The intraluminal middle cerebral artery occlusion (MCAO) model is the most frequent used model among experimental ischemic stroke models. Here we will demonstrate the entire model in detail with the guide of Laser Doppler flowmetry, and its representative results.
Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion in the United States in 20091,2. The intraluminal middle cerebral artery occlusion (MCAO) model was developed by Koizumi4 in 1986 to simulate this impactful human pathology in the rat. A modification of the MCAO method was later presented by Longa3. Both techniques have been widely used to identify molecular mechanisms of brain injury resulting from ischemic stroke and potential therapeutic modalities5. This relatively noninvasive method in rats has been extended to use in mice to take advantage of transgenic and knockout strains6,7. To model focal cerebral ischemia, an intraluminal suture is advanced via the internal carotid artery to occlude the base of the MCA. Retracting the suture after a specified period of time mimics spontaneous reperfusion, but the suture can also be permanently retained. This video will be demonstrating the two major approaches for performing intraluminal MCAO procedure in mice in a stepwise fashion, as well as providing insights for potential drawbacks and pitfalls. The ischemic brain tissue will subsequently be stained by 2,3,5-triphenyltetrazolium chloride (TTC) to evaluate the extent of cerebral infarction8.
This protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Florida and is in compliance with the “Principle of Laboratory Animal Care” (NIH publication No. 86-23, revised 1985).
1. Materials
2. Pre-surgical steps
3. MCA occlusion surgery:
Koizumi’s4 method
Longa’s3 method
The rest of the procedure is largely convergent:
4. Post-Op neurological evaluation
After the animal regains total consciousness, neurological deficits can be evaluated by a simple scale (five-point scale) as follows3:
5. Staining and quantitative measurement of brain infarct volume
6. Representative Results
After occlusion and reperfusion of the MCA via left CCA to establish transient cerebral ischemia, animals are sacrificed and their coronally sectioned brains are stained with TTC to evaluate the volume of infarct (Figure 1).
Figure 1. TTC-stained serial coronal brain sections (2 mm) from mice subjected to MCA occlusion.
The MCAO model is an established model that replicates cerebral ischemia in murine species. This model was first used in rats and thereafter
adapted to mice. Avoiding craniectomy to eliminate the influence of surgical manipulation on blood brain barrier permeability and intracranial pressure is an advantage
of this technique. However, many variables, such as strain-related differences10, size of suture tip11, duration of occlusion6,
body temperature12, anesthesia13, and other factors can lead to inconsistency in infarct volumes in this model.
There are specific technical details that warrant mention:
Koizumi’s Method | Longa’s Method | |
Site of suture insertion | Ipsilateral Common Carotid artery | Ipsilateral External Carotid artery |
Permanent arterial sacrifice | Common Carotid and External Carotid arteries | External Carotid artery |
Risk of lethal intraoperative arterial rupture/hemorrhage | Less concern | Greater concern |
Possibility of suture diversion into Pterygopalatine artery | Yes | Yes |
Procedural time | Generally quicker | Generally longer |
Laser Doppler Flowmetry: Percentage drop from baseline | >= 80% | >= 80% |
Laser Doppler Flowmetry: occlusion/reperfusion trend |
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Table 1. Comparison of two methods of intraluminal middle cerebral artery occlusion (MCAO) in mice
The authors have nothing to disclose.
Name of the reagent | Type | Company | Catalogue number | Comments (optional) |
---|---|---|---|---|
Male C57BL/6 mice | Animal | Harlan Laboratories, Indianapolis, IN, USA | 20-26 grs | |
Dissection microscope | Microscope | Leica stereo Microscope MZ6 | 6.3:1 zoom | |
Fiber optic illuminator | Illuminator | TechniQuip Corp., Livermore, CA, USA | FO1–150 | |
Isoflurane | Anesthesia | Baxter Pharmaceutics, Deerfield, IL | 1001936060 | |
SuperCut Iris scissor, straight | Surgical tool | World Precision Instruments, Inc., Sarasota, FL, USA | 14218 | |
Vannas micro-scissor, straight | Surgical tool | World Precision Instruments, Inc., Sarasota, FL, USA | 14003 | |
Dumont fine tip forceps | Surgical tool | World Precision Instruments, Inc., Sarasota, FL, USA | 503290 | |
Vessel clip | Surgical tool | World Precision Instruments, Inc., Sarasota, FL, USA | 14120 | |
High Frequency Desiccator 900 | Surgical tool | World Precision Instruments, Inc., Sarasota, FL, USA | 500397 | |
7-0 surgical Nylon monofilament suture | Suture | Ethicon, Inc., Somerville, NJ, USA | 1647G | Suture for occlusion |
7-0 surgical Silk suture | Suture | Ethicon, Inc., Somerville, NJ, USA | 7733G | Suture for ligation |
Silicone RTV adhesive | Silicone rubber | World Precision Instruments, Inc., Sarasota, FL, USA | 1571 | |
Vetbond | Tissue adhesive | 3M Company, St Paul, MN, USA | 70200746587 | |
Puralube Vet Ointment | Eye ointment | Pharmaderm, Melville, NY, USA | ||
Buprenorphine hydrochlorode | Analgesics | Hospira Inc., Lake Forest, IL, USA | NDC 0409-2012-32 | |
Thermal Cautery Unit | World Precision Instruments, Inc., Sarasota, FL, USA | 501292 | ||
PeriFlux System 5000 | TCD flowmetry | Perimed, Stockholm, Sweeden | ||
BAT-12 | Rectal thermometer | World Precision Instruments, Inc., Sarasota, FL, USA | BAT-12R | |
T/PUMP, TP600 | Thermal blanket | Gaymar, NY, USA | ||
2,3,5-triphenyltetrazolium chloride | Reagent | Sigma-Aldrich Co., St Louis, MO, USA | T8877 | |
Mice brain matrix slicer | Brain slicer | Zivic-Miller Lab., Inc., Allison Park, PA, USA | BSMAS001-1 |