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 been established. At present, two major types of rodent focal ischemia models by occluding MCA have been developed: suture MCAO model and embolic MCAO model. Although MCAO models via the intraluminal suture technique have been widely used in mechanism-driven stroke research, these suture models do not mimic human stroke, as up to 80% of human strokes are caused by thrombosis or embolism. However, embolic stroke model using blood clots closely mimics human stroke and is considered suitable for thrombolytic study. This embolic model was first developed in rats by Overgaard et al.¹ in 1992 and further characterized by Zhang et al. in 1997² and by Dinapoli et al. in 2006³. Although embolic MCAO has gained increasing attention, there are technical problems faced by many laboratories.

In this article, we demonstrate a standard method for producing embolic MCAO with homologous blood clots in the adult rat, which can develop a predictable infarction within the MCA territory. The techniques presented in this article should help investigators to overcome technical problems for establishing this model for stroke research.

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 the modified PE-50 tube into a 25 cm long segment with a 1 cm long tip (outside diameter: 0.30-0.34 mm).

2. Preparation of Homologous Blood Clots

1. Anesthetize the rat with isoflurane (5% for induction, 2-3% for maintenance) in 70% N₂O and 30% O₂ by a face mask prior to blood collection. Confirm anesthesia by a toe pinch.
2. Perform femoral arterial cannulation of a donor rat with published method⁴ and transfer femoral artery blood directly into a 20 cm long PE-50 tube. Place the tube for 2 hr at room temperature to clot blood, and then retain the tube for 22 hr at 4 °C. Note: Cannulation is performed using aseptic technique as described in step 3.1. The donor rat is recovered for further sampling.
3. Cut the PE-50 tube into 50 mm segments and flush out the clot from the tube into a sterile Petri dish containing normal saline.
4. Transfer the 50 mm long clot into a 60 mm long PE-10 tube and connect each end of the PE-10 tube to a 20 cm long PE-50 tube connected to 1 ml syringe containing normal saline with 23 G needle (Figure 2).
5. Shift the clot by continuous alternating movement from one syringe to the other for 5 min, which can remove blood cells from the clot and minimize the fragile of extravascular coagulated clot, without disrupting the fibrin core.
6. Cut the clot into a 40 mm long segment and transfer the clot segment into a modified PE-50 catheter as described in step 1.2 under aseptic conditions. Note: All PE tubing is sterilized with ethylene oxide.

3. Embolic Middle Cerebral Artery Occlusion (Figure 3A,B)

1. Sterilize all surgical tools by autoclaving (minimum 121 °C, 15 PSI, for 15 min). Sanitize the surgery table and associated surgical equipment using 70% ethanol.
2. Anesthetize the rat with isoflurane as described in step 2.1. Test the depth of anesthesia by performing a toe pinch on both rear feet, and any observed movement (withdrawing the paw) indicates that the animal is not sufficiently anesthetized to do surgery. Apply a small amount of vet ointment on both eyes to prevent dryness while under anesthesia. Shave the fur on the ventral neck and head regions with electric clippers to expose the skin areas.
3. Place the rat in the supine position on a heating pad. Insert a rectal probe, control and maintain body temperature between 36.5-37.5 °C using a homeothermic blanket control unit.
4. Disinfect the shaved skin and surrounding fur with 10% povidone-iodine followed by 70% ethanol.
5. Make a 2-cm-long midline incision on the neck under a dissecting microscope. Use retractors to expose the surgical field and dissect the right common carotid artery (CCA), external carotid artery (ECA), internal carotid artery (ICA), and pterygopalatine artery (PPA) free from surrounding nerves and fascia (Figure 3A). Note: Before surgery, change sterile gloves if no assistant helps to prepare the animal.
6. Dissect the CCA free from the surrounding nerves (without harming the vagal nerve) and place a sterile 5-0 silk suture under the artery. Tie a slip knot (#1) and grasp the suture using a small hemostat and pull taught toward the body.
7. Dissect the ECA and its two branches, the occipital artery (OA) and the superior thyroid artery (STA). Coagulate two branches using a veterinary electrosurgical unit. Place two pieces of sterile 6-0 silk sutures under the ECA.
8. Separate the two silks placed under the artery, one piece towards the head (distal end) and the other towards the body. Tie a tight ligature (#2) on the side closest to the head, grasp the silk with small hemostats and pull taught toward the head. Prepare a loose knot (#3) with the other silk to use later.
9. Dissect the ICA and PPA from the surrounding nerves (without harming the vagal nerve). Tie the PPA with a 6-0 suture (#4). Make a slip knot with 6-0 suture around the ICA (#5). Alternatively, clip the ICA using a microvascular clip.
10. Cut a small hole (1/2 thru) into the ECA between the tight (#2) and loose (#3) ligatures with a Vannas-style spring scissors. Insert the modified PE-50 tube containing blood clot into the incision and advance to the bifurcation of the CCA. Tighten the loose ligature (#3) around the lumen just enough to secure preserve mobility of the in-dwelling tube.
11. Cut off the ECA at the site of small hole to free the stump and position the stump below the bifurcation of the ECA and ICA; this will allow the modified tube easily slide into the ICA. Open the ICA and gently advance the tube from the lumen of the ECA into the ICA until the tip of the catheter reaches the origin of the MCA (~17 mm from the bifurcation). Note: Before insert the tip of tube into the ECA, sterile the outer surface of tube with 70% ethanol, and then wash with sterile normal saline.
12. Inject the clot through the modified PE-50 catheter along with 10 μl of saline over 10 sec using a 100 μl Hamilton syringe (Figure 3B).
13. Withdraw the catheter from the ECA 5 min later. Tie the ECA and reopen CCA. Suture the incision on the neck.

4. Monitoring Regional Cerebral Blood Flow (rCBF)

1. Prior to MCA occlusion, make a 1.2 cm long midline incision in the scalp to expose the skull bone. Remove the tissues on the skull bone with a dental scraper and sterile cotton swaps. Note: Before surgery, the exposed scalp area and surrounding fur are disinfected with 10% povidone-iodine followed by 70% ethanol.
2. Drill a 1.5 mm diameter burr hole located at 2 mm posterior and 5 mm lateral to the bregma using a 0.7 mm spherical stainless steel burr (Figure 4).
   NOTE: Keep the dura intact.
3. Place the probe 0.5 mm above the dura surface. Monitor the rCBF at 0 (baseline), 5, 15, 30, 60, 90, and 120 min after embolization and continue at 5, 15, 30, 45, and 60 min after intravenous administration of tPA. After the last measurement of rCBF, the scalp incision is closed by suture. Note: Prior to neck incision we measure rCBF as baseline before MCA occlusion. Post MCA occlusion we measure rCBF after suture closure of neck incision. Thus, the neck incision is kept sterile.

5. Post-operative Care

1. Inject 2.5 ml of saline solution subcutaneously to prevent dehydration and inject Buprenex (0.05 mg/kg, SC) immediately after the surgery and every 6-12 hr as needed for pain relief.
2. Stop isoflurane anesthesia. Place the rat in a 37 °C veterinary recovery chamber (keep animal warm) and keep observation. It generally takes 10 min for the rat to recover from anesthesia. Then, put the animal into a sterilized cage, place some wetted food in a Petri dish in the cage, and return the cage to animal sterilization room.
3. 24 hr after stroke, euthanize the rat with an overdose of sodium pentobarbital (100 mg/kg body weight, IP).

6. Neurological Deficit Score

1. Perform the Bederson score before and at 2 hr after embolization. Use a grading scale of 0-3 as previously described⁵: 0, raising the rat by tail, the animal extending both forelimbs to the floor and exhibiting no other deficits (normal movement); 1, raising the rat by tail, flex the contralateral forelimb; 2, decreased resistance to lateral push (and forelimb flexion) without circling; 3, the same behavior as grade 2 with circling.
   NOTE: Rats showing Bederson score = 0 (no deficit) at 2 hr after embolization are excluded from further study.

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

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