preparing middle cerebral artery occlusion reperfusion via the common carotid artery

Modified MCAO/R Model with Preserved ECA for Enhanced Reperfusion and Stability

According to the World Health Organization, stroke has remained the second leading cause of death worldwide for the past decade, with a high incidence rate, high mortality, and high disability rate1,2. As the global population ages, the incidence of stroke is expected to increase in developing countries, potentially becoming the leading cause of premature death and disability in adults. Additionally, there is a trend for strokes to occur at a younger age3. The loss of the labor force after a stroke also places a heavy burden on families and society4. Therefore, the development of safe and effective treatments poses a major challenge in stroke research.
Animal models serve as crucial tools for studying the prevention and treatment of human diseases. The successful translation of stroke treatment strategies relies on the reproducibility and reliability of stroke animal models5,6. The middle cerebral artery (MCA) is a common site for clinical stroke, making the MCAO model the closest model to human ischemic stroke. The MCAO model, prepared using the suture method, has been favored by researchers due to advantages such as no craniotomy and easy control of ischemic time. It has been utilized in over 40% of neuroprotective experiments7. However, despite its numerous advantages, the operational details of this model remain a controversial topic for many researchers.
For the suture-induced middle cerebral artery occlusion (MCAO) model, reperfusion occurs by withdrawing the suture. Currently, two main methods are used for suture insertion: Koizumi's method8 and Longa's method9. In Koizumi's method, the suture enters the internal carotid artery (ICA) mainly through the common carotid artery (CCA) incision, while in Longa's method, it passes through the severed external carotid artery (ECA) into the ICA. During reperfusion, the Koizumi method requires permanently ligating the CCA incision and relies on the circle of Willis for reperfusion10. However, some studies suggest that effective reperfusion cannot be achieved solely through the compensatory supply of the circle of Willis after losing the CCA supply. Moreover, the circle of Willis exhibits high anatomical variability, especially in C57Bl/6 mice, increasing infarction variability and reducing experimental data reliability. Consequently, this method has been increasingly questioned by researchers11.
Longa's method involves inserting a suture through the severed ECA and then permanently ligating the internal carotid artery (ICA) once the suture is withdrawn. This preserves CCA patency, allowing blood perfusion up to 100% of baseline values. However, this method necessitates separating the external carotid artery and small arterial branches, cutting them off, or electrocoagulating them, making the procedure challenging. It also disrupts the brain's complete blood flow structure, which differs from the clinical patient's state12. Importantly, studies indicate that cutting or ligating the ECA can cause ischemic lesions in the muscles controlling chewing and swallowing, affecting animal diet and leading to postoperative animal death and severe sensory and motor damage in rats13,14.
Hence, a modified model preparation method is urgently needed to address these issues. This study introduces a modified MCAO modeling method that repairs the CCA insertion incision and achieves effective reperfusion. The procedure is simple, practical, and feasible, inducing significant neurological damage and replicable infarct lesions and providing valuable guidance for stroke researchers.

Author Spotlight: A Novel Approach to Cerebral Ischemia Modeling &amp;#8211; Enhancing Reperfusion and Simplifying Procedure

A Modified Model Preparation for Middle Cerebral Artery Occlusion Reperfusion

Our research scope is modeling program for cerebral ischemia. We attempted to introduce silk thread from the common carotid artery and ligate the incision with fine silk thread, maintain reperfusion from the common carotid artery and the external carotid artery. The Koizumi method is considered to have insufficient reperfusion, but the Longa method is considered to affect the blood supply to the master muscle.The operation is complex and unfriendly to beginners. Our protocol address the above issues and provides an alternative approach. Our protocol makes it easier for more practitioners to master cerebral ischemic modeling, and quickly discover potential neuroprotective drugs.

Preparing Middle Cerebral Artery Occlusion Reperfusion via the Common Carotid Artery

After anesthetizing the rat and disinfecting the surgical area, make a two centimeter incision along the midline of the neck using a surgical blade. Employ retractors to laterally pull back the skin and salivary glands exposing the sternocleidomastoid, and cervical muscles. Then separate the sternocleidomastoid and cervical muscles to expose the carotid territory.Separate the common carotid artery and identify the vascular anatomy of the common internal and external carotid artery. Then separate the common carotid artery derived external carotid artery and internal carotid artery. Using a three oh silk thread tie an easily untangled knot over the external carotid artery and common carotid artery to temporarily block blood flow.Position a vessel clip on the common carotid artery approximately 0.5 centimeters from the first knot, and place a loose knot between the first vascular clip and the silk thread knot. Now dye the needle of a five milliliter syringe black. Then using the black dyed needle, create a small puncture in the common carotid artery and mark the pinholes in black.Insert the monofilament nylon suture into the common carotid artery through the black mark. Open the vascular clip and guide the nylon wire into the internal carotid artery until it halts with slight resistance. Then tighten the second knot securely to keep the monofilament nylon suture in place inside the artery preventing displacement from the blocking position.Untie the knots on the external carotid artery to allow blood reperfusion. After securing the monofilament suture and withdrawing it, release the knot on the common carotid artery. Apply a vascular clip ahead of the fixed monofilament suture to prevent bleeding.Then replace the first common carotid artery knot with a vascular clip. Use tweezers to rotate the vessel incision sideways. Clamp the incision with tweezers and ligate it using a six oh thread to repair the incision.Remove the vascular clip, check for leaks, and confirm complete reperfusion. Laser speckle flow imaging revealed that before occluding the middle cerebral artery or MCA, there was abundant blood flow in the MCA area. Upon occlusion of the MCA, the blood flow value on the ischemic side of the brain rapidly decreased.Blood flow on the ischemic side was rechecked before removing the suture, confirming MCA occlusion with only a slight change in blood flow. Upon withdrawing the suture, blood flow perfusion swiftly recovered.

preparing-middle-cerebral-artery-occlusion-reperfusion-via-common

Research

JoVE Journal

Medicine

The middle cerebral artery occlusion reperfusion (MCAO/R) model is crucial for understanding the pathological mechanisms of stroke and for drug development.However, among the commonly used modeling methods, the Koizumi method often faces scrutiny due to its ligation of the common carotid artery (CCA) and its inability to achieve adequate reperfusion. Similarly, the Longa method has been criticized for disconnecting and ligating the external carotid artery (ECA). This study aims to introduce a modified model preparation method that preserves the integrity of the ECA, involves inserting a monofilament nylon suture through the CCA, repairing the ligated CCA incision, and maintaining reperfusion from the CCA. Reperfusion of blood flow was confirmed using laser speckle flow imaging. Evaluation methods such as the Longa scale, Modified Neurological Severity Score, triphenyltetrazolium chloride (TTC) staining, and immunofluorescence labeling of neurons demonstrated that this approach could induce stable ischemic nerve damage. This modified MCAO/R model protocol is simple and stable, providing valuable guidance for practitioners in the field of cerebral ischemia.

This protocol describes the process of preparing for middle cerebral artery occlusion reperfusion via the common carotid artery.