics in a rat model of ischemic stroke.</a> Fundam Clin Pharmacol. (2), 194-200 (2017).</li><li>Khansari PS, ., Halliwell RF, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+Underlying+Neuroprotection+by+the+NSAID+Mefenamic+Acid+in+an+Experimental+Model+of+Stroke.">Mechanisms Underlying Neuroprotection by the NSAID Mefenamic Acid in an Experimental Model of Stroke.</a> (64), (2019).</li><li>Mishra, V., Verma, R., Raghubir, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+effect+of+flurbiprofen+in+focal+cerebral+ischemia:+the+possible+role+of+ASIC1a.">Neuroprotective effect of flurbiprofen in focal cerebral ischemia: the possible role of ASIC1a.</a> Neuropharmacology. 59 (7-8), 582-588 (2010).</li><li>Chen, T. Y., Goyagi, T., Toung, T. J., Kirsch, J. R., Hurn, P. D., Koehler, R. C., Bhardwaj, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+opportunity+for+ischemic+neuroprotection+with+selective+kappa-opioid+receptor+agonist+in+rats.">Prolonged opportunity for ischemic neuroprotection with selective kappa-opioid receptor agonist in rats.</a> Stroke. 35 (5), 1180-1185 (2004).</li><li>Tur&#243;czi, Z., 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=Muscle+fiber+viability,+a+novel+method+for+the+fast+detection+of+ischemic+muscle+injury+in+rats.">Muscle fiber viability, a novel method for the fast detection of ischemic muscle injury in rats.</a> PLoS ONE. 9 (1), e84783 (2014).</li><li>Im, K., Mareninov, S., Diaz, M. F. P., Yong, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+introduction+to+performing+immunofluorescence+staining.">An introduction to performing immunofluorescence staining.</a> Methods in Molecular Biology. , 299-311 (2019).</li><li>Zheng, 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=Protective+roles+of+adenosine+A1,+A2A,+and+A3+receptors+in+skeletal+muscle+ischemia+and+reperfusion+injury.">Protective roles of adenosine A1, A2A, and A3 receptors in skeletal muscle ischemia and reperfusion injury.</a> American Journal of Physiology-Heart and Circulatory Physiology. 293 (6), H3685-H3691 (2007).</li><li>Jiao, C., 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=Visualization+of+mouse+choroidal+and+retinal+vasculature+using+fluorescent+tomato+lectin+perfusion.">Visualization of mouse choroidal and retinal vasculature using fluorescent tomato lectin perfusion.</a> Translational Vision Science and Technology. 9 (1), (2020).</li><li>Simard, J. M., Sahuquillo, J., Sheth, K. N., Kahle, K. T., Walcott, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Managing+malignant+cerebral+infarction.">Managing malignant cerebral infarction.</a> Current Treatment Options in Neurology. 13 (2), 217-229 (2011).</li><li>Liu, X., 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=Osteoclasts+protect+bone+blood+vessels+against+senescence+through+the+angiogenin/plexin-B2+axis.">Osteoclasts protect bone blood vessels against senescence through the angiogenin/plexin-B2 axis.</a> Nature Communications. 12 (1), 1832 (2021).</li><li>Paro, M., Gamiotea-Turro, D., Blumenfeld, L., Bulsara KR, ., Verma, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Model+for+Encephalomyosynangiosis+Surgery+after+Middle+Cerebral+Artery+Occlusion-Induced+Stroke+in+Mice.">A Novel Model for Encephalomyosynangiosis Surgery after Middle Cerebral Artery Occlusion-Induced Stroke in Mice.</a> BioXriv. 10, (2021).</li><li>Venkat, 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=Treatment+with+an+Angiopoietin-1+mimetic+peptide+promotes+neurological+recovery+after+stroke+in+diabetic+rats.">Treatment with an Angiopoietin-1 mimetic peptide promotes neurological recovery after stroke in diabetic rats.</a> CNS Neuroscience &amp; Therapeutics. 27 (1), 48-59 (2021).</li><li>Cheng, X., 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=Acidic+fibroblast+growth+factor+delivered+intranasally+induces+neurogenesis+and+angiogenesis+in+rats+after+ischemic+stroke.">Acidic fibroblast growth factor delivered intranasally induces neurogenesis and angiogenesis in rats after ischemic stroke.</a> Neurological Research. 33 (7), 675-680 (2011).</li><li>Xu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+effects+of+mutant+of+acidic+fibroblast+growth+factor+against+cerebral+ischaemia-reperfusion+injury+in+rats.">Protective effects of mutant of acidic fibroblast growth factor against cerebral ischaemia-reperfusion injury in rats.</a> Injury. 40 (9), 963-967 (2009).</li><li>Tsai, M. 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=Acidic+FGF+promotes+neurite+outgrowth+of+cortical+neurons+and+improves+neuroprotective+effect+in+a+cerebral+ischemic+rat+model.">Acidic FGF promotes neurite outgrowth of cortical neurons and improves neuroprotective effect in a cerebral ischemic rat model.</a> Neuroscience. 305, 238-247 (2015).</li><li>Meller, R., 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=Neuroprotection+by+osteopontin+in+stroke.">Neuroprotection by osteopontin in stroke.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 25 (2), 217-225 (2005).</li><li>Meseguer, E., 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=Osteopontin+predicts+three-month+outcome+in+stroke+patients+treated+by+reperfusion+therapies.">Osteopontin predicts three-month outcome in stroke patients treated by reperfusion therapies.</a> Journal of Clinical Medicine. 9 (12), 4028 (2020).</li><li>Zhu, Z., 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=Plasma+osteopontin+levels+and+adverse+clinical+outcomes+after+ischemic+stroke.">Plasma osteopontin levels and adverse clinical outcomes after ischemic stroke.</a> Atherosclerosis. 332, 33-40 (2021).</li></ol>

The authors have no conflicts of interest to disclose.

This protocol describes a successful EMS procedure in a mouse model of MCAo-induced stroke. The data show that grafted tissue remains viable and can form bonds with brain cortex long after EMS surgery. These findings support the rationale for using a cerebral muscle graft to gradually develop a richly vascular trophic environment at the site of stroke. EMS is a promising therapy for potentially repairing infarcted cerebral tissue in the same environment.
The critical steps of the protocol include step 2.2.4: this step causes unavoidable trauma to the TM, which may reduce its ability to bond with the cortex and release trophic factors. Take care to limit TM trauma to the extent possible. An alternative strategy to reduce tissue trauma is to bluntly dissect the TM from the skull only at its dorsal border and forego the myotomy. In this case, the TM would be lifted away from the skull (rather than fully reflected), and the craniotomy would be performed with the craniotomy drill underneath the muscle. This reduces the amount of space available to perform this step but again may reduce TM trauma. Further, extreme care and practice is necessary in steps 2.2.5 and 2.2.6, for preventing injury to the underlying brain cortex during craniotomy and manipulation of the dura.
This EMS model is a natural adjunct to the well-established MCAo model. Because the MCAo model closely simulates the pathophysiology of ischemia and vascular network damage, that is common in human patients, the MCAo + EMS model is likely to have a high level of translatability to humans. The EMS model presented here is the first therapeutic intervention that has been studied for ischemic stroke in the preclinical setting that relies only on autologous tissue. Moreover, because the TM graft is organic and autologous, it may demonstrate paracrine signaling interactions with the adjacent injured brain that serve to regulate the release of trophic factors to optimum levels at various time points.
While stroke creates a proangiogenic environment and stimulates angiogenesis itself26, the intrinsic post-stroke response is not sufficient to improve vascular supply in the damaged region due to subthreshold levels of angiogenic factors. Here, EMS further enhanced FGF-acidic protein expression compared to stroke-only animals. This protein indirectly controls neovascularization in concert with other growth factors. FGF-acidic also works as a neurotrophic factor, promoting neuroprotection and neurogenesis27,28. Some of the neuroprotective effects of FGF-acidic are mediated by activation of AKT and MAPK/EPK pathways29. In addition to FGF, there was also reduced expression of the protein osteopontin. Osteopontin is a pro-inflammatory, pleotropic cytokine that is increasingly being recognized for its role in multiple neuropathologies and tissue remodeling processes, among other functions. The role of osteopontin in stroke is still uncertain30. However, recent studies in humans point to osteopontin as a poor prognostic factor after stroke. A decrease in serum osteopontin levels after stroke was shown in one study to predict favorable outcomes (modified Rankin scale score &#60; 2 at 90 days) in human patients with stroke31. Another study showed a dose-dependent relationship between higher levels of plasma osteopontin and outcomes of death and disability in human patients after stroke32. In line with these clinical studies, the data here suggest that reduced osteopontin after EMS may promote an anti-inflammatory environment to increase neo-vessel formation. Overall, the differential expression of FGF-acidic and osteopontin points toward mechanisms governing the angiogenesis following EMS in this mouse model and increases the likelihood that the procedure that can also bring about neuroprotection and neuro-regeneration in addition to angiogenesis.
There are some potential limitations of this procedure. Measuring cerebral flow due to increased blood vessel density is challenging in this procedure, as commonly used procedures of laser Doppler or laser speckle flowmeter are affected by the presence of temporalis muscle on the top of cortex which prevent true blood measurement on cortical surface. Thus, this procedure may need more sophisticated, but rarely available, small rodent MRI scan if real time flow measurement is required. However, the use of blood vessel density measurement indirectly supports the success of EMS procedure in improving angiogenesis as suggested by our data. Another limitation is the invasive nature of EMS interventions on the top of MCAo, which itself is an invasive procedure. While there was no increased mortality with EMS in this study compared to MCAo only, the requirement for hemicraniectomy may limit its future translatability for all type of stroke. However, in clinical practice, &#62;10% of patients with large ischemic stroke require hemicraniectomy to manage increased intracranial pressure23, and this EMS model may have translational value for this subgroup of stroke patients in particular. Finally, the time point of 4 h post-MCAo for performance of EMS was chosen to fall within the standard treatment window of rT-PA for most human patients, though future studies will use later time points to evaluate the therapeutic window for EMS.
Overall, the EMS model provides a well-tolerated option for inducing angiogenesis after ischemic stroke, and in addition to its potential clinical translation, may be used in future studies examining the pathophysiology of stroke and angiogenesis.
The EMS model described here offers a safe method of achieving cerebral angiogenesis for preclinical study, obviating the need for pharmacologic interventions, which often lead to unwanted side effects or uncontrolled angiogenesis. Many patients with large ischemic strokes require a hemicraniectomy during their clinical course to manage increasing intracranial pressure. This EMS procedure, which also includes hemicraniectomy in mice for muscle grafting, may provide preclinical proof of concept for translational application of EMS in ischemic stroke. Therefore, this model has the potential to expand the knowledge of neurovascular recovery after an ischemic stroke and to facilitate the development of innovation, which are the need of the hour, in therapeutics for stroke survivors.

Ischemic stroke is an acute neurovascular injury with devastating chronic sequelae. Most of the stroke survivors, 650,000 per year, in the US suffer from permanent functional disability1. None of the available treatments confer neuroprotection and functional recovery after the acute phase of ischemic stroke. After an acute ischemic stroke, both direct and collateral blood supplies are diminished which leads to dysfunction of brain cells and networks, resulting in sudden neurological deficits2,3. Restoration of blood supply to the ischemic region remains the foremost goal of stroke therapy. Thus, enhancing angiogenesis to promote blood supply in the ischemic territory is a promising therapeutic approach; however, previously studied methods for promoting post-stroke angiogenesis, including erythropoietin, statins, and growth factors, have been limited by unacceptable levels of toxicity or translatability4.
Encephalomyosynangiosis (EMS) is a surgical procedure that enhances cerebral angiogenesis in humans with moyamoya disease, a condition of narrowed cranial arteries that often leads to stroke. EMS involves partial detachment of a vascular section of the patient's temporalis muscle from the skull, followed by craniotomy and grafting of the muscle onto the affected cortex. This procedure is well tolerated and induces cerebral angiogenesis, reducing the risk of ischemic stroke in patients with moyamoya disease5,6. Thus, the procedure serves largely a preventative role in these patients. The angiogenesis brought about by this procedure may also have a role in promoting neurovascular protection and recovery in the setting of ischemic stroke. This report supports the hypothesis that angiogenesis brought about by EMS has the potential to expand the understanding of and therapeutic options for cerebral ischemia.
Beside EMS, there are several pharmacological and surgical approaches to improve angiogenesis, but they have several limitations. Pharmacological approaches such as vascular endothelial growth factor (VEGF) administration has been found to be insufficient or even detrimental due to several limitations, including the formation of chaotic, disorganized, leaky, and primitive vascular plexuses, which resemble those found in the tumor tissues7,8 and have no beneficial effects in clinical trials9. 
Surgical approaches include direct anastomosis such as superficial temporal artery-middle cerebral artery anastomosis, indirect anastomosis such as encephalo-duro arterio-synangiosis (EDAS), encephalomyosynangiosis (EMS), and combinations of direct and indirect anastomosis10. All these procedures are very technically challenging and demanding in small animals, except for EMS. Whereas the other procedures require complex vascular anastomosis, EMS requires a relatively simple muscle graft. Moreover, the proximity of the temporalis muscle to the cortex makes it a natural choice for grafting, as it does not need to be completely excised or disconnected from its blood supply, as would be necessary if a more distant muscle were used for grafting. 
EMS has been studied in chronic cerebral hypoperfusion models in rats7,11. However, EMS using a temporalis muscle graft has never been studied in acute ischemic stroke in rodents. Here, we describe a novel protocol of EMS in mice after an ischemic stroke via middle cerebral artery occlusion model (MCAo). This manuscript serves as a description of methods and early data for this novel approach of EMS in mice after MCAo.

<table><tbody><tr><td>6-0 monocryl suture</td><td>Ethilon</td><td>697G</td><td></td></tr><tr><td>70% ethanol to sanitize operating surface</td><td>Walgreens</td><td></td><td></td></tr><tr><td>Bupivacaine 0.25% solution</td><td>Midwest Vet</td><td></td><td></td></tr><tr><td>Clamps for tissue retraction</td><td>Roboz</td><td></td><td></td></tr><tr><td>Doccal suture with silicone coating</td><td>Doccal Corporation</td><td>602145PK10Re</td><td></td></tr><tr><td>Electric heating pad for operating surface</td><td></td><td></td><td></td></tr><tr><td>Isoflurane anesthesia</td><td>Piramal Critical Care Inc</td><td></td><td></td></tr><tr><td>Isoflurane delivery apparatus</td><td>B6Surgivet (Isotech 4)</td><td></td><td></td></tr><tr><td>Micro drill</td><td>Harvard Apparatus</td><td></td><td></td></tr><tr><td>Microdissecting tweezers, curved x2</td><td>Piramal Critical Care Inc</td><td></td><td></td></tr><tr><td>mouse angiogenesis panel arrat</td><td>R&amp;amp; D biotech</td><td>ARY015</td><td></td></tr><tr><td>Needle driver</td><td>Ethilon</td><td></td><td></td></tr><tr><td>Ointment for eye protection</td><td>Walgreens</td><td></td><td></td></tr><tr><td>Operating microscope</td><td>Olympus</td><td></td><td></td></tr><tr><td>Operating surface</td><td>Olympus</td><td></td><td></td></tr><tr><td>Povidone iodine solution</td><td>Walgreens</td><td></td><td></td></tr><tr><td>Rectal thermometer</td><td>world precison instrument</td><td></td><td></td></tr><tr><td>Saline or 70% ethanol for irrigation</td><td>Walgreens</td><td></td><td></td></tr><tr><td>Small electric razor to shave operative site</td><td>Generic</td><td></td><td></td></tr><tr><td>Surgical scissors</td><td>Roboz</td><td></td><td></td></tr></tbody></table>

a model for encephalomyosynangiosis treatment after middle cerebral artery occlusion-induced stroke in mice

There is no effective treatment available for most patients suffering with ischemic stroke, making development of novel therapeutics imperative. The brain's ability to self-heal after ischemic stroke is limited by inadequate blood supply in the impacted area. Encephalomyosynangiosis (EMS) is a neurosurgical procedure that achieves angiogenesis in patients with moyamoya disease. It involves craniotomy with placement of a vascular temporalis muscle graft on the ischemic brain surface. EMS has never been studied in the setting of acute ischemic stroke in mice. The hypothesis driving this study is that EMS enhances cerebral angiogenesis at the cortical surface surrounding the muscle graft. The protocol shown here describes the procedure and provides initial data supporting the feasibility and efficacy of the EMS approach. In this protocol, after 60 min of transient middle cerebral artery occlusion (MCAo), mice were randomized to either MCAo or MCAo + EMS treatment. The EMS was performed 3-4 h after occlusion. The mice were sacrificed 7 or 21 days after MCAo or MCAo + EMS treatment. Temporalis graft viability was measured using nicotinamide adenine dinucleotide reduced-tetrazolium reductase assay. A mouse angiogenesis array quantified angiogenic and neuromodulating protein expression. Immunohistochemistry was used to visualize graft bonding with brain cortex and change in vessel density. The preliminary data here suggest that grafted muscle remained viable 21 days after EMS. Immunostaining showed successful graft implantation and increase in vessel density near the muscle graft, indicating increased angiogenesis. Data show that EMS increases fibroblast growth factor (FGF) and decreases osteopontin levels after stroke. Additionally, EMS after stroke did not increase mortality suggesting that protocol is safe and reliable. This novel procedure is effective and well-tolerated and has the potential to provide information of novel interventions for enhanced angiogenesis after acute ischemic stroke.

A total of 41 mice were used for this study. After three mortalities, one in MCAo and two in MCAo + EMS, a total of 38 mice were used for obtaining the results shown.
Statistics 
Data from each experiment are presented as mean &#177; standard deviation (S.D.). Significance was determined using either unpaired student&#39;s t-test for comparing two groups or one-way ANOVA for more than two groups, with a Newman&#8722;Keuls post-hoc test to correct for multiple comparisons.
Nicotinamide adenine dinucleotide (reduced)-tetrazolium reductase (NADH-TR) staining 
This staining was done to assess the long-term viability of the grafted muscle as in Turoczi et al.19. Briefly, at the time of sacrifice, the grafted muscle flap was carefully excised, fixed with 4% paraformaldehyde for 30 min, and cryopreserved in optimum cutting temperature (OCT) medium at -80 &#176;C. Several 12 &#181;m thick cryosections of temporalis muscle tissue were stained for NADH-TR enzyme-histochemical reaction. Slides were incubated for 30 min at 37 &#176;C in a solution of nitroblue tetrazolium (1.8 mg/dL) and NADH (15 mg/dL) in 0.05 M Tris buffer (pH 7.6). Unused tetrazolium reagent was removed using increasing followed by decreasing concentrations of acetone. Quantitative assessment of NADH-tetrazolium-stained muscle was performed on muscle images taken at 40x magnification.
Immunostaining studies 
Immunostaining was used to visualize muscle graft bonding with cortex and blood vessel density at the junction of muscle and cortex20,21. For visualization of muscle bonding with brain tissue, mice that had undergone EMS surgery were used here. At the end of each respective time point, mice were anesthetized with an avertin injection (50 mg/kg body weight), followed by perfusion with 1x PBS containing 5 mM Ethylenediaminetetraacetic acid (EDTA) and fixation with 4% paraformaldehyde. The skull was carefully cut to prevent accidental detachment of temporalis muscle (TM) graft from brain cortex. TM graft above the brain cortex was then separated from the remaining temporalis muscle. The brain was carefully removed and post-fixed in 4% paraformaldehyde overnight. The fixed brain was then dehydrated with 30% sucrose in 1x PBSuntil the brain sunk to the bottom of the vial (approximately 1-3 days). Tissue sections of 30 &#181;m size were cut with freezing microtome and mounted on slides.
For immunostaining of blood vessels in the ipsilateral brain cortex, MCAo and MCAo + EMS mice were sacrificed, perfused, fixed, and processed as above. Brain slices of 30 &#181;m size were sectioned on a freezing microtome and mounted on a glass side. Antigen retrieval was done using citrate buffer (pH 6.0) and sections were incubated with blocking buffer followed by incubation overnight with primary antibodies, anti-alpha skeletal muscle actin 1:200, and Lectin-Dy59421,22.&#160;Three coronal brain sections per mouse (n = 5 mice/group; total = 15 sections) were taken between 0.45 mm and 0.98 mm from bregma, stained, and visualized for quantification at 20x magnification at the junction of the ischemic core and penumbra regions. A blinded observer quantified lectin positive vessel density in the brain parenchyma using ImageJ software.
Muscle graft remains viable at 21 days after EMS 
One prerequisite for success of this surgery is long-term viability of the grafted temporalis muscle. The TM graft showed transient damage of muscle cells at 7 days after surgery in grafted muscle vs. control muscle (71.32% muscle cell survival &#177; 16.64% vs. 97.19% &#177; 3.81%). However, this difference between grafted and the control muscle vanished, and muscles recovered completely 21 days after surgery (98.22% &#177; 3.965 vs. 96.87% &#177; 2.27%; Figure 2A).
Muscle grafts make loose bonds with brain tissue 
Successful grafting of the temporalis muscle onto the brain cortex surface is a foremost requirement for the success of this model. In both the EMS + MCAo and EMS-only model, the temporalis muscle grafts adhered to the cortical surface 21 days after EMS, suggesting successful surgery, graft implantation, and bonding (Figure 1B and Figure 2B).
Blood vessel density increases in the perilesional cortex after EMS 
Acute stroke leads to acute reduction in cerebral blood flow, impeded recruitment of collateral vessels, abnormal vascular sprouting, and dysfunctional angiogenesis, which contribute to poor stroke outcomes23.&#160;EMS significantly increases blood vessel surface area and integrated density in perilesional cortex after stroke (p &#60; 0.05 vs. MCAo-only; Figure 3).
Analysis of angiogenic and neuromodulating proteins 
A mouse angiogenesis array was used to compare expression of angiogenic and neuromodulating proteins 7 days and 21 days after MCAo in MCAo-only vs. MCAo + EMS mice as per manufacturer&#39;s instructions24. ImageJ software was used to quantify pixel density for each data point of the protein dot blot. Data were recorded as the ratio of the density of each analyzed protein to the averaged density of the standards for each blot.
Fibroblast growth factor (FGF)-acidic is upregulated and osteopontin is downregulated after EMS 
Protein array results showed a significant increase in protein levels of FGF-acidic (0.677 &#177; 0.007 vs. 0.585 &#177; 0.014, p = 0.045), a potent angiogenic factor, and decrease in osteopontin levels, a multifunctional molecule expressed in inflammatory conditions (0.692 &#177; 0.007 vs. 0.758 &#177; 0.014, p = 0.048) in the MCAo + EMS group 21 days after stroke, suggesting improved angiogenesis and neuroprotection (Figure 4A).
Mortality outcomes for EMS after stroke 
Both MCAo and EMS are invasive surgical techniques that may cause some mortality in mice. In this experiment, there was between 10%-11% mortality in mice 21 days after MCAo surgery, which is an accepted death rate for mice subjected to 60 min of MCAo14. Performing EMS on mice after MCAo did not increase mortality (Figure 4B) suggesting tolerance of EMS surgery even after MCAo.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63951/63951fig01.jpg" /> 
Figure 1. Stepwise EMS procedure after middle cerebral artery occlusion (MCAo): (A) Step 1. A skin incision is made over the right middle cerebral artery territory. The skin and subcutaneous tissues are reflected, exposing the skull and temporalis muscle. Step 2. The temporalis muscle is dissected away from the skull and reflected ventrally. Step 3. A craniotomy is performed (4-5 mm) and the dura is gently removed. Step 4. The temporalis muscle is placed directly on brain surface to cover the exposed cortex. Step 5. The dorsal edge of the temporalis muscle is sutured to the subcutaneous tissue of the dorsal skin flap, flush with the brain surface. Step 6. The incision is closed, and the mouse is removed from anesthesia and returned to its cage.This part of figure has been modified from25. (B) Conceptual schematic for encephalomyosynangiosis (EMS) treatment of MCAo-induced stroke. Abbreviations: FGF = Fibroblast growth factor. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63951/63951fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63951/63951fig02.jpg" /> 
Figure 2. Immunostaining studies. (A) Temporalis muscle grafts maintain viability. Temporalis muscle grafts (EMS) on ischemic cortex tissue maintain high viability. (Left) Representative image of nicotinamide adenine dinucleotide (reduced)-tetrazolium reductase-stained muscle tissue cells from control (na&#239;ve muscle from contralateral side) and grafted muscle at 7 days after middle cerebral artery occlusion (MCAo) + encephalomyosynangiosis (EMS) surgery. Black arrow () shows damaged cells. (Right) Quantification of live/dead muscle cells. Muscle cells at 7 days after EMS show some mild damage (p &#60; 0.1; t-test) that completely recovered at 21 days. (n = 5 mice/time points = total 10 mice in this group) Data are mean &#177; S.D. Scale bar = 20 &#181;m. (B)&#160;Bonding of grafted temporalis muscle with brain cortex 21 days after EMS surgery. EMS tissues stained with anti-alpha skeletal muscle actin (green) and Lectin-Dy594 (red; blood vessel marker) antibody (n = 3 mice). Scale bar = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63951/63951fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63951/63951fig03.jpg" /> 
Figure 3: Encephalomyosynangiosis (EMS) surgery increases blood vessel density in ischemic lesions 21 days after stroke. (A) Representative images of coronal brain sections from mice subjected to (left) middle cerebral artery occlusion (MCAo) or (Right) MCAo + EMS and stained with L. esculentum (Tomato) Lectin-Dy594, which binds to glycoproteins in the basal membrane of endothelial cells. Graphs are quantified areas. MCAo + EMS mice showed higher endothelium network using parameters viz. vascular fraction area (B) and integrated density (C). **p &#60; 0.01 (unpaired t-test), while MCAo-only mice showed damage close to the ischemic lesion (dashed line). N = 5 mice/group= 10 mice total. Data are mean &#177; S.D. Scale bar = 100 &#181;m. Abbreviations: Contra = contralateral side; Ipsi =ipsilateral side. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63951/63951fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63951/63951fig04.jpg" /> 
Figure 4: Encephalomyosynangiosis modulates angiogenic proteins after stroke. (A) A mouse angiogenesis array (ARY015) was used to simultaneously assess the relative levels of 53 mouse angiogenesis-related proteins after middle cerebral artery occlusion (MCAo) and MCAo + EMS (day 21 after MCAo) in brain tissue lysates from the perilesional cortex. Quantitative analysis shows that EMS surgery significantly reduced osteopontin and increased fibroblast growth factor (FGF)-acidic protein after stroke (*p &#60; 0.05 or **p &#60; 0.01) vs ipsilateral MCAo. Data are mean &#177; S.D.; n = 3 mice/group/time point = total 15 mice. (B) EMS did not increase mortality after stroke (MCAo). Kaplan Meier survival curve shows that EMS + MCAO did not change post-stroke mortality vs. MCAO alone (p = 0.54). For EMS n = 3; for MCAo n = 11; and for MCAo + EMS n = 21. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63951/63951fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Model for Encephalomyosynangiosis Treatment after Middle Cerebral Artery Occlusion-Induced Stroke in Mice at JoVE.com

A Model for Encephalomyosynangiosis Treatment after Middle Cerebral Artery Occlusion-Induced Stroke in Mice

The protocol aims to provide methods for encephalomyosynangiosis-grafting of a vascular temporalis muscle flap on the pial surface of ischemic brain tissue-for the treatment of non-moyamoya acute ischemic stroke. The approach's efficacy in increasing angiogenesis is evaluated using a transient middle cerebral artery occlusion model in mice.

There is no effective treatment available for most patients suffering with ischemic stroke, making development of novel therapeutics imperative. The ...

a-model-for-encephalomyosynangiosis-treatment-after-middle-cerebral

Nanyang Technological University

Research

JoVE Journal

Neuroscience

2.7K Views.  UConn School of Medicine. The protocol aims to provide methods for encephalomyosynangiosis-grafting of a vascular temporalis muscle flap on the pial surface of ischemic brain tissue-for the treatment of non-moyamoya acute ischemic stroke. The approach's efficacy in increasing angiogenesis is evaluated using a transient middle cerebral artery occlusion model in mice.

Video: A Model for Encephalomyosynangiosis Treatment after Middle Cerebral Artery Occlusion-Induced Stroke in Mice

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

Medicine

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 ...

Mouse Model of Intraluminal MCAO: Cerebral Infarct Evaluation by Cresyl Violet Staining

Stroke is the third cause of mortality and the leading cause of disability in the World. Ischemic stroke accounts for approximately 80% of all strokes. However, the thrombolytic tissue plasminogen activator (tPA) is the only treatment of acute ischemic stroke that exists. This led researchers to develop several ischemic stroke models in a variety of species. Two major types of rodent models have been developed: models of global cerebral ischemia or focal cerebral ischemia. To mimic ischemic stroke in patients, in whom approximately 80% thrombotic or embolic strokes occur in the territory of the middle cerebral artery (MCA), the intraluminal middle cerebral artery occlusion (MCAO) model is quite relevant for stroke studies. This model was first developed in rats by Koizumi et al. in 1986 1. Because of the ease of genetic manipulation in mice, these models have also been developed in this species 2-3.
	Herein, we present the transient MCA occlusion procedure in C57/Bl6 mice. Previous studies have reported that physical properties of the occluder such as tip diameter, length, shape, and flexibility are critical for the reproducibility of the infarct volume 4. Herein, a commercial silicon coated monofilaments (Doccol Corporation) have been used. Another great advantage is that this monofilament reduces the risk to induce subarachnoid hemorrhages. Using the Zeiss stereo-microscope Stemi 2000, the silicon coated monofilament was introduced into the internal carotid artery (ICA) via a cut in the external carotid artery (ECA) until the monofilament occludes the base of the MCA. Blood flow was restored 1 hour later by removal of the monofilament to mimic the restoration of blood flow after lysis of a thromboembolic clot in humans. The extent of cerebral infarct may be evaluated first by a neurologic score and by the measurement of the infarct volume. Ischemic mice were thus analyzed for their neurologic score at different post-reperfusion times. To evaluate the infarct volume, staining with 2,3,5-triphenyltetrazolium chloride (TTC) was usually performed. Herein, we used cresyl violet staining since it offers the opportunity to test many critical markers by immunohistochemistry. In this video, we report the MCAO procedure; neurological scores and the evaluation of the infarct volume by cresyl violet staining.

The intraluminal middle cerebral occlusion model in mice is herein presented. The extent of cerebral infarct is evaluated by a neurologic score and cresyl violet staining, an alternative staining to TTC, offering the great advantage to test in parallel many interest markers.

Stroke is the third  cause of mortality and the leading cause of disability in the World. Ischemic  stroke accounts for approximately 80% of all strokes. ...

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 ...

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 ...

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

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

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

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

Modeling Stroke in Mice: Transient Middle Cerebral Artery Occlusion via the External Carotid Artery

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options are limited to a small proportion of stroke patients within the first hours after stroke. Novel therapeutic strategies are being extensively investigated, especially to prolong the therapeutic time window. These current investigations include the study of important pathophysiological pathways after stroke, such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Over the last decade, there has been increasing concern about the poor reproducibility of experimental results and scientific findings among independent research groups. To overcome the so-called &#8220;replication crisis&#8221;, detailed standardized models for all procedures are urgently needed. As an effort within the &#8220;ImmunoStroke&#8221; research consortium (https://immunostroke.de/), a standardized mouse model of transient middle cerebral artery occlusion (MCAo) is proposed. This model allows the complete restoration of the blood flow upon removal of the filament, simulating the therapeutic or spontaneous clot lysis that occurs in a large proportion of human strokes. The surgical procedure of this &#8220;filament&#8221; stroke model and tools for its functional analysis are demonstrated in the accompanying video.

Different models of middle cerebral artery occlusion (MCAo) are used in experimental stroke research. Here, an experimental stroke model of transient MCAo via the external carotid artery (ECA) is described, which aims to mimic human stroke, in which the cerebrovascular thrombus is removed due to spontaneous clot lysis or therapy.

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options ...

Establishing a Transcranial Middle Cerebral Artery Occlusion Mice Model

A Modified Transcranial Middle Cerebral Artery Occlusion Model to Study Stroke Outcomes in Aged Mice

In experimental stroke research, middle cerebral artery occlusion (MCAO) with an intraluminal filament is widely used to model ischemic stroke in mice. The filament MCAO model typically exhibits a massive cerebral infarction in C57Bl/6 mice that sometimes includes brain tissue in the territory supplied by the posterior cerebral artery, which is largely due to a high incidence of posterior communicating artery atresia. This phenomenon is considered a major contributor to the high mortality rate observed in C57Bl/6 mice during long-term stroke recovery after filament MCAO. Thus, many chronic stroke studies exploit distal MCAO models. However, these models usually produce infarction only in the cortex area, and consequently, the assessment of post-stroke neurologic deficits could be a challenge. This study has established a modified transcranial MCAO model in which the MCA at the trunk is partially occluded either permanently or transiently via a small cranial window. Since the occlusion location is relatively proximal to the origin of the MCA, this model generates brain damage in both the cortex and striatum. Extensive characterization of this model has demonstrated an excellent long-term survival rate, even in aged mice, as well as readily detectable neurologic deficits. Therefore, the MCAO mouse model described here represents a valuable tool for experimental stroke research.

Author Spotlight: Innovative Techniques and Future Directions in Stroke Research 

This protocol demonstrates a unique mouse stroke model with a medium-sized infarct and an excellent survival rate. This model allows preclinical stroke researchers to extend the ischemia duration, use aged mice, and assess long-term functional outcomes.

In experimental stroke research, middle cerebral artery occlusion (MCAO) with an intraluminal filament is widely used to model ischemic stroke in mice. ...

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 ...