A Rat Model of Middle Cerebral Artery Occlusion/Reperfusion without Damaging the Anatomical Structure of Cerebral Vessels

Abstract

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.

Introduction

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.

Protocol

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

1. House the animals in an environment maintaining a 12 h light/dark cycle, controlled temperature, and humidity. Ensure they have unrestricted access to food and water.

2. Anesthesia

1. Anesthetize the rats via intraperitoneal injection with a mixture containing ketamine (80 mg/kg) and xylazine (20 mg/kg).
2. Confirm the induction of anesthesia by assessing the withdrawal reflex of the hind paw and the blinking reflex. If adequate depth of anesthesia is not achieved, supplement with isoflurane during the surgical procedure.

3. Ischemic modeling procedure

1. Sterilize all surgical tools, operating tables, and related equipment using 70% ethanol.
2. Prepare the surgical site by shaving the fur around the throat and left neck area, excluding the anticipated incision site. Disinfect the skin at the surgical site three times with 70% ethanol.
3. Make a midline incision on the ventral side, approximately 1.5 cm long, and open the superficial fascia.
4. Perform sharp and blunt dissection to expose the external carotid artery (ECA), internal carotid artery (ICA), and common carotid artery (CCA) as per the previously described method⁷.
5. Place three 4-0 silk sutures around the CCA and another silk suture through the ECA near the ICA bifurcation. Tie a releasable slip knot on the proximal CCA. Tighten the suture on the distal CCA using a hemostat to block blood flow. Loosely tie another slip knot around the CCA during suturing to ensure the vessel is not occluded.
   NOTE: Ensure that the slip knot is not overly tight, only sufficient to block blood flow, as it will need to be released during vascular suturing.
6. Make a 0.5 mm incision between the proximal silk suture and the middle suture on the CCA using scissors.
   NOTE: Ensure that the incision allows the filament (silicon-coated tip) to enter the CCA.
7. Introduce a 3 cm length of 4-0 filament (silicon-coated tip) into the CCA. Tighten the silk sutures around the CCA to secure the intraluminal filament and prevent bleeding; then, remove the suture from the distal CCA.
8. Continue to insert the filament into the lumen of the MCA, controlling the sutures around the ECA to ensure the filament does not enter the ECA. The insertion length is typically 18 mm to 20 mm.
9. Start a timer and record the time when occlusion begins. Suture the neck incision with 3-0 silk sutures and carefully place the animal in a recovery cage. Administer 0.5 mL of normal saline intraperitoneally.

4. Microsurgical suturing of the CCA

1. Re-anesthetize the rat 1.5 h post-ischemia. Disinfect the incision site three times with 70% ethanol and reopen the incision.
2. Gently withdraw the occluding filament until the white tip is clearly visible near the CCA incision. Place a micro clamp horizontally close to the secured intraluminal suture knot near the CCA bifurcation.
   NOTE: Avoid clamping the white filament tip to prevent silicone detachment and subsequent thrombosis.
3. Release the secured intraluminal suture knot and completely remove the filament.
4. Place another micro clamp near the heart-end side of the proximal CCA knot. Subsequently, remove the suture knot around the proximal CCA.
   NOTE: Proper placement of clamps is crucial for elevating fixed vessels in later steps to facilitate microvascular suturing. Place clamps horizontally on surrounding muscles to prevent slippage.
5. Rinse the incision site with normal saline and dry the area with a sterile cotton ball to remove the blood and achieve a clear surgical field.
6. Under a microscope (4x magnification), suture the CCA incision using 11-0 microsurgical sutures in an interrupted pattern, typically requiring two stitches depending on the incision size.
7. After suturing, remove the micro clamps from both ends of the CCA to restore blood flow.
8. Apply pressure with a sterile cotton ball to the CCA suture site for 30 s. Observe the CCA for 2 min to ensure no bleeding occurs. Suture the rat's neck incision and place the animal in a 35 °C care box for anesthesia recovery before returning it to the cage.

5. Staining with 2,3,5-triphenyltetrazolium chloride (TTC)

1. Euthanize the animals under deep anesthesia with 4% isoflurane 3 days following ischemia-reperfusion.
2. Remove the brains and cut them into 2 mm-thick coronal sections using a scalpel blade⁸.
3. Incubate the brain sections with 2% TTC for 20 min at 37 °C.
4. Rinse the brain sections twice with 10% formalin, then fix them with 10% formalin for 30 min at room temperature.
5. Capture digital photographs of consecutive slices and quantitatively measure the infarction areas using Image J software.

6. Neurological function test

NOTE: Neurological function was assessed using the Garcia test, following a previous report⁹.

1. Assess spontaneous activity, symmetry in the movement of all four limbs, forepaw outstretching, climbing, body proprioception, and response to vibrissae touch. Assign scores based on the Garcia test criteria, with a maximum score of 18 points indicating normal neurological function.

7. Statistical analysis

1. Utilize GraphPad Prism to conduct statistical analysis using Student's t-test. Express the data as the mean ± standard deviation. Consider differences between means statistically significant if P < 0.05.

Representative Results

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.

Discussion

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 ischemia^10,11,12. 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 perfusion^13,14. Additionally, ischemia of the ECA can impair the function of facial muscles and nerves, impacting the assessment of various neurological functions^15,16.

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.

Materials

Name	Company	Catalog Number	Comments
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