Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats

Summary

In this paper, we demonstrate a standard method for producing an embolic middle cerebral artery occlusion with homologous blood clots (fibrin-rich) in adult rat. This model closely mimics human ischemic stroke and is suitable for preclinical study of thrombolytic therapy for ischemic stroke.

Abstract

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic stroke. However, the use of tPA is limited by its narrow therapeutic window and by increased risk of hemorrhagic transformation. There is an urgent need to develop suitable stroke models to study new thrombolytic agents and strategies for treatment of ischemic stroke. At present, two major types of ischemic stroke models have been developed in rats and mice: intraluminal suture MCAO and embolic MCAO. Although MCAO models via the intraluminal suture technique have been widely used in mechanism-driven stroke research, these suture models do not mimic the clinical situation and are not suitable for thrombolytic studies. Among these models, the embolic MCAO model closely mimics human ischemic stroke and is suitable for preclinical investigation of thrombolytic therapy. This embolic model was first developed in rats by Overgaard et al.¹ in 1992 and further characterized by Zhang et al. in 1997². Although embolic MCAO has gained increasing attention, there are technical problems faced by many laboratories. To meet increasing needs for thrombolytic research, we present a highly reproducible model of embolic MCAO in the rat, which can develop a predictable infarct volume within the MCA territory. In brief, a modified PE-50 tube is gently advanced from the external carotid artery (ECA) into the lumen of the internal carotid artery (ICA) until the tip of the catheter reaches the origin of the MCA. Through the catheter, a single homologous blood clot is placed at the origin of the MCA. To identify the success of MCA occlusion, regional cerebral blood flow was monitored, neurological deficits and infarct volumes were measured. The techniques presented in this paper should help investigators to overcome technical problems for establishing this model for stroke research.

Introduction

Stroke is the third leading cause of death in the United States, but treatment options for acute stroke remain limited. At present, intravenous infusion of recombinant tissue plasminogen activator (tPA) to dissolve the blood clots is the most efficient therapy for acute ischemic stroke. However, the use of tPA is limited by its narrow therapeutic window and by increased risk of intracerebral hemorrhage. Therefore, a model of stroke suitable for thrombolytic research is urgently needed.

The middle cerebral artery (MCA) is the artery most often occluded in stroke in humans. Focused on this artery, many animal models of ischemic stroke have be....

Protocol

Ethics Statement: Male adult Sprague-Dawley rats (weighing 330-380 g) were used in this protocol. This protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at the LSU Health Science Center-Shreveport and is in compliance with the 'Guide for the Care and Use of Laboratory Animals' (eighth edition, National Academy of Sciences, 2011).

1. Preparation of Modified PE-50 Tube

1. Hold a 30 cm long PE-50 tube above a gas fire by hands, gradually soften the tube. Gently stretch the tube.
2. Select a point on the stretched tube using a digital caliper (Figure 1), mark it, and cut th....

Results

Laser Doppler flowmetry (LDF) was used to monitor rCBF during induction of cerebral ischemia^6,7. Many laboratories including our laboratory have been using rCBF to identify animals with successful MCA occlusion, but the thresholds of baseline varied between laboratories which are related to the site of measurement. The probe of the LDF is positioned at 2 mm posterior and 5 mm lateral to the bregma as described previously⁶. rCBF was monitored at 0, 5, 15, 30, 60, 90, and 120 min after injection of th.......

Discussion

In this study, we demonstrated a standard method for performing an embolic MCAO stroke model in the rat, in which the origin of the MCA is occluded by a fibrin-rich clot. The major advantage of this model is: the occlusion of the stem of MCA with a fibrin-rich blood clot is similar to thromboembolic stroke in humans, the embolic stroke model is suitable for performing preclinical investigation of fibrinolytic therapy, and that this model can develop a reproducible and predictable infarct volume within the territory suppl.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
rh-tPA	Chemical	Genentech
2,3,5-triphenyltetrazolium chloride	Chemical	Sigma	T8877
Anesthesia vaporizer	Equipment	Soma Technology	Drager Vapor 19.1
Rechargeable high speed micro drill	Equipment	Fine Science Tools	18000-17
Curved scissors	Equipment	Fine Science Tools	14117-14
Dumont forceps (Medical #7)	Equipment	Fine Science Tools	11270-20
Dumont forceps (Medical #5)	Equipment	Fine Science Tools	11251-35
Vannas-style spring scissors	Equipment	Fine Science Tools	15000-03
Veterinary recovery chamber	Equipment	Peco Services	V1200
Genie plus syringe pump	Equipment	Kent Scientific Corporation
Rat brain matrix	Equipment	Kent Scientific Corporation	RBMA-310C
Digital caliper	Equipment	World Precision Instruments	501601
Dissecting microscope	Equipment	World Precision Instruments	PZMTIII-BS-LWD
Hamilton syringe	Equipment	Hamilton	model 710
Homeothermic blanket control unit	Equipment	Harvard Apparatus
Electric clipper	Equipment	Braintree Scientific	CLP-9931
Veterinary electrosurgical unit	Equipment	MACAN Manufacturing Company	MV-9
Blood flowmeter	Equipment	Adinstruments
PowerLab 4/30	Equipment	Adinstruments
LabChart 7.2	software	Adinstruments
1 ml syringe	Consumable	Becton, Dickinson and Company	309659
23 G needle	Consumable	Becton, Dickinson and Company	305143
30 G needle	Consumable	Becton, Dickinson and Company	305106
PE-50 tubing	Consumable	Becton, Dickinson and Company	427517
PE-10 tubing	Consumable	Becton, Dickinson and Company	427400
6-0 Silk suture	Consumable	Harvard apparatus	723287
5-0 Silk suture	Consumable	Harvard Apparatus	517607