Standardization of The Rat Intraluminal MCAO Model: A Detailed Protocol with Quality Control Measures

Summary

This protocol demonstrates an optimized 60-min middle cerebral artery occlusion (MCAO) technique in rats. The protocol is validated using multimodal approaches, achieving an 87.5% success rate with minimal mortality and yielding reproducible infarct and behavioral outcomes for stroke research.

Abstract

The rat middle cerebral artery occlusion (MCAO) model remains the reference standard for elucidating ischemic stroke pathophysiology and validating neuroprotective therapies. Subtle surgical deviations, however, can profoundly alter infarct volume and behavioral outcomes. Using a "visualize-then-systematize" framework, we re-examined every step of model establishment and validation. Detailed photographic documentation of key surgical landmarks and critical steps was performed to standardize anatomical orientation and filament placement. 2,3,5-Triphenyltetrazolium chloride (TTC) staining was used to precisely map the volume and topology of infarcts at 24 h and 72 h after reperfusion. Neurological deficits were graded with the Zea-Longa, Bederson, and modified Garcia scales. Twenty-four male Sprague-Dawley rats underwent 60 min transient MCAO; infarct volume and neurological scores were assessed at 24 h and 72 h. Approximately 87.50% of animals achieved successful cerebral blood-flow blockade (Zea-Longa ≥ 1 and unequivocal TTC-defined infarct), with an overall mortality of 12.50%, attesting to the model's high fidelity. This standardized protocol provides a technically refined, reproducible foundation for preclinical stroke research, facilitating inter-laboratory consistency in studies investigating stroke pathophysiology and therapeutic interventions.

Introduction

Stroke is the second leading cause of death and the third leading cause of disability worldwide^1,2. Stroke disproportionately affects low- and middle-income countries, where it occurs, on average, 15 years earlier than in high-income countries¹. The two main types are ischemic stroke (85%) and intracerebral hemorrhage (15%)³. Ischemic stroke, accounting for the vast majority of strokes, results from cerebral blood-flow blockage, leading to cell death and brain infarction⁴. The ischemic cascade involves multiple biochemical events, including significant pro-oxidative processes^5,6.

Despite extensive pre-clinical and clinical efforts, only a handful of therapies—most notably intravenous thrombolysis with tissue plasminogen activator and mechanical thrombectomy—have proven efficacious, and these treatments are limited by narrow therapeutic windows and strict patient-selection criteria. Consequently, there is an urgent need to elucidate the cellular and molecular mechanisms underlying ischemic injury, neurovascular repair, and post-stroke recovery. A reproducible, clinically relevant animal model is indispensable for achieving this goal.

The middle cerebral artery occlusion (MCAO) model is broadly used to explore ischemic stroke in rodents^7,8. The intraluminal filament occlusion method is one of the most classic and widely used MCAO techniques⁹. This technique can model both permanent and transient occlusions without requiring craniectomy. A newer transfemoral technique using endovascular wires has shown promise for inducing transient MCAO in rats, offering more consistent outcomes¹⁰. This model involves occluding the middle cerebral artery, typically using an intraluminal filament technique¹¹. MCAO causes focal cerebral hypoperfusion, leading to ischemia and reperfusion injury¹².

While MCAO is valuable for investigating stroke pathophysiology and potential treatments, it can produce variable lesion volumes⁷. Therefore, improvements to the MCAO model—such as refining reperfusion techniques, establishing standardized operating protocols, and developing unified evaluation criteria—can reduce variability in infarct location and volume, thereby enhancing its utility in ischemic stroke research.

This protocol requires a learning curve of approximately 10–15 surgeries to achieve consistent results¹³. Success rates may vary based on operator experience and rat strain¹⁴. The model is suitable for studying acute ischemic stroke but may not fully replicate human comorbidities such as hypertension or diabetes¹⁵. Accordingly, we sought to devise and rigorously validate an optimized MCAO protocol that aims to reduce experimental variability through standardized procedures and rigorous quality control measures. Four complementary innovations were integrated to achieve dual gains in standardization and data reliability: (1) Dual-time-point longitudinal assessment—neurological scores from the Zea-Longa, Bederson and Modified Garcia scales were obtained in parallel with 2,3,5-Triphenyltetrazolium chloride (TTC)-derived infarct volumes at 24 h and 72 h post-reperfusion, delineating the temporal relationship between edema resolution and functional recovery; (2) Triple-scale cross-validation—blinded, dual-investigator scoring with integrated tri-scale calibration significantly increased the sensitivity and precision of behavioral evaluation; (3) Whole-course hypothermic rapid-sectioning—a standard operating procedure (SOP) of "90 s ice bath extraction followed by single-step 2-mm coronal slicing in pre-chilled molds" was established to minimize autolysis and human error, ensuring high-fidelity volumetric measurements; (4) Integrated quality-control framework—a comprehensive SOP spanning preoperative fasting, anesthesia depth control, preoperative thermoregulation, and cross-contamination prevention was constructed to deliver a reproducible and readily transferable MCAO standard operating procedure, laying a solid foundation for inter-laboratory consistency and cross-study comparability.

Protocol

The experiment was approved and supervised by the Institutional Animal Experiments and Experimental Animal Welfare Committee of Capital Medical University. All protocols complied with the NIH Guide for the Care and Use of Laboratory Animals (NIH Publ. 80-23, 1996 rev.), and sterile equipment and aseptic techniques are required.

1. Animal selection

1. Use male Sprague-Dawley rats (aged 6–8 weeks, weighing 240–270 g) obtained from a certified laboratory animal supplier (License No. SCXK (Beijing) 2021-0011) to establish a model of MCAO.
2. House the animals conventionally with ad libitum food and water; all procedures followed the International Code of Ethics.
3. Use the following criteria based on animal body weight for filament size selection to ensure consistent occlusion:
   - 220–250 g: 0.33–0.35 mm diameter
   - 250–280 g: 0.35–0.38 mm diameter
   - 280–310 g: 0.38–0.40 mm diameter
   NOTE: These specifications account for age-related increases in vessel diameter and reduce the incidence of SAH or incomplete occlusion¹³.

2. Sample size

1. Calculate sample size using a sample size calculation software. Based on preliminary data showing an expected infarct volume of 35% ± 10% (mean ± SD) in the MCAO group versus 0% ± 2% in the SHAM group, estimate an effect size (Cohen's d) . For a two-tailed Mann-Whitney U test (non-parametric alternative to t-test), with α = 0.05 and power (1-β) = 0.80, calculate a minimum sample size.
   NOTE: The effect size was estimated as 3.5 and the minimum sample size was 6 animals per group.
2. To account for anticipated mortality (12.5%) and exclusion rate (5–10%), include 24 rats in the MCAO group and 6 rats in the SHAM group, providing adequate power (>0.90) for the primary endpoint (infarct volume at 24 h).

3. Inclusion and exclusion criteria

1. Include the animals if they met the following criteria: (1) male Sprague-Dawley rats, 240–270 g, 6–8 weeks old; (2) absence of pre-existing neurological deficits (preoperative neurological score = 0); (3) successful induction of anesthesia without complications.
2. Exclude animals from analysis if they exhibited: (1) subarachnoid hemorrhage (SAH) evident at necropsy; (2) premature death (<24 h post-reperfusion) unrelated to experimental intervention; (3) failed occlusion (neurological score = 0 and absence of TTC-defined infarct); (4) technical surgical failure (e.g., CCA rupture).
   NOTE: These criteria were prespecified prior to study commencement to minimize selection bias.
3. Animal replacement policy: Replace only animals excluded due to technical surgical failure or intraoperative death to maintain statistical power. Do not replace animals with unsuccessful modeling (failed occlusion) but report them as part of the success rate calculation to avoid artificially reducing variability.

4. Pre-surgical preparations

1. Prepare autoclaved tools, sterile filament, and a draped field.
2. No routine fasting is required; when necessary, limit the fasting duration to 2–4 h. Induce anesthesia with isoflurane (3–4%) and maintain it at 1.5–2% in 30–40% O₂ during the procedure. Confirm the depth of anesthesia by the loss of the righting reflex and toe-pinch.
3. Maintain body temperature at 37 ± 0.5 °C using a feedback-controlled heating pad with rectal temperature monitoring.
4. Shave the neck and scalp, then clean the area with povidone-iodine, followed by 70% ethanol.
5. Administer buprenorphine (0.05 mg/kg, subcutaneous) 30 min prior to surgery and every 12 h postoperatively for 48 h, or meloxicam (1 mg/kg, subcutaneous) once daily for 3 days¹⁶. Assess pain by monitoring grooming behavior, posture, and spontaneous activity; provide additional analgesia if signs of discomfort persist.

5. Occlusion of the MCA

1. Place the rat in dorsal recumbency with the head gently extended to expose the ventral neck.
2. Make a midline cervical incision (1.5–2.0 cm) using iris scissors.
3. Bluntly separate the sternomastoid and omohyoid muscles using curved forceps to expose the left common carotid artery (CCA) bifurcation.
4. Ligate the external carotid artery (ECA) distally with a permanent 4-0 silk suture and proximally with a temporary ligature. Create a small incision between the two ECA ligatures using microscissors.
5. Temporarily occlude the CCA and internal carotid artery (ICA) using microvascular clamps (60 g pressure).
6. Insert a 4-0 silicone-coated monofilament (0.35–0.40 mm diameter) through the ECA incision. Advance it retrogradely into the CCA, then redirect it into the ICA past the CCA bifurcation.
   NOTE: Ensure the filament enters the ICA toward the cranial base, not the pterygopalatine artery (PPA) which branches laterally. If resistance is encountered at <10 mm, retract the filament, rotate the animal's head 30° toward the contralateral side, and re-advance to redirect past the PPA origin.
7. Advance the filament 18–20 mm from the CCA bifurcation until slight resistance indicates occlusion of the MCA origin. Verify the depth using the pre-marked reference line on the filament (positioned 2 mm proximal to the silicone stopper), measured with a caliper from the ECA stump insertion point. Release the CCA clamp once the filament is in position.

6. Reperfusion

1. After 60 min of occlusion, reopen the incision and loosen the temporary ECA ligature.
2. Gently withdraw the filament until its tip clears the ECA stump, thereby restoring blood flow. Promptly place a permanent ligature proximal to the stump to prevent back-bleeding. Close the wound in layers.
3. Transfer the rat to a warmed recovery cage for continuous monitoring.

7. Sham surgery

1. Prepare the animal following the same procedure described above for MCAO (fasting, anesthesia, temperature control, sterile draping).
2. Perform cervical incision and CCA bifurcation exposure as above.
3. Dissect and expose the vessel. Do not insert the filament.
4. Let the arteries remain patent; do not induce cerebral ischemia.
5. Close the incision in layers after 10 min of exposure. Transfer the animal to a warmed recovery cage for monitoring.

8. Postoperative neurological scores

1. Evaluate at 24 h and 72 h after reperfusion, immediately before sacrifice for infarct volume measurement.
2. Habituate the animal in a quiet room, 25 °C for 30 min.
3. Have two independent investigators blinded to group allocation. Have each investigator record scores on separate sheets without consultation; any inter-rater difference ≥1 point triggers re-evaluation.
4. Scales (in order of administration): Perform Zea-Longa¹⁷(Table 1), Bederson¹⁸ (Table 2), and Modified Garcia¹⁹ (Table 3) scoring, following the latest published protocols for consistent criteria.
5. Use the mean of the two observers for statistical analysis. Exclude data from any animal if the mean scores differ by >10% and replace with a new subject.

9. Measurement of brain infarct volume

1. At 24 h and 72 h post-reperfusion, deeply anesthetize rats with 5% isoflurane. Confirm deep anesthesia by loss of righting reflex and toe-pinch response, together with stable respiration, before proceeding.
2. Rapidly remove the brain within 90 s of deep anesthesia confirmation, rinse with ice-cold PBS, and cut into 2 mm coronal slices using a chilled rat brain matrix. During slicing, adhere to the following precautions
   1. Keep the entire procedure on ice at 0–4 °C; harvest the brain within 90 s and position it flat in a pre-chilled matrix.
   2. Use a sharp blade to make single, clean cuts for precise 2 mm coronal sections, collecting each slice in rostro-caudal order into ice-cold PBS.
   3. Immediately clean the matrix and blade with 75% ethanol between animals to prevent cross-contamination.
3. After pre-warming 2% TTC to 37 °C, immerse the slices completely and incubate in the dark for 20 min, gently swirling every 5 min at 20 min.
4. Promptly stop the reaction by rinsing three times with 4 °C PBS. Then fix in 4% paraformaldehyde at 4 °C overnight.
5. At 300 dpi, photograph both sides of every slice once.
   1. Use fixed illumination against a white background to eliminate shadows and reflections.
   2. Lay slices flat without overlap, and include a scale bar and ID label in each frame. Hold the camera perpendicular to the sections and keep the focal length and white balance constant throughout.
6. Quantify infarct volume using ImageJ.
   1. Import the 300 dpi image (RGB format) and convert to 8-bit grayscale (Image > Type > 8-bit).
   2. Apply Otsu's auto-thresholding (Image > Adjust > Auto Threshold > select Otsu) to separate the infarct (white) from viable tissue (red).
   3. Use the Freehand selections tool to outline the ipsilateral hemisphere boundary.
   4. Measure the area (Analyze > Measure) and record the pixel count.
   5. Calculate infarct volume: Σ (infarct area × slice thickness [2 mm]) × (contralateral hemisphere volume/ipsilateral hemisphere volume), expressed as a percentage of the contralateral hemisphere.

10. Statistical analysis

1. Present the data as mean ± standard deviation (SD) or median [interquartile range] as appropriate.
2. Perform between-group comparisons using the unpaired Student's t-test or Mann-Whitney U test for non-normally distributed data.
3. Set statistical significance at P < 0.05. Conduct all analyses using an appropriate statistical analysis software.

Results

Animals were randomly allocated to either 24 h (n = 10) or 72 h (n = 10) post-reperfusion survival groups for terminal TTC staining. An additional 4 rats underwent MCAO but died before the scheduled endpoint (mortality rate 12.5%). The SHAM group comprised n = 6. Thus, the total number of animals used was 30 (24 MCAO + 6 SHAM).

Rats underwent 60-min MCAO to induce cerebral ischemia (Figure 1). Reperfusion was initiated by removing the intraluminal filament and rel...

Discussion

It is important to clarify that the primary objective of this study was methodological standardization rather than biological discovery. Developing a standardized MCAO protocol is vital for reducing inter-laboratory variability and enhancing reproducibility in ischemic stroke research. This protocol enhances procedural transparency through rigorous quality control measures and addresses common technical deficiencies in MCAO modeling, including standardized filament insertion, temperature regulation, and histological proc...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2% TTC staining solution	Beijing Solarbio Science & Technology Co., Ltd.	T8170	25 g/bottle, ≥98 % purity
4% PFA	Beijing Solarbio Science & Technology Co., Ltd.	P1110	500 mL ready-to-use
Absorbable suture	Shandong Weigao Group Medical Polymer Co., Ltd.	VCP345H	5-0 Vicryl
Adson forceps	Shanghai Jinzhong Surgical Instrument Co., Ltd.	JD-11010	1×2 teeth, 12 cm
Dumont curved forceps	Shanghai Jinzhong Surgical Instrument Co., Ltd.	JD-11052	11 cm length, 0.05 mm fine tip
Feedback heating pad	Shenzhen Reward Life Science & Technology Co., Ltd.	69001	37 ± 0.3 °C closed loop
ImageJ	National Institutes of Health	version 1.53k
Iris scissors	Shanghai Jinzhong Surgical Instrument Co., Ltd.	JD-14060	Curved tip, 10 cm
Micro-vascular clips	Shenzhen Ruiwode Lift Technology Co.,Ltd.	JD-16010	1.5 cm curved clip
Non-absorbable silk	Shanghai Pudong Jin-Huan Medical Products Co., Ltd.	S-2304	6-0 Black silk thread
PASS software	NCSS, LLC, Kaysville, UT, USA	version 15.0	Sample size calculation software
Rat brain matrix	Shenzhen Ruiwode Lift Technology Co.,Ltd.	68610	2 mm coronal slots
Silicone-coated nylon filament	Shenzhen Ruiwode Lift Technology Co.,Ltd.	60-1250	0.35 mm OD, 30 mm silicone tip, EO-sterile
Small-animal anesthesia machine	Shenzhen Ruiwode Lift Technology Co.,Ltd.	R500IE	Isoflurane vaporizer, induction chamber & mask included
SPSS	IBM Corp., Armonk, NY, USA	version 26.0	Statistical analysis software
Vannas micro-scissors	Shanghai Jinzhong Surgical Instrument Co., Ltd.	JD-14058	Curved tip, 8 cm