A Modified Model Preparation for Middle Cerebral Artery Occlusion Reperfusion

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 rate^1,2. 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 models^5,6. 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 rats^13,14.

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.

The experimental protocol was conducted in compliance with the Use of Laboratory Animals and Institutional Animal Care and Use Committee guidelines at Chengdu University of Traditional Chinese Medicine (Record number: 2019-DL-002). All animal research data have been documented following the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines. Male Sprague Dawley (SD) rats weighing 250 g ± 20 g and aged 6-8 weeks were utilized for this study. The specifics regarding the animals, reagents, and equipment employed are listed in the Table of Materials.

1. Animal preparation

1. Induce and maintain deep anesthesia in rats using Zoletil 50 (50mg/kg, IM) and Xylazine Hydrochloride (40mg/kg, IP). Ensure the body temperature is maintained at 37 ± 0.5 °C using a rectal probe connected to a heating pad throughout the surgical procedure. Apply veterinary ointment to the rat's eyes to prevent them from drying out.
2. Shave the hair from the head and neck of the rats. Use depilatory cream to remove fur from the head and neck, then wash off the cream with normal saline. Disinfect the skin at the surgical site by applying ethanol and povidone-iodine three times using sterile cotton balls.
3. Make a 2 cm incision in the middle of the rat's head along the direction of the sagittal suture using a scalpel, and carefully remove the muscles covering the skull.
4. Thin the skull on the ischemic side of the rat using a skull drill. Use normal saline to cool down and remove debris during the grinding process. Record the baseline blood flow of the rats using laser speckle contrast imaging (LSCI).
5. Apply ethanol and povidone-iodine three times to the skin of the neck using sterile cotton balls. Make a 2 cm incision along the midline of the neck using a surgical blade. Use retractors to laterally pull back the skin and salivary glands, exposing the sternocleidomastoid and cervical muscles.
6. Separate the sternocleidomastoid and cervical muscles to expose the carotid territory. Identify the vascular anatomy of the common carotid artery (CCA), the internal carotid artery (ICA), and the external carotid artery (ECA). Based on vascular anatomy, separate the CCA, as well as the CCA-derived ECA and ICA¹⁵.
   NOTE: The anatomical structure of the rat indicates that the CCA region is mainly covered by sternocleidomastoid and cervical muscles, with the ICA and ECA derived from the CCA. After separation exposes the CCA, a Y-shaped fork can be seen along the CCA, representing the ICA and ECA. Be cautious not to damage the vagus nerve, which runs parallel to the CCA.

2. Occlusion of the MCA

1. Tie an easily untangled knot over the external carotid artery (ECA) and common carotid artery (CCA) using a 3-0 silk thread to temporarily block blood flow. Position a vessel clip on the CCA approximately 0.5 cm from the first knot.
   1. Dye the needle of a 5 mL syringe black. Create a small puncture in the CCA using the black-dyed needle and mark the pinholes in black.
2. Insert the monofilament nylon suture into the CCA through the black mark. Open the vascular clip and guide the nylon wire into the Internal Carotid Artery (ICA) until it halts with slight resistance. Tighten the second knot securely to keep the monofilament nylon suture in place inside the artery, preventing displacement from the blocking position.
3. Note the time of ischemia at this point and measure the blood flow value on the ischemic side using LSCI. Administer drops of bupivacaine to the neck wound for analgesia and wound closure. Remove the anesthesia mask and allow the rat to recover.
   NOTE: If the monofilament nylon suture encounters difficulty entering the ICA, retract it slightly and attempt insertion again.

3. Reperfusion of MCAO

1. Following 90 min of ischemia, re-anesthetize the rats with isoflurane. Measure cerebral blood flow from the ischemic side using LSCI and ensure the monofilament nylon suture has not shifted.
2. Untie the knots on the external carotid artery (ECA) to allow blood reperfusion. Release the knot on the common carotid artery (CCA), secure the monofilament suture, and withdraw the suture. Apply a vascular clip ahead of the fixed monofilament suture to prevent bleeding.
3. Replace the first CCA knot with a vascular clip. Use tweezers to rotate the vessel incision sideways (Figure 1). Clamp the incision with tweezers and ligate it using 6-0 thread to repair the incision. Remove the vascular clip, check for leaks, and confirm complete reperfusion (Video 1).
   NOTE: Perform ligation to repair the incision to avoid excessive vessel clamping, which could lead to CCA stenosis.
4. Record cerebral cortical blood flow values using LSCI after incision ligation to confirm successful reperfusion. Close the neck incision with sutures and administer 1,00,000 units of penicillin and 2 mL of saline to prevent infection and dehydration.
5. Maintain warmth until the rats regain consciousness. Offer soft food post-surgery and monitor the animals' vital signs.

4. Evaluation of nerve function and cerebral ischemic injury

1. Once the rats have fully awakened, their neural function will be assessed by researchers unaware of the animal grouping. Utilize the Longa score scale (Table 1) and the modified neurological severity score (mNSS) scale (Table 2) to evaluate the neurological function of all rats.
2. After 24 h post-surgery, euthanize the rats under isoflurane-induced anesthesia (4% isoflurane at 4 L/min oxygen) via cervical dislocation¹⁶ (following institutionally approved protocols). Rinse the brain with ice water to remove any remaining blood.
3. Divide the brain tissue into 5 sections and stain them with TTC. Capture images of both sides of the brain tissue slices, measure the ischemic area of the front and back sides using Image J software, and calculate the infarction rate using the following formula:
   
   NOTE: S₁ and S₂ represent the infarct area of the proximal forebrain and proximal brainstem sides of the brain slices, respectively. The infarct area of each brain slice is corrected using Swanson's formula, where h represents the thickness of the brain slice. This formula, previously outlined by the author in prior research¹⁷, allows for a more accurate calculation of the infarct lesion size.
4. Analyze the data using statistical and graphing software to assess the model's success rate and stability.
5. Employ the immunofluorescence method to label cortical neurons in rats, further validating the neuronal damage caused by this method. After 24 h of ischemia-reperfusion in rats, cardiac perfusion using pre-cooled PBS was performed to obtain brain tissue.
6. Embed and freeze the brain tissue into sections. Use neuronal antibodies such as the immature neuronal marker Doublecortin (DCX)¹⁸, neuronal nucleus marker Neuronal Nuclei (NeuN)¹⁸, and neuron dendritic marker Microtubule-associated protein-2 (MAP-2)¹⁹. Observe the expression of ischemic neurons using laser confocal microscopy.

Laser speckle flow imaging demonstrated that prior to the occlusion of the monofilament nylon suture, there was abundant blood flow in the middle cerebral artery (MCA) area, and the baseline blood flow values of the rats were recorded. Following the occlusion of the MCA, the blood flow value on the ischemic side of the brain rapidly decreased. Before withdrawing the suture, the blood flow values on the ischemic side were rechecked to confirm whether the suture was occluding the MCA. The results indicated only a slight change in blood flow. Upon withdrawing the suture, the blood flow perfusion swiftly recovered (Figure 2). Video 1 also shows the robust filling pulse of the common carotid artery, suggesting that this method can achieve sufficient blood flow perfusion post-cerebral ischemia.

The results of the neurological function scores (Figure 3) revealed that compared to the sham surgery group, the Longa score of the model group was 1.80 ± 0.27 (p < 0.05), and the mNSS score was 7.4 ± 0.89 (p < 0.05). These scores indicate that the middle cerebral artery occlusion/reperfusion (MCAO/R) model prepared using this method led to significant neurological damage. Video 2 demonstrates the characteristic behavior of model group rats rotating while crawling, a hallmark of stroke-induced injury, further confirming the validity of this method.

TTC staining demonstrated that the infarct rate in the model group rats was 5.58 ± 1.79 (p < 0.05), with the infarct area primarily affecting the striatum and cortex, and the infarct size remained relatively stable. Immunofluorescence staining illustrated the loss of cortical neurons following ischemia-reperfusion (Figure 4). These findings collectively indicate that this method can establish a stable MCAO/R model suitable for evaluating preclinical drug efficacy.

Figure 1: Schematic diagram of CCA's connector ligation. The yellow circle denotes the insertion point and ligation site of the wire plug, while the ligation position (yellow arrow) indicates where the common carotid artery (CCA) was ligated with the wire. Please click here to view a larger version of this figure.

Figure 2: Laser speckle blood flow images at the times of rats on the ischemic side pre-, post-occlusion, and pre, and post-reperfusion. (A) Cerebral blood flow images of rats, where blue areas indicate low blood flow values and red areas indicate high blood flow values. Scale bars: 200 mm. (B) Changes in cerebral ischemia-reperfusion in rats, presented as mean ± standard deviation, with a sample size of n = 5. Please click here to view a larger version of this figure.

Figure 3: Results of infarct area and neurological function scores in the sham group and model group. (A) TTC staining depicted the infarcted areas of brain tissue in both the sham group and the model group, with white areas indicating infarcted tissue and red areas indicating normal tissue. (B) Quantitative results of the Longa and mNSS scores, as well as the infarction rate, are presented. Statistical significance compared to the sham group is denoted by *p < 0.05, with a sample size of n = 5. Please click here to view a larger version of this figure.

Figure 4: Results of cortical neuron injury in sham group and model group. Expression of NeuN (A), DCX (B), and MAP-2 (C) in cortical neurons. Scale bars: 100 µm. Please click here to view a larger version of this figure.

Figure 5: Diagram of MCA occlusion and reperfusion by Koizumi, Longa, and the modified method. Please click here to view a larger version of this figure.

Table 1: Modified neurological severity score.

Table 2: Zea-Longa scores.

Video 1: Key steps for ligature repair of CCA incision during reperfusion in rats. Please click here to download this Video.

Video 2: Behavioral videos of rats undergoing MCAO/R. Please click here to download this Video.

The middle cerebral artery occlusion (MCAO) model induced by a monofilament nylon suture is the most common method used for preparing MCAO models. This approach is widely adopted in preclinical studies and has gained recognition from many practitioners due to its simplicity, lack of need for craniotomy, minimal surgical trauma, and ability to achieve reperfusion.

There are two classical surgical techniques for intraluminal filament MCAO: the Koizumi method⁸ and the Longa method⁹. The main difference between these methods lies in how the suture is introduced to occlude the MCA. In the Koizumi method, the suture is inserted through the common carotid artery (CCA) incision into the internal carotid artery (ICA). The front end of the suture is coated with silicone to block the starting point of the MCA. Conversely, in the Longa method, the suture is inserted into the ICA through the severed external carotid artery (ECA). The front end of the suture is expanded into a spherical shape to block the starting point of the MCA (Figure 5). During reperfusion, the suture is gently withdrawn to restore blood flow.

The Longa method involves introducing the suture into the internal carotid artery (ICA) through the severed external carotid artery (ECA). This process requires the separation and disconnection of the ECA, as well as the separation and removal of small branches, which are then cut off using an electrocoagulation pen to prevent bleeding. Consequently, this method demands careful and precise operation. Moreover, the short residual end of the severed ECA can make it easy for the suture to slip off during insertion, further increasing the complexity of the procedure. This challenge is particularly pronounced when preparing a mouse model of cerebral ischemia, as it often necessitates the use of a microscope to ensure accuracy, thus adding to the overall complexity of the operation.

The primary concern with the Koizumi method lies in the ligation of the common carotid artery (CCA), which results in compensatory reperfusion of brain tissue on the ischemic side solely through the Circle of Willis. The CCA is a crucial branch of the aortic arch from the heart and serves as the main artery supplying cerebral blood flow perfusion. Therefore, the cerebral ischemia-reperfusion model created by the Koizumi method often experiences a state of hypoperfusion. This hypoperfusion is not limited to the middle cerebral artery region but extends to the entire ischemic hemisphere on the same side. Even after 35 days of modeling, the blood flow value does not return to baseline, suggesting that the Koizumi method should be regarded as an ischemic model with chronic hypoperfusion rather than an ischemia-reperfusion model¹⁰.

In contrast, the Longa method can achieve a blood flow value of 100% in MCAO/R animals. However, it is crucial to note that achieving complete recanalization does not necessarily mean that the Koizumi method should be abandoned. When considering clinical translation, the Koizumi method may be more suitable for patients with spontaneous slow recanalization after artery occlusion. However, this phenomenon is relatively rare in clinical practice. Only a small percentage of ischemic stroke patients meet the criteria for thrombolysis and receive effective treatment within the treatment time window^20,21. Furthermore, some patients do not achieve complete revascularization even after thrombolytic therapy²². Hence, the Koizumi method can be utilized to simulate the conditions of such patients.

On the other hand, the Longa method allows vessels to rapidly recanalize after occlusion, making it more suitable for simulating patients who receive effective thrombolytic therapy or thrombectomy in clinical practice. For simulating patients who have not undergone thrombolytic therapy, the permanent cerebral ischemia model prepared by the Koizumi method may offer a more complete occlusion and a simpler surgical procedure.

The Longa method for inducing middle cerebral artery occlusion/reperfusion (MCAO/R) can lead to more significant reperfusion injury by preserving common carotid artery (CCA)-derived vascular recanalization^23,24. However, it's important to note that the external carotid artery (ECA) needs to be cut off and permanently ligated during the Longa method, which disrupts the complete blood flow structure of the brain and is not consistent with clinical patients. Moreover, the lingual, maxillary, and occipital arteries are branches of the ECA¹³. Thus, cutting or permanently ligating the ECA can cause eating and drinking disorders, resulting in weight loss²⁵. Previous studies have indicated that the deaths of mice following MCAO modeling are primarily due to inadequate food or water intake rather than cerebral ischemic injury^14,26. Additionally, ligation of the ECA can reduce retinal blood flow and induce retinal damage in the form of cell death²⁷.

In addition, the most important indicator for stroke research is infarct volume; however, the volume of infarcts induced by the suture method has been proven to have a significant standard deviation, even following strict surgical and data collection protocols. In previous reports, the infarct volume was associated with the degree of reperfusion²⁸, and the reperfusion of the stroke model prepared by Koizumi's method depends on the Willis circulation^29,30,31. However, it has been proven that the Willis cycle has high anatomical variability, especially in C57Bl/6 mice, where 90% of animals may lack one or two posterior communicating arteries³², which is the key determinant of high variability in ischemic injury³³. Therefore, preserving the perfusion of CCA can reduce the variability of infarct volume³⁴. It was reported that the average coefficient of variation in infarct size in stroke trials was 29.5%³¹, which contributed to the low statistical power of stroke trials. It also poses a huge challenge for potential drug discovery. This undoubtedly increases the cost of development and clinical translation and can lead to researchers missing out on potentially effective drugs. It also increases the number of animals to be included in the statistical analysis of the experiment, which further violates the 3R principle of animal ethics.

Therefore, in this study, modeling methods were modified to ensure the integrity of brain tissue blood flow structure and sufficient reperfusion after cerebral ischemia. The main modifications include two steps: (1) inserting the suture into CCA using a 5 mL syringe needle instead of making a small incision; this step reduces the incision of the CCA and is more conducive to ligating the incision; (2) withdrawing the suture and use finer silk threads to ligate the needle opening of the CCA, without affecting the blood flow perfusion of the CCA. Of course, this also requires careful surgical operation, but is different from the introduction of suture via ECA; this method preserves the integrity of the cerebral blood flow structure while allowing blood flow to be fully reperfused through CCA, making the operating procedure simpler and reducing the variability of infarct volume. Some researchers have used similar research methods to repair incisions with tissue glue, but this has the potential for thrombosis, which will bring uncontrollable effects. And it takes longer to stabilize the incision²⁵.

In conclusion, this study provides a modified approach to the preparation of the cerebral ischemia-reperfusion model that allows for adequate blood perfusion after filament withdrawal and can result in significantly reproducible infarct volumes and neurological damage. All of the above results demonstrate that the modified method is feasible and reproducible and may provide a reference for practitioners to prepare models to facilitate the effective clinical translation of cerebral ischemic drugs.