Two-vessel Occlusion Mouse Model of Cerebral Ischemia-reperfusion

Summary

A mouse model of cerebral ischemia-reperfusion is established to investigate the pathophysiology of stroke. We distally ligate the right middle cerebral artery and right common carotid artery and restore blood flow after 10 or 40 min of ischemia.

Abstract

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse model is useful for investigating the pathophysiology of cerebral ischemia-reperfusion and determining potential therapeutic strategies for patients with stroke. Variations in the anatomy of the circle of Willis of C57BL/6 mice affects their infarct volume after cerebral-ischemia-induced injury. Studies have indicated that distal MCA occlusion (MCAO) can overcome this problem and result in a stable infarct size. In this study, we establish a two-vessel occlusion mouse model of cerebral ischemia-reperfusion through the interruption of the blood flow to the right MCA. We distally ligate the right MCA and right common carotid artery (CCA) and restore blood flow after a certain period of ischemia. This ischemia-reperfusion injury induces an infarct of stable size and a behavioral deficit. Peripheral immune cells infiltrate the ischemic brain within the 24 h infiltration period. Additionally, the neuronal loss in the cortical area is less for a longer reperfusion duration. Therefore, this two-vessel occlusion model is suitable for investigating the immune response and neuronal recovery during the reperfusion period after cerebral ischemia.

Introduction

The cerebral ischemia-reperfusion mouse model is one of the most widely used experimental approaches for investigating the pathophysiology of ischemia-induced brain injury¹. Because cerebral ischemia-reperfusion activates the peripheral immune system, peripheral immune cells infiltrate into the ischemic brain and cause neuronal damage². Thus, a reliable and reproducible mouse model that mimics cerebral ischemia-reperfusion is required to understand the pathophysiology of stroke.

C57BL/6J (B6) mice are the most commonly used strain in stroke experiments because they can easily be genetically manipulated. Two common models of MCAO/reperfusion that mimic the condition of cerebral ischemia-reperfusion are available. The first is the intraluminal filament model of proximal MCAO, where a silicon-coated filament is employed to intravascularly occlude the blood flow in the MCA; the occluding filament is subsequently removed to restore blood flow³. A short occlusion duration results in a lesion of the subcortical region, whereas a longer occlusion duration causes infarcts in the cortical and subcortical areas. The second model is the ligation model of distal MCAO, which involves extravascular ligation of the MCA and CCA to reduce the blood flow through the MCA, after which blood flow is restored through the removal of the suture and aneurysm clip⁴. In this model, an infarct is caused in the cortical areas, and the mortality rate is low. Because the ligation of MCAO/reperfusion model requires craniectomy to expose the site of the distal MCA, the site can be easily confirmed, and examining whether the blood flow in the distal MCA is disrupted during the procedure is straightforward.

B6 mice exhibit considerable variations in the anatomy of their circle of Willis; this might affect the infarct volume following cerebral ischemia-reperfusion^5,6,7. Currently, this problem can be overcome through ligation of the distal MCA⁸. In this study, we establish a method for occluding the MCA blood flow and enabling reperfusion after a predetermined period of ischemia. Two-vessel occlusion of the cerebral ischemia-reperfusion model induces transient ischemia of the MCA territory through ligation of the right distal MCA and right CCA, with blood flow restored after a certain period of ischemia.This MCAO/reperfusion model induces an infarct of stable size, a bulk of brain-infiltrating immune cells in the ischemic brain, and a behavioral deficit after cerebral ischemia–reperfusion⁴.

Protocol

The institutional animal care and use committees of Academia Sinica and Taipei Medical University approved this protocol for the use of experimental animals.

1. MCAO/reperfusion model

1. Provide the mice with free access to water and chow until the surgery.
2. Autoclave the surgical tools and sanitize the surgery table and equipment using 70% ethanol. Wear a surgical mask and sterile gloves. Use a dry bead sterilizer to resterilize the surgical tools if multiple mouse surgeries will be conducted in one experiment.
3. Anesthetize an 8- to 12-week-old mouse (mass: 25–30 g) by using 0.8% chloral hydrate, via an intraperitoneal injection. Make sure the anesthetized mouse does not have a pedal reflex (as tested using a firm toe pinch) after the anesthetization.
4. Use vet ointment to prevent eye dryness for the mouse while it is under anesthesia.
5. Use a noninvasive blood pressure system to monitor the mouse’s blood pressure.
6. Use a physiological monitoring system to monitor its rectal temperature and arterial blood gases. Maintain the body temperature at 36.5 ± 0.5 °C.
7. Subcutaneously inject the mouse with a prophylactic antibiotic (25 mg/kg cefazolin)⁸.
8. Place the mouse in the supine position on the heating pad.
9. Use electric clippers to expose the skin by shaving the mouse’s fur on the ventral neck region, as well as in the region between the right eye and right ear.
10. Use epilating cream to clear the fur from the mouse’s body and disinfect the surgical site alternating scrbus with povidione-iodine and  70% ethanol.
11. Use iris scissors to cut a 1 cm-long midline incision at the neck.
12. Use iris forceps to carefully dissect the CCA free from the vagus nerves without causing physical injury.
13. Use 5-0 silk sutures to isolate the CCA.
14. Make a 0.3 cm incision in the scalp at the midpoint between the right eye and right ear.
15. Use microscissors to cut the temporalis muscle to expose the zygomatic and squamosal bone.
16. Under a stereo dissecting microscope, use a microdrill to create a 2 mm-diameter hole directly over the right-side distal MCA.
17. Ligate the trunk of the right-side distal MCA using a 10-0 suture.
18. Occlude the right-side CCA using a nontraumatic aneurysm clip.
19. After either 10 or 40 min of ischemia, remove the aneurysm clips and suture to restore blood flow to the MCA and CCA.
20. Use a suture clip to seal the skin incision on the head.
21. Seal the cervical skin incisions using a single suture followed by closing neck skin with suture or staple⁹. 
22. Subcutaneously inject buprenorphine (0.1 mg/kg) for pain relief⁹.
23. Maintain the mouse’s body temperature at 36.5 ± 0.5 °C on the heating pad until it has fully recovered from the anesthesia. Do not return the animal that has undergone surgery to the company of other animals until it has fully recovered. Do not leave the animal unattended until it regains sufficient consciousness.
24. Place the mouse into the autoclaved cage so that it can freely access water and chow after it has fully recovered.

2. Staining with 2,3,5-triphenyltetrazolium chloride

1. Anesthetize the mouse with 0.8% chloral hydrate via an intraperitoneal injection.
2. Use operating scissors to decapitate the animal.
3. Expose the skull by using iris scissors to make an incision in the skin of the head.
4. Use operating scissors to cut the anterior of the frontal bone.
5. Use iris scissors to cut the skull along the sagittal suture.
6. Use a bone rongeur to push aside the frontal and parietal bone and expose the brain.
7. Use iris forceps to dissect the brain.
8. Use a mouse brain matrix and razor blades to obtain 2 mm coronal slices.
9. Stain the brain slices for 10 min at 37 °C with 2% 2,3,5-triphenyltetrazolium chloride (TTC) in 1x phosphate-buffered saline.
10. Rinse the brain 2x with 10% formalin.
11. Fix the brain in 10% formalin at room temperature for 24 h. 

3. Measurement of infarct size

1. Arrange the sections on a clean plastic slide and orient the sections from rostral to caudal.
2. Scan the slide using a scanner. Place a metric ruler and make sure it is visible in the scanned image. Flip the slide over and scan the reverse side.
3. Calculate the infarction area of each section using ImageJ software.
   1. Open the image file and set up the scale for the image.
   2. Use freehand selection to select the infarct area.
   3. Use the regions of interest (ROI) manager to measure the area of interest.
4. Sum the infarction areas for each section and multiply the result by the section thickness to estimate the total infarction volume.

4. Statistical analysis

1. Use GraphPad Prism 6 to determine the statistical significance with Student’s t-test.
   NOTE: The error bars on the bar graphs represent standard errors of the mean (SEMs).
2. Use G*Power 3.1 to calculate the appropriate sample size and perform a power analysis¹⁰.

Results

This MCAO/reperfusion procedure produced a cortical infarct in the vicinity of the right MCA and caused a behavioral deficit. Different degrees of ischemia-induced infarct volume (Figure 1A,B) and neuronal loss (Figure 1C,D) were created in the cerebral cortex of the right MCA area through an increase in ligation duration. This MCAO/reperfusion injury decreased the animal's locomotor activity...

Discussion

The MCAO/reperfusion mouse model is an animal model commonly employed to mimic transient ischemia in humans. This animal model can be applied to transgenic and knockout mice strains to investigate the pathophysiology of stroke. Several steps in the protocol are especially critical. (1) The microdrill must be carefully used when creating a hole in the skull, with inappropriate action easily causing bleeding from the MCA. (2) The MCA should not be damaged, and bleeding must be avoided before and after the ligation procedur...

Materials

Name	Company	Catalog Number	Comments
Bone rongeur	Diener		Friedman
Buprenorphine	Sigma	B-044
Cefazolin	Sigma	1097603
Chloral hydrate	Sigma	C8383
Dissection microscope	Nikon	SMZ-745
Electric clippers	Petpro
10% formalin	Sigma	F5304
Germinator dry bead sterilizer	Braintree Scientific
Iris Forceps	Karl Klappenecker		10 cm
Iris Scissors	Diener		9 cm
Iris Scissors STR	Karl Klappenecker		11 cm
Microdrill	Stoelting	FOREEDOM K.1070
Micro-scissors-Vannas	HEISS	H-4240	blade 7mm, 8 cm
Mouse brain matrix	World Precision Instruments
Non-invasive blood pressure system	Muromachi	MK-2000ST
Operating Scissors STR	Karl Klappenecker		14 cm
Physiological Monitoring System	Harvard Apparatus
Razor blades	Ever-Ready
Stoelting Rodent Warmers	Stoelting	53810	Heating pad
Suture clip	Stoelting
Tweezers	IDEALTEK		No.3
Vetbond	3M	15672	Surgical glue
10-0 suture	UNIK	NT0410
2,3,5-Triphenyltetrazolium chloride	Sigma	T8877