Selective and Permanent Occlusion of the Middle Cerebral Artery in Rats: An Experimental Approach for Studying Motor and Spatial Memory Deficits

Summary

The objective of this study was to establish a reproducible method for performing a craniectomy followed by permanent, selective middle cerebral artery occlusion in rats for application in experimental studies of motor and spatial memory deficits.

Abstract

With major clinical relevance, ischemic stroke (IS) demands experimental approaches that enable the development of novel adjuvant therapies. Due to the similarity of the cerebral vascular anatomy to humans, rats are widely used as experimental models of IS. However, there remains a need for methods capable of generating an ischemic environment that more closely resembles that observed in humans, employing an approach that prioritizes selective arterial occlusion. In this context, the aim of this study was to establish a reproducible method for performing a craniectomy followed by permanent and selective occlusion of the middle cerebral artery (MCA) in rats, to standardize the model and reduce technical challenges for application in studies of motor and spatial memory deficits. Wistar rats (N = 28) were equally divided into two groups, one undergoing craniectomy alone (SHAM) and the other craniectomy followed by selective MCA occlusion (MCAO). On the seventh and eighth postoperative days, the postural test and the object location recognition memory test were performed, respectively. After euthanasia, brain specimens were collected, and Nissl-stained histological sections were prepared. To demonstrate the feasibility and effectiveness of the method, a detailed surgical protocol was developed, including preoperative preparation, anesthesia, positioning, craniectomy, MCAO, and closure. The applied method demonstrated deficits in the MCAO group, as evidenced by the postural test (p = 0.015) and the object location recognition memory test (p < 0.001). Histological analysis confirmed the selectivity of the approach, with injury involving hippocampal-associated areas such as the perirhinal, entorhinal, and piriform cortices, in addition to the external capsule, while deeper brain structures were preserved. This protocol proved to be a reproducible and effective method for inducing selective MCA ischemia in rats and represents a valid and technically accessible tool for experimental studies investigating pathophysiological and therapeutic mechanisms in ischemic stroke.

Introduction

The ischemic penumbra surrounding the infarcted area in ischemic stroke (IS) is capable of recovery if treatments are administered in a timely manner, aiming to prevent or mitigate motor and memory impairments¹. However, therapies such as chemical thrombolysis and mechanical thrombectomy remain poorly accessible in clinical practice for a large segment of the population², supporting the need for experimental models to investigate more widely available treatment strategies. Animal studies are indispensable due to their reproducibility and the ability to analyze collateral circulation³, with rats being widely used in IS models⁴. Owing to its close correlation with human IS⁵ and its vessel diameter, the middle cerebral artery (MCA) is the primary target vessel in experimental studies of arterial occlusion^6,7,8.

One of the most widely used animal models to induce cerebral ischemia is filament-based intraluminal occlusion followed by reperfusion^9,10. Nevertheless, the variability associated with the precise positioning of the filament within the MCA, inadvertent occlusion of adjacent vessels of the circle of Willis, collateral effects due to external carotid artery (ECA) ligation required for the procedure, and the duration of occlusion all contribute to conflicting results in meta-analyses^11,12.

In the clinical setting, most patients suffering from IS do not achieve cerebral reperfusion and do not exhibit hypothalamic damage or sequelae associated with ECA occlusion, highlighting the need for experimental approaches that induce selective ischemia without subsequent reperfusion. An alternative is permanent MCA occlusion (MCAO) performed via craniectomy. With direct visualization of the MCA, selective occlusion prevents damage associated with ECA ligation and injury to the anterior choroidal, hypothalamic, and posterior cerebral arteries¹³, resulting in an ischemic effect more closely resembling that observed in lacunar infarction.

Recent studies indicate that animal models of IS should exhibit a closer correlation between the ischemic area and unfavorable clinical outcomes^14,15. Developing technical alternatives that enable targeted induction of ischemia with minimal complications interfering with the results appears to be the most suitable strategy. Within this context, the aim of this protocol was to establish a reproducible method for performing craniectomy followed by permanent and selective MCAO in rats, with the purpose of standardizing the model and reducing technical challenges, for use in studies of motor and spatial memory deficits. This approach was adapted from classical studies^16,17,18,19 that demonstrated high survival rates, allowing long-term evaluations.

Using the inferior cerebral vein (ICV) as an anatomical landmark, this protocol seeks to demystify the technical difficulty historically cited as the major limitation of this approach, enabling the investigation of selective cerebral ischemia in both acute and chronic phases. Motor function and spatial memory were assessed to verify the deficits induced by the proposed lesion model, in addition to histological analysis of ischemic brain regions.

Protocol

The protocol followed the guidelines for the care and use of laboratory animals established by the National Council for the Control of Animal Experimentation (CONCEA) and was approved by the Ethics Committee on Animal Experimentation (CEUA) of the State University of Western Paraná (UNIOESTE), protocol number 25-23.

1. Preoperative preparation

1. Prepare surgical instruments, gauze, cotton pads, and position the stereomicroscope (Figure 1).
2. Clean the surgical table and equipment with 70% ethanol. Prepare sterile surgical instruments and perform all surgical procedures using aseptic technique.

2. Anesthesia

1. Induce anesthesia with a mixture of ketamine (3.75 mL), xylazine (2.5 mL), and 0.9% sodium chloride (3.75 mL). Administer 0.2 mL of the mixture per 100 g of body weight intraperitoneally. Confirm anesthesia depth using a hind paw or tail pinch.
2. If necessary, administer additional doses corresponding to one-third of the maximum dose to avoid overdose.
3. Administer the prophylactic antibiotic enrofloxacin (20 mg/kg) and the analgesic tramadol hydrochloride (12 mg/kg) intraperitoneally.
4. Measure rectal temperature and maintain body temperature at 37°C (± 2°C) using a heating pad throughout the procedure.

3. Positioning

1. Place the rat in lateral decubitus on a cotton pad on the microscope stage (Figure 1A). Support the head with a small soft roll and align it at 180° to expose the left lateral surface of the skull. Secure the head with adhesive tape (Figure 2A).
2. Apply lubricating eye drops and tape the eyelids closed.
3. Recheck rectal temperature and adjust the heating source if needed.

4. Craniectomy

1. Perform trichotomy between the lateral canthus and the tragus (approximately 1.6 cm × 0.5 cm) using a mini trichotomizer or razor blade (Figure 1B).
2. Disinfect the shaved area with 2% chlorhexidine scrub and then 0.2% aqueous chlorhexidine.
3. Make a 1-cm skin and subcutaneous incision using straight scissors (Figure 1C), maintaining a 0.3-cm margin posterior to the lateral canthus and anterior to the tragus (Figure 2B).
   ​NOTE: Retract this layer with Adson forceps (Figure 1D) or hemostatic forceps.
4. Identify and coagulate the middle temporal branch of the superficial temporal vessels using electrocautery (Figure 1E).
5. Palpate the zygomatic arch using a freer elevator (Figure 1F) and incise the temporal muscle fascia longitudinally.
6. Dissect the temporal muscle cranially and caudally to expose the temporal and frontal bones and part of the masseter muscle. Isolate the zygomatic arch longitudinally and remove an approximately 0.5-cm segment (Figure 2C).
   1. Precautions:
      1. Control bleeding from the anterior deep temporal branch of the maxillary vessels using compression or electrocautery.
      2. Avoid posterior oblique cuts to prevent damage to the temporomandibular joint.
      3. Avoid injury to the extraorbital lacrimal gland located beneath the arch.
      4. Avoid opening the orbital cavity.
7. Drill the lateral temporal bone immediately posterior to the zygomatic arch removal site (Figure 2D) using a 3.5-mm diameter round diamond-coated burr mounted on a low-speed rotary motor (Figure 1G). Intermittently irrigate with 0.9% saline to prevent thermal injury. Leave small bone fragments attached to the dura mater (Figure 2E) and remove them gently with Halstead forceps (Figure 1H).
   ​NOTE: A craniectomy using a smaller drill bit is an alternative but increases the risk of dural and brain injury.

5. MCA occlusion (Figure 3)

1. Open the dura mater using a 6 mm × 0.25 mm ultrafine needle.
   NOTE: Avoid damaging adjacent cortical vessels.
2. Identify the inferior cerebral vein (ICV) below the craniectomy window and the MCA crossing perpendicularly above it (Figure 3A,C). Identify the basal and thickest MCA branch located in the center of the craniectomy field.
3. Pass a non-absorbable 8-0 polypropylene suture around the MCA using a fine needle holder (Figure 3B and Figure 1I). Tie three knots to occlude the vessel (Figure 3C).
   NOTE: Other non-absorbable sutures such as nylon, cotton, or silk may be used.
4. Confirm the absence of downstream blood flow. If an additional MCA branch is present, ligate it as well.
5. Irrigate the cavity with saline and perform hemostasis with electrocautery or gentle compression, as needed.
   ​NOTE: Avoid coagulating brain tissue to prevent histological or neuroinflammatory artifacts.

6. Closure

1. Reapproximate the dura mater when possible.
2. Reapproximate the deep muscle layer and suture the temporal muscle fascia with absorbable 4-0 suture (Figure 3D).
3. Close the subcutaneous and skin layers using absorbable 4-0 polyglactin 910 sutures in a continuous pattern.
   NOTE: Avoid leaving exposed suture ends. Continuous suturing is recommended.
4. Clean the surgical site with 0.2% aqueous chlorhexidine.
5. Keep the animals warm until recovery from anesthesia. Monitor temperature before returning them to their cages. The total surgical duration, from skin incision to wound closure, typically ranges between 30 min and 45 min.
6. Postoperatively, administer a second dose of tramadol hydrochloride (12 mg/kg, intraperitoneally) 12 h after surgery for postoperative analgesia. Administer a second prophylactic antibiotic dose the day after surgery.

Results

A total of 28 male Wistar rats (10–12 weeks old, mean weight 349 g) were randomly and equally divided into two groups: MCAO (submitted to the complete protocol described) and SHAM (submitted only to the craniectomy stage). In this study, 3 animals were excluded intraoperatively and replaced during surgery due to venous bleeding (2 MCAO) or minimal cortical injury during craniectomy (1 SHAM). One additional MCAO animal died on postoperative day 5. The overall mortality rate of the model was 3.6%. The final analyzed sample...

Discussion

The MCAO model in rats has been used since 1975^16,17 as an experimental paradigm for cerebral ischemia, and has been refined over the years, demonstrating efficacy in inducing neurological damage^{8,22,23,24,25,26,27,}

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Adson toothed forceps	ABC stainless	L47-P4-164	Other options may be available from different companies
Castroviejo needle holder	Golgran	135-3	Other options may be available from different companies
Electrocautery	Chunguang medical	128	Other options may be available from different companies
Freer elevator	ABC stainless	47P4-819	Other options may be available from different companies
Halsted curved	ABC stainless	AI1055	Other options may be available from different companies
Micro drill	Nail drill	ZS-710	Other options may be available from different companies
Mini trichotomizer	Kemei	KM-666	Other options may be available from different companies
Prolene	Ethicon	8730H	8-0 size suture
Stereomicroscope	Alltion	ASM-112	Other options may be available from different companies
Straight scissor	ABC stainless	L23-W4	Other options may be available from different companies
Surgical compresse	Cremer	158595	Other options may be available from different companies
Surgical gauze	Cremer	198669	Other options may be available from different companies
Thermometer	G-Tech	DMT-2027	Other options may be available from different companies
Vicryl	Ethicon	VCP346H	4-0 size suture