Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice

Summary

In order to understand the pathophysiology of stroke, it is important to use reliable models. This paper will describe one of the most frequently used stroke models in mice, termed the middle cerebral artery occlusion (MCAo) model (also termed the intraluminal filament or suture model) with reperfusion.

Abstract

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ischemic in origin and occur in the territory of the middle cerebral artery (MCA). Currently the only Food and Drug Administration (FDA) approved drug for the treatment of this devastating disease is tissue plasminogen activator (tPA). However, tPA has a small therapeutic window for administration (3 - 6 hr), and is only effective in 4% of the patients who actually receive it. Current research focuses on understanding the pathophysiology of stroke in order to find potential therapeutic targets. Thus, reliable models are crucial, and the MCA occlusion (MCAo) model (also termed the intraluminal filament or suture model) is deemed to be the most clinically relevant surgical model of ischemic stroke, and is fairly non-invasive and easily reproducible. Typically the MCAo model is used with rodents, especially with mice due to all the genetic variations available for this species. Here we describe (and present in the video) how to successfully perform the MCAo model (with reperfusion) in mice to generate reliable and reproducible data.

Introduction

Stroke is the fifth leading cause of death worldwide, with one person dying from the disease every 4 minutes. Over 800,000 Americans suffer a stroke every year, which is not only devastating for the patient, but also for their families. Stroke is the main cause of adult disability and the annual expenditure is estimated to be in the order of $ 36.5 billion¹ despite very few treatment options being available.

Tissue plasminogen activator (tPA) is the only Food and Drug Administration (FDA) licensed drug for ischemic stroke. However, it is only effective if administered to patients within 3-6 hours from the onset of the stroke, and....

Protocol

This protocol and the experiments reported in the video were approved by the LSUHSC-S Institutional Animal Care and Use Committee and are in compliance with the guidelines of NIH.

NOTE: Male C57BL/6 mice weighing 25 - 29 g were used in this study. The mice were maintained on a standard chow pellet diet with free access to water, under a 12 hr light/dark cycle in individually ventilated cages. The procedure will be performed under sterile conditions using sterile techniques (e.g., sterile gloves, sterile instruments).

1. Pre-surgical Preparations

1. Induce anesthesia using a combination of ketamine (150 mg/kg) and xylazine (10 mg/kg) inj....

Results

Mice underwent 30-min MCAo-induced brain ischemia (Figure 1) followed by a period of reperfusion (24 hr and 1 wk are presented here, but the length of reperfusion can be varied). The mortality during MCAo was minimal (approximately 2%). Post ischemia, the mortality rate (within the first 24 hr) was around 26%.

Laser Doppler flowmetry was used to confirm blood flow perfusion in the MCA territory before and after .......

Discussion

Since its conception 20 years ago, the MCAo model for human stroke involving insertion of a filament has been used in a huge number of studies. This is mainly due to the fact that it mimics what happens clinically in the most common form of stroke (i.e., ischemic stroke). The striatum is more sensitive to ischemia than the cerebral cortex, and as such, the length of ischemic time will translate into whether both the striatum and the dorsolateral cortex will be affected, or just the striatum. Both infarct and rep.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Male C57BL/6 mice	Jackson Laboratory, Bar Harbor, ME	#000664
Ketamine Hydrochloride	Morris & Dickson, Shreveport, LA	67457-108-10
Xylazine	Akorn, Inc, Lake Forest, IL	NADA# 139-236
DC temperature control system	FHC, Bowdoin, ME	40-90-8D
Mini rectal thermistor probe	FHC, Bowdoin, ME	40-80-5D-02
Heating pad	FHC, Bowdoin, ME	40-90-2-06
Clippers	Amazon, Bellevue, WA	#64800
70% ethanol	Worldwide Medical Products, Bristol, PA	#51011023
Dissecting microscope	Olympus, Center Valley, PA	SZ40
Iris scissors (straight)	Fine Science Tools, Foster City, CA	11251-20
Dumont forceps (45° bent tip)	Fine Science Tools, Foster City, CA	11297-00
Micro vessel clip	Fine Science Tools, Foster City, CA	18055-05
Micro dissecting spring scissors (straight)	Fine Science Tools, Foster City, CA	14088-10
Retractors (blunt)	Fine Science Tools, Foster City, CA	18200-11 (Helen used 17022-13)
Cotton tipped applicators	Fisher Scientific, Waltham, MA	23-400-100
Gauze sponges	Covidien, Mansfield, MA	#9023
7-0 silk braided surgical suture	Braintree Scientific, Braintree, MA	SUT-S103
0.9% sodium chloride	Morris & Dickson, Lake Forest, IL	0409-4888-20
6-0 medium MCAO suture (silicon rubber coated monofilament)	Doccol Corporation, Sharon, MA	6023PKRe
Sofsilk 6-0 silicone coated braided silk	Covidien, Mansfield, MA	SUT-14-1
Carprofen	Pfizer, New York, NY	NADA# 141-199
Puralube	Dechra, Norwich, UK	NDC 17033-211-38
Physitemp temperature controller	Harvard Apparatus, Holliston, MA	TCAT-2AC
Heat lamp	Harvard Apparatus, Holliston, MA	HL-1
Laser doppler probe	AD Instruments, Colorado Springs, CO	MSP100XP
24-well plates	Fisher Scientific, Waltham, MA	#353226
Phosphate buffered saline (PBS)	Life Technologies, Carlsbad, CA	20012-050
Single edge razor blades	Fisher Scientific, Waltham, MA	12-640
2,3,5-triphenyltetrazalium chloride (TTC)	Sigma Aldrich, St. Louis, MO	T8877-50G
Mouse brain matrix slicer	Braintree Scientific, Braintree, MA	BS-A 5000C
Water bath	VWR, Radnor, PA	#182
10% formalin	Sigma Aldrich, St. Louis, MO	HT501128-4L
ImageJ analysis software	NIH, Bethesda, MD	free download
Retractor	Medical Device Purchase, Newcastle, CA	MP-740