Name: a model for encephalomyosynangiosis treatment after middle cerebral artery occlusion-induced stroke in mice
Uploaded: 2022-06-22T15:00:00
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.
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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.

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	<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....

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 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

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans2,3. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.

Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable ...

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

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

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

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

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

A Preclinical Model to Assess Brain Recovery After Acute Stroke in Rats

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery occlusion/reperfusion (MCAO/R) model in male Sprague-Dawley rats was chosen. By changing the rat&#39;s weight (260&#8722;330 g), the thread bolt type (2636/2838/3040/3043) and the brain infarct time (2-3 h), a higher Longa&#39;s score, a larger infarct volume and a greater model success ratio were screened using the Longa&#39;s score and TTC staining. The optimum model condition (300 g, 3040 thread bolt, 3 h brain infarct time) was acquired and used in a 1-90 day observation period after reperfusion via assessment of sensorimotor functions and infarct volume. At these conditions, the bilateral asymmetry test had a significant difference from 1 to 90 days, and the grid-walking test had a significant difference from 1 to 60 days; both differences could be a suitable sensorimotor functional test. Thus, the most appropriate condition of a novel rat model in the recovery and sequela stages of brain ischemia was found: 300 g rats that underwent MCAO with a 3040 thread bolt for a 3 h brain infarct and then reperfused. The appropriate sensorimotor functional tests were a bilateral asymmetry test and a grid-walking test.

The&#160;purpose of this study is to establish and validate an animal model for research in the recovery and sequela stages of brain ischemia by testing brain infarction and sensorimotor function after middle cerebral artery occlusion/reperfusion (MCAO/R) after 1-90 days in rats.

The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery ...

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 ...

Occlusion of Distal Middle Artery to Develop an Ischemic Stroke Mouse Model

Induction of Acute Ischemic Stroke in Mice Using the Distal Middle Artery Occlusion Technique

Ischemic stroke remains the predominant cause of mortality and functional impairment among the adult populations globally. Only a minority of ischemic stroke patients are eligible to receive intravascular thrombolysis or mechanical thrombectomy therapy within the optimal time window. Among those stroke survivors, around two-thirds suffer neurological dysfunctions over an extended period. Establishing a stable and repeatable experimental ischemic stroke model is extremely significant for further investigating the pathophysiological mechanisms and developing effective therapeutic strategies for ischemic stroke. The middle cerebral artery (MCA) represents the predominant location of ischemic stroke in humans, with the MCA occlusion serving as the frequently employed model of focal cerebral ischemia. In this protocol, we describe the methodology of establishing the distal MCA occlusion (dMCAO) model through transcranial electrocoagulation in C57BL/6 mice. Since the occlusion site is located at the cortical branch of MCA, this model generates a moderate infarcted lesion restricted to the cortex. Neurological behavioral and histopathological characterization have demonstrated visible motor dysfunction, neuron degeneration, and pronounced activation of microglia and astrocytes in this model. Thus, this dMCAO mouse model provides a valuable tool for investigating the ischemiastroke and worth of popularization.

Author Spotlight: Establishing a Reliable Distal MCA Occlusion Model in Mice for Stroke Research

Here, we present a protocol to establish a distal middle cerebral artery occlusion (dMCAO) model through transcranial electrocoagulation in C57BL/6J mice and evaluate the subsequent neurological behavior and histopathological features.

Ischemic stroke remains the predominant cause of mortality and functional impairment among the adult populations globally. Only a minority of ischemic ...

Middle Cerebral Artery Occlusion (MCAO) to Induce Stroke in a Murine Model

To begin, lay an eight to 10 weeks anesthetized C57 black six male mouse on the surgical bench. Using blunt forceps, pull out the tongue and hold it with fingers. Insert a laryngoscope into the mouth and visualize the vocal cord.Stabilize the mouse chin on the laryngoscope and hold the 20 gauge intravenous, or IV catheter with a guide wire inserted in it. Then slightly insert the guide wire into the vocal cord and slowly push the 20 gauge IV catheter into the trachea until its wing part is even with the nose tip. Once the mouse is connected to the ventilator, keep it in a lateral position with the right temporal area facing up.Maintain the rectal temperature at 37 degrees Celsius using a heating pad and a heat lamp. After locating the zygomatic arch and incising the temporal muscle, use two forceps to dissect the underlying zygomatic arch and expose the joint between the maxilla and zygomatic bones. Then using scissors, cut a three millimeter portion of the zygomatic arch and remove it.Finally, separate the masseter muscle from the skull base. Position four small retractors in different directions and expose the cranial skull base with one retractor pulling the trigeminal nerve branches laterally. Apply a saline drop to the skull above the middle cerebral artery, or MCA trunk and proximal to the rhinal cortex branch.Use an electrical grinder to thin the skull until a small fracture is visible. And with the tip of the forceps, remove the thin skull. Place a single-strand loop of black braided silk on top of the MCA.Then insert an 8-O microsurgical needle to lift the MCA trunk and tie the suture under the needle, leaving the needle's two ends on top of the silk thread loop knot. For transient middle cerebral artery occlusion, or MCAO, slightly tighten the silk thread knot under the needle to block arterial blood flow, representing MCAO onset. Use the forceps to hold the suture and slowly remove the needle at the end of the ischemia.The laser speckle contrast imaging confirmed the reduced blood supply in the right MCA. For transient MCAO after suture removal, the cerebral blood flow reperfusion was evident, and further improved after 24 hours. TTC staining of the brain after 24 hours of stroke demonstrated the generation of infarcted tissue in both the cortical and lateral striatum areas in young and aged mice.The infarct size was moderate compared to filament MCAO.

Surgical Procedure for Transient Middle Cerebral Artery Occlusion

To begin, position the anesthetized mouse on the operating table. Maintain body temperature using a rectal probe connected to a heating pad. Make an incision on the skin of the head in the direction of the sagittal suture, starting from the ears to the area between the eyes.Retract the skin and remove the periosteum on the right side of the skull. Then find the coordinates, 2.5 millimeters lateral from bregma. Apply cyanoacrylate to the doppler holder and attach it for laser-doppler flowmetry.After the glue has dried, connect the doppler probe. Then, check for the correct signal readout for assessing the cerebral blood flow. To perform artery occlusion, turn the mouse over to the supine position and fix it to the surgical table with medical tape.Then make a midline incision on the neck. Pull the skin laterally, followed by pulling away the salivary glands using retractors. This exposes the carotid territory.Identify the vascular anatomy of the common carotid artery, or CCA, the ICA, and the ECA, as well as the different arteries derived from them. For easier handling, detach CCA, followed by ECA from the connective tissue. Then cauterize the superior thyroid artery and detach ICA from the connective tissue.Check again to make sure the main arteries are fully detached from the connective tissue. Next, wrap 6-0 silk sutures around the ECA at the maxillary lingual bifurcation. Then suture around the CCA.Place a vascular clip interrupting blood circulation from the ICA. Make a small incision in the ECA close to the area where the tight knot is located. Insert the monofilament until the thick coating has completely entered the arterial lumen.Tighten the second knot to hold the monofilament inside the artery and to prevent the pressure exerted by the blood from pushing it out. Cut the ECA below the first knot and rotate the stump to orient it in the direction of the ICA. Advance the monofilament via the ICA until the point where the MCA branches out.The occlusion is reflected in an abrupt blood flow drop in the LDF readout. After 40 minutes, anesthetize the mouse again and place it back on the surgical table. Remove the stitches from the neck.After 45 minutes of occlusion, loosen the knot holding the monofilament in place. Pull slowly and gently on the filament and check that tissue recanalization occurs. Then pull out the filament and tighten the knot to prevent blood loss, clean the blood and untie the CCA knot.Ensure that there is no arterial wall damage. Remove the retractors and reposition the muscles, glands, and skin. Suture the skin using a 6-0 suture and apply disinfectant.Disconnect the Doppler probe and detach the holder. Finally, suture and disinfect the skin of the head. The outcome of the transient middle cerebral artery occlusion procedure was evaluated using in-vivo MRI neuroimaging.Lesion evolution assessed at different time points after reperfusion showed that progression of the lesion volume takes approximately two days to complete. Behavioral tests, such as grip strength test, showed a loss of strength in mice after 24 hours of transient middle cerebral artery occlusion. Also, upon stimulation in the corner test, the mice showed a preference to turn to the right side, which was ipsilateral to the lesion, indicating a successful middle cerebral artery occlusion on the right side.

Distal Middle Artery Occlusion Procedure to Induce Ischemic Stroke in Mice

To begin, wipe the heating surgery board with 75%ethanol and set the temperature to 37 degrees Celsius. Place the anesthetized mouse in a right lateral position on the board with the head positioned away. To protect the cornea from drying, apply eye ointment.After shaving the fur from the left orbit to the left ear canal, use a depilatory cream to remove the remaining fur. Disinfect the surgical area three times with povidone iodine and 75%ethanol. Perform a toe-pinch assessment.After making a vertical incision between the left orbit and the left ear canal, retract the soft skin tissue to expose the temporal muscle. Using straight micro forceps, separate the apical and dorsal segments of the temporal muscle from the skull. Identify the Y-shaped bifurcation of the middle cerebral artery, or MCA, beneath the temporal bone.Then using an electric cranial drill, thin out the skull to make a bone window until the dura mater becomes visible. Remove the dura mater above the MCA with curved micro forceps. Using electric coagulation forceps, coagulate the artery at the site's proximal and distal to the bifurcation.Monitor the blood flow of the left MCA cortical branch in a laser speckle blood-flow meter. Then suture the muscle and the skin separately with polyglycolic acid sutures, and apply diclofenac sodium gel and mupirocin ointment to the skin incision. Place the mouse in a recovery chamber.