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 in these cases it benefits only 4% of patients². Therefore, it is imperative that reproducible, clinically relevant animal models of stroke are used to aid in the development of potential therapeutic strategies and treatments for this disease. It is important to note that in vitro models, whilst useful in modeling certain aspects of cerebral dysfunction, are not capable of recapitulating the complex physiological interactions that occur in brain and periphery following a stroke. Consequently, in vivo models are essential.

The most common type of stroke is ischemic in origin, accounting for 87% of total strokes. Other strokes are intracerebral hemorrhage (9%) and subarachnoid hemorrhage (4%), and are caused most often by an emboli to the middle cerebral artery (MCA). This is attributable to the prominent curve at the root of MCA, which causes laminar blood flow entering the brain to become disrupted. The MCA arises from the internal carotid artery (ICA) and routes along the lateral sulcus, where it branches and projects to the basal ganglia and the lateral surfaces of the frontal, parietal and temporal lobes, including the primary motor and sensory cortex. The Circle of Willis is created by posterior cerebral arteries being connected to the cerebral arteries and the posterior communicating arteries.

The intraluminal filament or suture model of MCAo is one of the most widely used in stroke research. However, there are a couple of different variations to this model, and these are based on whether the microfilament is inserted into the external carotid artery (ECA, termed the Longa method)³, or whether it is inserted into the ICA (termed the Koizumi method)⁴. In Koizumi's method, the common carotid artery (CCA) on the side of the surgery must be permanently tied if the filament is removed to prevent bleeding from the incision in the CCA, while in Longa's method it is the ECA that must be permanently tied⁵. Here the Longa method will be used as we feel this is a far superior and a more clinically relevant surgical model of ischemic stroke. Furthermore, the use of a silicon-tipped monofilament, especially with the Longa method, produces very reproducible MCAo as opposed to the flame-blunted monofilaments, which often produce incomplete occlusion and/or subarachnoid hemorrhage⁶.

The intraluminal filament method can be used as a model of permanent or transient occlusion^4,6. To perform the transient model, the filament is removed after a period of ischemia (e.g., 30 min, 60 min, or 2 hr), and reperfusion is allowed to happen. This model, to some extent, simulates the restoration of blood flow after spontaneous or therapeutic intervention (e.g., tPA administration) to lyse a thromboembolic clot in humans. For the permanent model, the filament is simply left in place for a period of time (e.g., 24 hr), so no reperfusion occurs. Another advantage of the intraluminal filament method is the fact that a craniotomy does not need to be performed, allowing the skull to be left intact and avoiding any changes in intracranial pressure and temperature.

In this video we demonstrate how to perform the Longa intraluminal filament method to induce MCAo and reperfusion. We also show how to perform the 18-point neurological score and determine the infarct volume using 2,3,5-triphenyltetrazalium chloride (TTC) staining.

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) injected intraperitoneally (i.p.). Monitor depth of anesthesia by foot pinch initially every 3 - 5 min and every 10 min once anesthesia is achieved. While the animal is under anesthesia, administer a sterile ocular ointment to prevent dryness. The initial dose of ketamine/xylazine usually lasts about 30-40 minutes.  An additional dose can be administered if needed, which is verified by the animal’s response to a foot pinch.
2. Place mice in a supine position on a temperature regulated heat mat and maintain body temperature at 36.5 ± 0.1 °C, which is verified using a rectal probe.
3. Shave the neck and disinfect the skin with 70% ethyl alcohol. 

2. Occlusion of the MCA (Figure 1)

1. Make a midline neck incision using Iris straight scissors and retract (using retractors) the soft tissues to expose the vessels.
2. Dissect the CCA and the ECA from surrounding tissue using Dumont forceps without damaging the vagus nerve.
3. Make a temporary suture by tying a loose knot around the CCA using 6-0 silk.
4. Make a permanent suture around the ECA and the smaller vessels extending from it by tightly ligating the vessels (distal to the bifurcation of the CCA).
5. Make a suture around the ECA proximal to the CCA bifurcation.
6. Place a microvessel clip around the internal carotid artery (ICA) and the pterygopalatine artery (PPA).
7. Make a small incision in the ECA using micro dissecting spring scissors and insert a 180 μm silicon-tipped monofilament. Make the cut as close to the permanent suture as possible for easier manipulation of the filament.
8. Tighten the temporary suture around the ECA with the filament inserted and remove the microvessel clip.
9. Cut the ECA between the permanent, distal suture and the entry point of the filament using micro dissecting spring scissors.
10. Guide the filament through the ICA until resistance is felt (approx. 9 - 10 μm beyond the bifurcation of the CCA) in the MCA.
    NOTE: In the event too much resistance is felt while the majority of the filament is still visible, the filament may have entered the PPA. If this happens, pull the filament back to the bifurcation, gently push forward into the ICA using Dumont forceps, and advance the filament until it can be visualized in the ICA.

3. Reperfusion

1. After a 30-min occlusion period, remove the filament by gently pulling it back using Dumont forceps and secure the suture around the open end of the ECA.
2. Remove the temporary suture around the CCA by carefully loosening the ligature using Dumont forceps and blood flow is resumed through the CCA.
3. Close the incision with a continuous surgical suture. Closure of the skin can be accomplished by either continuous or interrupted sutures.  Skin staples are also an acceptable method. 
4. Inject mice with 1 ml of saline subcutaneously as volume replenishment and with the analgesic carprofen (5 mg/Kg, s.c.) for relief of pain and discomfort from the surgical procedure, Signs that indicate additional pain relief is needed include hunched back, ungroomed coat, decreased activity, abnormal posturing, and decreased appetite.
5. Observe mice throughout the recovery from the anesthesia in a heated 30 °C cage using a heat lamp regulated using a temperature controller and place mashed chow in a petri dish on the floor of the cage to encourage eating. The mice will be housed one per cage during the reperfusion period.

4. Sham Surgery

1. Subject mice to the same procedure without the monofilament insertion.

5. Post-operative Neurological Scores (Table 1; Figure 2)

1. Neurologically evaluate mice after the appropriate reperfusion period using an 18-point scoring system assessing the General; Motor; Sensory; Proprioception. A higher neurological score corresponds to decreased neurological function.
   NOTE: Mice that are judged unresponsive and unable to walk will be euthanized. Other criteria for humane euthanasia will include a weight loss of greater than 20%, respiratory distress, and infection around the surgical area. A CO₂ chamber will be used for euthanasia and the physical method to confirm death will be cervical dislocation or thoracotomy.

6. Measurement of Brain Infarct Volume (Figure 3)

1. Induce anesthesia using a combination of ketamine (150 mg/kg) and xylazine (10 mg/kg) injected i.p. Monitor depth of anesthesia by foot pinch initially every 3 - 5 min and every 10 min once anesthesia is achieved.
2. Place mice in a supine position and cut the skin from the abdomen to the neck followed by the peritoneum using iris straight scissors.
3. Lift up the sternum using Dumont forceps and cut the ribs to expose the heart.
4. Open the left and the right side of the chest using two pairs of hemostats, exposing the heart.
5. Insert a 26 G needle attached to a 5 ml syringe into the left ventricle and cut the right atrium. Perfuse with room temperature normal saline or phosphate buffered saline (PBS) until the fluid becomes clear (usually 3 - 5 min).
6. Remove the skull carefully away from the brain using iris straight scissors and Dumont forceps.
7. Cut off the olfactory bulbs and the cerebellum using a razor blade so that the brain will fit in the matrix. Use this matrix to slice the brain into even segments. Chill the matrix before use to keep the tissue cool. Place the brain into the matrix and set it on ice.
8. Slice the brain into 2 mm coronal segments using two razor blades. Make the first cut using a razor blade beginning 2 mm from the top and leave this razor blade in place. Make another 2 mm cut behind the first. Remove the first razor blade with the tissue attached. Repeat this process until all tissue has been sliced.
   NOTE: There should be 4 - 5 segments when finished.
9. Place the segments into 24-well plate containing 2% 2,3,5-triphenyltetrazalium chloride (TTC), which is then placed in a shallow water bath at 37 °C for 20 min. Placing each segment into an individual well helps to keep them in the order in which they were cut. Ensure that the TTC solution completely covers the tissue segments. After 10 min turn all the slices over.
10. Place a small amount of 10% formalin into the wells of a new 24-well plate and transfer the segments to this plate in the order in which they were cut.
11. Scan the segments into the computer and analyze the infarct size as a percentage of the whole brain slice⁶ using ImageJ analysis software (NIH 1.57 Image Software)⁶.
    1. Outline the infarcted area to generate an area measurement. Next measure the entire contralateral hemisphere to determine the area. Divide the infarct area by the area of the contralateral hemisphere and multiply by 100 to determine the infarct volume.

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