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

Summary

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.

Abstract

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.

Introduction

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 deficits^2,3. 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 disease^5,6. 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 tissues^7,8 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 rats^7,11. 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.

Protocol

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 °F and 30%-70% relative humidity with a 12 h light/dark cycle.

1. Pre-surgery preparation

1. Sterilize all instruments by autoclaving prior to surgery. Sanitize the operating surface with 70% ethanol and warm the operating surface to 37 °C with an electric heating pad.
2. 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.
3. Place the mouse on its left side on the operating surface and apply eye ointment to protect both eyes.
4. Shave hair over the surgical field (i.e., right lateral cranium between eye and ear) with electric clippers. Clean the surgical field in concentric circles outward from the middle of the surgical site, with 70% ethanol followed by povidone solution, and repeat these steps 2x.
   NOTE: 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.
5. 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.
6. Set up a surgical microscope at 4x magnification. The microscope is used for all surgical steps.

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.

1. MCAo surgery
   NOTE: MCAo is a well-characterized model of ischemic stroke in rodents, as described by us and others^12,13,14. 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.
   1. 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.
   2. Measure rectal temperatures using a temperature control system, maintaining the temperature at ~37 °C during surgery with an automatic heating pad.
   3. 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.
   4. 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.
2. EMS surgery
   1. 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 MCAo and were re-anesthetized before the EMS surgery.
   2. 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.
   3. Retract skin flaps using clamps and visually identify the temporalis muscle and the skull.
   4. 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.
   5. Perform a craniotomy ~5 mm in diameter at the skull underneath the reflected temporalis muscle using a micro drill.
   6. Remove the dura mater with tweezers to expose the pial surface of the brain. Use extreme caution to avoid accidental injury to the brain.
   7. 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.
   8. 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.

3. Post-operative considerations

1. 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.
2. Monitor for severe dehydration (loss of body weight >20%) until 7 days after surgery. Administer an additional bolus of subcutaneous normal saline 1% volume by body weight if >20% weight loss.
3. 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 committee^15,16,17,18. 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.

Results

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 ± standard deviation (S.D.). Significance was determined using either unpaired student's t-test for comparing two groups or one-way ANOVA for more than two groups, with a Newman−Keuls post-hoc test to correct for multiple comparisons....

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
6-0 monocryl suture	Ethilon	697G
70% ethanol to sanitize operating surface	Walgreen
Bupivacaine 0.25% solution	Midwest Vet
Clamps for tissue retraction	Roboz
Doccal suture with Nylon coating	Doccal corporation Sharon MA	602145PK10Re
Electric heating pad for operating surface
Isoflurane anesthesia	Piramal Critical Care Inc
Isoflurane delivery apparatus	--B6Surgivet (Isotech 4)
Micro drill	Harvard Apparatus
Microdissecting tweezers, curved x2	Piramal Critical Care Inc
mouse angiogenesis panel arrat	R& D biotech	ARY015
Needle driver	Ethilon
Ointment for eye protection	walgreen
Operating microscope	Olympus
Operating surface	Olympus
Povidone iodine solution	walgreen
Rectal thermometer	world precison instrument
Saline or 70% ethanol for irrigation	walgreen
Small electric razor to shave operative site	generic
Surgical scissors	Roboz