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, includin....

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 w....

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