Induction of Ischemic Stroke and Ischemia-reperfusion in Mice Using the Middle Artery Occlusion Technique and Visualization of Infarct Area

Summary

We describe a mouse model of stroke induced by the occlusion of the middle cerebral artery using a silicone coated suture. The protocol can be applied to induce permanent occlusion or a temporary ischemia, followed by reperfusion.

Abstract

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of stroke on tissue injury and to evaluate the effectiveness of therapeutic interventions, several experimental models in a variety of species were developed. They include complete global cerebral ischemia, incomplete global ischemia, focal cerebral ischemia, and multifocal cerebral ischemia. The model described in this protocol is based on the middle cerebral artery occlusion (MCAO) and is related to the focal ischemia category. This technique produces consistent focal ischemia in a strictly defined region of the hemisphere and is less invasive than other methods. The procedure described is performed on mice, given the availability of several genetic variants and the high number of tests standardized for mice to aid in the behavioral and neurodeficit evaluation.

Introduction

The study of cardiovascular disease, such as stroke, relies on the use of in vivo models. To understand the possible implication of ischemia, drug toxicity, and/or treatment, there is a need to use a suitable, standardized, reliable, and reproducible model of the disease, which enables comparative studies between treatment groups. In this manuscript, we are using mice, given the availability of a large number of transgenic mice and standardized assessment models. Raking scores to assess motor and behavior deficits following experimental ischemic stroke and the following recovery have been developed^1,2....

Protocol

All procedures were approved by the University of Miami Institutional Animal Care and Use Committee (IACUC) in accordance with the National Institutes of Health (NIH) guidelines. The use to sterile equipment and aseptic techniques is required.

1. Preparing the Occlusion Suture

1. Use a suture of 0.21 mm diameter for mice between 20 - 25 g and 0.23 mm for mice between 25 - 35 g of body weight. The choice of the type of sutures for the MCAO procedure depends on animal weight.
2. Using a silver pen, mark the suture at 9 mm starting from the silicone coated tip. This will serve as a guide for insertion length.

Results

The insertion route for the occlusion suture is demonstrated in Figure 1. The MCAO suture is to be routed to the occlusion area, bifurcating in the ICA. Successful occlusion of the MCA will lead to tissue injury, visible by TTC staining. Figure 2 presents images of staining from sham treated animal (Figure 2A) and from a 60 min MCAO ischemia reperfusion animal (staining at 90 min or 24 hr post-occlusion, Figure 2B). To de.......

Discussion

The successful utilization of the described MCAO method is highly dependent on an understanding of cerebral blood flow anatomy. Since the correct placement of the suture is hard to discern due to the lack of direct visual clues, repeated practice is important to master the procedure before using it for investigative studies. Stroke volume should be analyzed to ensure consistent results. The addition of a laser Doppler system can help to determine the successful occlusion of blood flow and should be used periodically to e.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
MCAO suture 0.23 mm	Doccol	702345PK5Re
MCAO suture 0.21 mm	Doccol	702145PK5Re
Silver pen	staples	503205
Anesthesia machine	Vetequip	901806
Surgical scissors	Fine science tool	14558-09
Surgical forceps straight tip	Fine science tool	00108-11
Surgical forceps angled tip	Fine science tool	00109-11
Spring scissors	Fine science tool	15000-08
Nylon suture	Braintree scintific	SUT-S 104
Closing suture	VWR	95057-036
Isoflurane	Piramal
2,3,5-Triphenyltetrazolium chloride	FisherSci	50-121-8005
Brain block	Braintree scintific	BS-A 5000C
Cryostat blade	VWR	89202-606
Optional:
Periflux Laser doppler system	Perimed	Periflux 5000
Monitoring unit	Perimed	PF 5010 - LDPM