The current protocol describes a rat model of middle cerebral artery occlusion/reperfusion that preserves the anatomical structure of cerebral vessels without causing damage.
Ischemic stroke stands as the primary cause of long-term disability and mortality among adults worldwide. Animal models of ischemic stroke have significantly contributed to our understanding of its pathological mechanisms and the development of potential treatments. Presently, there are two common methods involving filament (endovascular suture) techniques to induce animal models of cerebral ischemia. However, these methods have inherent limitations, such as reduced blood perfusion to the brain, damage to the external carotid artery system, impaired food and/or water intake, and sensory dysfunction of the face. This article introduces a new method for inducing a rat ischemic stroke model without compromising the cerebral vascular anatomy. In this study, the common carotid artery (CCA) of Sprague-Dawley rats was exposed, and an incision was made. A filament was then inserted through the incision into the internal carotid artery to occlude the middle cerebral artery. After 1.5 h of induced ischemia, the occluding filament was fully removed from both the internal carotid artery and the CCA. The incision in the CCA was subsequently sutured using 11-0 microsurgical sutures under a microscope (magnification 4x). Through the utilization of microsurgical techniques to repair the CCA, this study successfully developed a unique method to induce an ischemic stroke model in rats while preserving the anatomical integrity of cerebral blood vessels.
Stroke, the leading cause of death and long-term neurological dysfunction in adults worldwide, encompass various types, with ischemic stroke accounting for approximately 81.9% of cases, while cerebral hemorrhage and subarachnoid hemorrhage account for 14.9% and 3.1%, respectively1. Ischemic stroke often results from atherosclerosis or occlusion of the middle cerebral artery (MCA), leading to local cerebral blood flow reduction and subsequent brain damage. This reduction in nutrient supply to ischemic cells triggers energy depletion, causing membrane integrity loss, cell swelling, and eventual lysis. Additionally, factors like increased intracellular calcium, excitatory amino acid release, heightened free radical production, and inflammatory cell activation contribute to brain tissue injury post-stroke2.
The treatment goals for ischemic stroke are multifaceted: reduce ongoing neurological damage and lower mortality and long-term disability; prevent complications arising from immobility and neurological dysfunction; and minimize the risk of stroke recurrence. Given the complexity of ischemic stroke pathophysiology, a robust stroke model is essential for the research and development of treatments targeting ischemic stroke.
Currently, middle cerebral artery occlusion (MCAo) is a common animal model of ischemic stroke3. MCAo is typically performed by inserting a silicon-tipped or flame-blunted monofilament through the cervical vessels until it reaches the origin of the middle cerebral artery (MCA). In 1986, Koizumi and colleagues developed a technique involving the insertion of a silicon-tipped monofilament through an incision in the common carotid artery (CCA), allowing it to reach the entrance of the MCA and block its blood flow4. In 1989, Longa’s group reported another MCAo method that involved introducing a monofilament through an incision in the external carotid artery (ECA). The monofilament then traverses the bifurcation of the internal carotid artery (ICA) and ECA before reaching the starting point of the MCA5.
The limitations of the two existing methods for inducing MCAo are evident. Koizumi’s method necessitates the permanent blockage of the ipsilateral CCA, leading to reduced MCA perfusion since reperfusion solely relies on the circle of Willis4. Conversely, Longa’s method involves cutting the ECA, resulting in damage to the ECA system and subsequent impairment of facial muscle function5. This damage can impact food and water intake, as well as cause sensory dysfunction of the face, affecting rehabilitation and the assessment of neurological function post-ischemic stroke6.
The current protocol seeks to address these limitations by developing a new method to induce an ischemic stroke model without damaging the anatomical structure of cerebral vessels. This novel approach utilizes microsurgical techniques to repair the CCA, thus avoiding the drawbacks associated with previous methods.
The experimental procedure received approval from the Institutional Animal Care and Use Committee at Yantai University (Approval Number: YTDX20230410) and adhered to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Male Sprague-Dawley rats weighing between 300-350 g were utilized for this study. A comprehensive list of the animals, reagents, and equipment used is provided in the Table of Materials.
1. Animal preparation
2. Anesthesia
3. Ischemic modeling procedure
4. Microsurgical suturing of the CCA
5. Staining with 2,3,5-triphenyltetrazolium chloride (TTC)
6. Neurological function test
NOTE: Neurological function was assessed using the Garcia test, following a previous report9.
7. Statistical analysis
The process of microsuture
The filament was withdrawn near the incision of the CCA until the white filament tip was visible (Figure 1A). Micro clamps were then placed on the CCA (Figure 1B). Under a microscope at 4x magnification, the incision of the CCA was sutured using microsurgical sutures in an interrupted pattern (Figure 1C). Subsequently, the microclamps were removed from the CCA, restoring the anatomical structure and blood flow (Figure 1D).
TTC staining and mortality rate
A total of 12 adult male rats were randomly divided into two groups: sham and model (n = 6 in each group). Unfortunately, one animal in the sham group died from an anesthetic overdose, and one animal in the model group died within 24 h. The infarcts resulting from cerebral ischemia were observed in both the striatum and the cortex (Figure 2A). At 3 days post-ischemic stroke, the total infarct percentage was measured to be 29.3% ± 1.6% of the hemisphere (Figure 2B), with a mortality rate of 16.7% after surgery.
Evaluation of neurological function
At 1 and 3 days post-ischemic stroke, the scores of the model groups significantly decreased compared to those of the sham groups (17.8 ± 0.4 vs. 8.2 ± 1.5, Figure 3A; 17.6 ± 0.9 vs. 9.6 ± 1.3, Figure 3B, respectively).
Figure 1: The process of microsuture. (A) The filament is withdrawn from the CCA. Blue arrows indicate the microvascular clamps. (B) Black arrow indicates the incision after the filament is completely removed. Blue arrows indicate the microvascular clamps. (C) Under a microscope (magnified 4x), the CCA incision was sutured using 11-0 microsurgical sutures in an interrupted pattern. Blue arrows indicate the microvascular clamps. Black arrow indicates the sutured incision. (D) The micro clamps are removed and the blood flow of CCA is restored. Black arrow indicates the sutured site of CCA. Please click here to view a larger version of this figure.
Figure 2: TTC staining results of the ischemia model. (A) Representative images of 2,3,5-triphenyltetrazolium chloride (TTC)-stained brain slices after cerebral ischemia. In living tissue, TTC is enzymatically reduced by dehydrogenases to 1,3,5-triphenylformazan, which has a red color. The tissues in necrotic areas remain white because of the absence of such enzymatic activity. (B) Percentage of ischemic area in rats at 3 days after cerebral ischemia. Compared with the Sham group, **P < 0.01. Data are expressed as the mean ± standard deviation. There were five rats in each group. Please click here to view a larger version of this figure.
Figure 3: Evaluation of neurological function. (A) Garcia scores 1 day after cerebral ischemia. (B) Garcia scores 3 days after cerebral ischemia. Compared with the Sham group, **P < 0.01. Data are expressed as mean ± standard deviation. There were five rats in each group. Please click here to view a larger version of this figure.
MCAo is a widely used method to create animal models of stroke, effectively replicating the stroke process observed in patients by restoring blood flow after ischemia10,11,12. Our laboratory has developed an ischemic stroke model that enables reperfusion from the CCA without causing damage to the ECA. Following the induction of ischemia, microsurgical repair of the CCA is performed. Although direct measurement of CCA blood flow was not conducted, experimenters observed vascular pulsation in the CCA before proceeding with reperfusion. This technique preserves the anatomical integrity of the neck vessels while closely resembling the stroke process in patients. Furthermore, evaluations of neurological function and the area of brain ischemia have confirmed the validity of the stroke induced by this surgical approach.
The previously described ischemic stroke models can be affected by various factors. In the method by Koizumi et al.4, blood reperfusion heavily relies on the circle of Willis due to the ligation of the CCA. However, variations in cerebral vasculature can influence the final cerebral infarction volume, potentially increasing the mortality rate of the stroke model due to low blood perfusion13,14. Additionally, ischemia of the ECA can impair the function of facial muscles and nerves, impacting the assessment of various neurological functions15,16.
The current method addresses these challenges through three key steps: (1) Keeping the CCA incision as small as possible, only large enough for filament insertion, reducing time and damage associated with vascular suturing. (2) Placing microvascular clamps as close to the CCA bifurcation as possible when clamping the distal CCA to prevent clamping the white filament tip, which could lead to silicone detachment and thrombosis. (3) Observing the CCA for resumed pulsation and lack of substantial bleeding before suturing the midline neck incision after vascular repair.
The current method does have several limitations. Firstly, there is a possibility of forming emboli at the suture site, which could lead to further brain ischemia if they detach. This risk should be carefully considered and managed in future experiments. Secondly, the study did not monitor the body weight of the rats, which makes it challenging to determine if the method affected their food intake. Food intake is a direct indicator of the method's advantages, and its assessment could provide valuable insights. Lastly, the current method requires technical proficiency in microsurgical operations, which may limit its widespread use without proper training and expertise.
In summary, while the MCAo modeling method offers high physiological relevance and structural integrity, it does come with limitations that need to be addressed for more robust experimentation. Preserving the anatomical integrity of neck vessels and closely simulating stroke processes in patients are significant strengths of the method. This approach will undoubtedly contribute significantly to ischemic stroke research, improving the reliability and accuracy of experimental models. Ultimately, this may lead to fewer experimental groups needed to test therapeutic effects, resulting in more reliable experimental outcomes.
The authors have nothing to disclose.
This work was supported by the Hebei Province Introducing Foreign Intelligence Project in 2023. We thank Lisa Kreiner, PhD, from Liwen Bianji, (Edanz) (www.liwenbianji.cn), for editing the English text of a draft of this manuscript.
10% formalin | Beijing Labgic Technology Co.,Ltd. | BL913A | |
11-0 microsurgical sutures | Ningbo Medical Suture Needle Co., Ltd. | 3/8 1×5 | |
2,3,5-triphenyltetrazolium chloride (TTC) | SIGMA | T8877 | |
3-0 silk suture | Ningbo Medical Suture Needle Co., Ltd. | 3-0 | |
4% isoflurane | Tianjin Ringpu Bio-Technology Co., Ltd. | 20221102 | |
6-0 silk suture | Ningbo Medical Suture Needle Co., Ltd. | 6-0 | |
70% ethanol | Shandong Lierkang Medical Technology Co., Ltd. | 20230408 | |
Filament | Xinong Technologies Co Ltd. | 2838-A5 | |
Ketamine | Jiangsu Hengrui Pharmaceutical Co., Ltd. | 230613BL | |
Male Sprague Dawley rats | Jinan Pengyue Experimental Animal Breeding Co., Ltd. | SCXK (Lu) 20190003 | |
Microclips | Shanghai Jinzhong Medical Device Factory | W40150 | |
Microscope | ZEISS | S100 OPMI pico | |
Nursing box | Beijing Fuyi Electrical Appliance Co., Ltd. | FYL-YS-100L | |
Xylazine | SIGMA | PHR3263 |
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