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.
<strong...

<ol>
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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. 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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 incl...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>Walgreen</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 Nylon coating</td>
			<td>Doccal corporation Sharon MA</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&#38; 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>walgreen</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>walgreen</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>walgreen</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....

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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 stroke injury destroy vascular network in the brain and early revival of vascular supply is necessary for a stroke recovery. EMS surgery swiftly restores vascular networks by increasing angiogenesis in the brain. EMS surgery offers a safe method of achieving cerebral angiogenesis, which ultimately will improve blood flow to the ischemic tissue.This method may reduce the need for pharmacological interventions. EMS has therapeutic potential as a treatment for ischemic stroke. In particular, it may be applied in the setting of malignant stroke where hemicraniectomy is performed routinely.EMS may facilitate prompt stroke recovery. EMS surgery can provide insights into the mechanism of neuroangiogenesis, as well as it application can be extended to a study of neurogenesis, which often follow angiogenesis. Although any surgical procedure needs substantial practice, however, I believe any researcher who has some experience of rodent surgery can learn and perform EMS surgery easily.Sterilize all instruments by autoclaving prior to the surgery. Sanitize the operating surface with 70%ethanol and warm it to 37 degrees Celsius with an electric heating pad. After isofluorine induced anesthesia, place the mouse on its left side on the operating surface.Verify the depth of anesthesia by toe pinching with blunt forceps. Then lubricate both eyes with ophthalmic ointment. Next, shave the hair over the surgical field with an electric razor and clean the surgical field with 70%ethanol, followed by providone solution.Administer a single dose of 0.25%bupivacaine by subcutaneous injection as preoperative analgesia at the surgery site. Then set up a surgical microscope at the desired magnification. After 60 minutes of middle cerebral artery occlusion, randomize the mice into middle cerebral artery occlusion only or middle cerebral artery occlusion along with encephalomyosynangiosis groups.For groups receiving encephalomyosynangiosis make a 10 to 15 millimeter skin incision with scissors, extending from one to two millimeters rostral to the right ear and one to two millimeters caudal to the right eye. Then retract the skin flaps using clamps and visually identify the temporalis muscle and the skull. Bluntly dissect the temporalis muscle away from the skull using scissors with a spreading technique.Next, perform a two to three millimeter myotomy directed ventrally along the caudal border of the muscle to facilitate ventral reflection. Afterward, perform a craniotomy of approximately five millimeters in diameter at the skull underneath the reflected temporalis muscle using a micro drill. Next, remove the dura mater with tweezers to expose the pile surface of the brain with extreme caution to avoid accidental injury to the brain.After suturing the incision as described in the text, place the mouse back into its cage and monitor until recovery from anesthesia. Then return the mouse to its housing facility. The temporalis muscle graft showed transient damage of the muscle cells seven days after surgery in the grafted and control muscle, with a muscle cell survival of approximately 71%and 97%However, this difference between the grafted and control muscle vanished and the muscles recovered completely 21 days after surgery.In the encephalomyosynangiosis only and middle cerebral artery occlusion model, the temporalis muscle grafts adhered to the cortical surface, suggesting successful surgery, graft implantation, and bonding. The encephalomyosynangiosis significantly increases blood vessel surface area and integrated density in the perilesional cortex after stroke. Protein array results showed a significant increase in the FGF acidic protein levels and a decrease in the osteopontin levels in the middle cerebral artery occlusion with encephalomyosynangiosis group 21 days after the stroke, suggesting improved angiogenesis and neuroprotection.21 days after the middle cerebral artery occlusion surgery, the mice had 10%to 11%mortality. However, encephalomyosynangiosis after the occlusion surgery did not increase mortality, suggesting its tolerance. Take precautions, number one, when you dissect temporalis muscle to avoid damage.Number two, with the use of micro drill to open the skull. And number three, during the removal of dura mater with tweezers. EMS can be applied for other acute injuries such as hematic brain injuries.If successful EMS will not only improve the stroke outcome but also may improve benefits in other acute brain injuries. EMS will open doors to the novel treatment approach for malignant stroke. It will provide novel method for improving angiogenesis and may shed light on adult neurogenesis, which often depends well perfused tissue.

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

Research

JoVE Journal

Neuroscience

2.5K Views.  UConn School of Medicine. A stroke injury destroy vascular network in the brain and early revival of vascular supply is necessary for a stroke recovery. EMS surgery swiftly restores vascular networks by increasing angiogenesis in the brain. EMS surgery offers a safe method of achieving cerebral angiogenesis, which ultimately will improve blood flow to the ischemic tissue.This method may reduce the need for pharmacological interventions. EMS has therapeutic potential as a treatment for ischemic stroke. In partic...