Middle cerebral artery (MCA) ligation is a technique to study focal cerebral ischemia in animal models. In this method, the middle cerebral artery is exposed by craniotomy and ligated by cauterization. This method gives highly reproducible infarct volumes and increased post-operative survival rates compared to other methods available.
Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to develop suitable animal models to study the events that unfold during ischemic insult. These techniques include transient or permanent, focal or global ischemia models using many different animal models, with the most common being rodents.
The permanent MCA ligation method which is also referred as pMCAo in the literature is used extensively as a focal ischemia model in rodents 1-6. This method was originally described for rats by Tamura et al. in 1981 7. In this protocol a craniotomy was used to access the MCA and the proximal regions were occluded by electrocoagulation. The infarcts involve mostly cortical and sometimes striatal regions depending on the location of the occlusion. This technique is now well established and used in many laboratories 8-13. Early use of this technique led to the definition and description of “infarct core” and “penumbra” 14-16, and it is often used to evaluate potential neuroprotective compounds 10, 12, 13, 17. Although the initial studies were performed in rats, permanent MCA ligation has been used successfully in mice with slight modifications 18-20 .
This model yields reproducible infarcts and increased post-survival rates. Approximately 80% of the ischemic strokes in humans happen in the MCA area 21 and thus this is a very relevant model for stroke studies. Currently, there is a paucity of effective treatments available to stroke patients, and thus there is a need for good models to test potential pharmacological compounds and evaluate physiological outcomes. This method can also be used for studying intracellular hypoxia response mechanisms in vivo.
Here, we present the MCA ligation surgery in a C57/BL6 mouse. We describe the pre-surgical preparation, MCA ligation surgery and 2,3,5 Triphenyltetrazolium chloride (TTC) staining for quantification of infarct volumes.
This protocol was approved by the University of Rochester committee devoted to the ethical use of animals in research (UCAR). Aseptic techniques should be followed during the protocol. The use of sterile gloves and a mask is required.
All the equipment, materials, chemicals, and tools that are used during the protocol are described in Table 1.
1. Pre-surgical Preparation
2. Surgical Procedure and MCA Ligation
3. TTC Staining and Determination of Stroke Volume
4. Directions to use Image J software
5. Representative Results:
The infarcts obtained by permanent MCA ligation in the mouse are mostly cortical. However, it is possible to obtain subcortical lesions if the MCA is ligated proximal to the lenticulostriate branch. The stroke volumes after MCA ligation might vary from 10mm³ to 35mm³ 19, 23. The stroke volumes calculated with TTC staining in Moyanova et al. 19 are between 10mm³ to 22mm³ while the stroke volumes determined from MRI images in Filiano et al 23 are between 20mm³ to 35mm³. The possible reason for these differences maybe the exact location of the ligation and the different methods used to measure the stroke volumes.
In Figure 1, a TTC stained brain 24 hr post permanent MCA ligation in a wild type C57/BL6 mouse is shown. The stroke site is white in appearance (Fig. 1A, B). Figure 1A and B illustrate the infarct site from different angles. Figure 2 shows 1mm thick slices of the TTC stained stroke brain from anterior to posterior (Fig. 2A-G). Infarct volume was calculated 24 h post-surgery. The volume of the infarct is ˜23mm³.
Figure 1. Representation of stroke site. A-B, TTC stained whole brain images, 24 h after MCA ligation. White area represents the infarct.
Figure 2. Representative results. (A>G), TTC stained 1mm brain sections, 24 h after MCAL. Slices are aligned from anterior to posterior. White area represents the infarct.
The permanent MCA ligation method gives highly reproducible infarct volumes and increased post-operative survival rates compared to other methods available. The ease and the short duration (˜30 min) of the procedure make it even more practical. The method is widely used in both mice and rats.
This technique requires an invasive surgery under a stereomicroscope. Therefore, experience in operating under a microscope and perfecting a successful craniotomy is essential. It is best to establish consistent infarcts in the lab before experiments are performed. To attain reproducible results it is important to ligate the MCA at the same exact location each time. The MCA should be ligated proximal to lenticulostriate branches if subcortical infarcts are desired. The artery is fully ligated and the occlusion is permanent. Therefore this model does not allow reperfusion via the MCA. Although not included in this demonstration, it is best if a doppler probe is used for measuring the blood flow in the affected area to verify complete ligation of the artery.
The operator should be very careful not to damage or puncture MCA while exposing and coagulating the artery. Craniotomy should be performed with extensive care to prevent any damage to the zygomatic bone. Because the surgical area is very close to the infarct area, any damage to the cortical surface should be avoided during craniotomy or cauterization. FST 18015-00 cateurizer unit or a bipolar cateurizer can be used instead of the FST 18000-00 used in this demonstration. FST 18015-00 allows the user to adjust the temperature of the cauterizer tip to low heat which might prevent any damage to the cortical tissue. FST 18015-00 also avoids the temperature fluctuations seen in battery operated cauterization tools. During cauterization, the user should be very careful to keep the cauterizer out of the oxygen flow and can momentarily shut down the O2/isofluorane to avoid risk of ignition. This will not affect the anesthesia state of the mouse. To prevent this risk, we use a ventilation pipe extended to the surgical area which increases the air circulation sufficiently, as well as draws off any excess O2/isofluorane in the air.
The permanent MCA ligation model is extremely useful in studying ischemic stroke. It can be used to test neuroprotectants or study molecular mechanisms of ischemia in vivo on transgenic mouse models. The obvious benefit for using this in vivo approach is that this allows for the study of ischemic insult on intact neuronal networks and the behavioral response post-insult, in addition to the neuroinflammatory processes that are present after ischemic damage. Therefore, this in vivo stroke model provides a critical complementary approach to in situ models that are used to mimic ischemia in cell culture.
The authors have nothing to disclose.
The surgical technique was originally acquired in the lab of Dr. William D. Hill at the Medical College of Georgia. The authors would also like to thank Dr. David A. Rempe and Landa Prifti for the use of the dissection camera. This research was supported by NIH NS041744, NS051279, F31 NS064700 and AHA 30815697D.
NAME | COMPANY AND CATALOGUE NUMBER |
---|---|
A. Solutions and Chemicals | |
Povidone-Iodine Solution, Surgical Scrub | Aplicare, 82-209 |
70% Ethanol | Koptec, V1001 |
2,3,5 Triphenyltetrazolium chloride (TTC) | Sigma, T8877 |
4% Paraformaldehyde | Electron Microscopy Sciences, 159SP |
Phosphate Buffered Saline, pH:7.4 | Made and sterilized in the lab. |
Mineral Oil | PML Microbiologicals, R6570 |
Isofluorane | The Butler Company, 029405 |
Buprenorphine (0.3mg/ml) | Hospira, NDC 0409-2012-32 |
Artificial tears | The Butler Company, 007312 |
Recovery Gel | Clear H2O, 72-01-5022 |
B. Surgical Materials and Equipment | |
C57/BL6 mouse, 10-12 weeks old | The colony was bred in the university vivarium |
100% oxygen tank | Airgas |
Anesthetic Vaporizer and Flow meter | Surgivet, Model 100 |
Heating Pad with Rectal Probe | Fine Science Tools, TR200 |
Dissecting microscope | Zeiss, Stemi 2000 |
Heating panel | Petco Services Ltd. 0307-013 |
Small Vessel Cauterizer Set | Fine Science Tools, 18000-00 |
Mini Peristaltic Pump | Harvard Apparatus, MPII |
1ml syringe | BD, 329650 |
18G needle | Becton Dickinson, 305196 |
Surgical 5.0 Nylon Suture | Ethilon, 698H |
Sectioning Block | Kent Scientific, 1mm |
Single Edge Razor Blades | Electron Microscopy Sciences, 71962 |
Label Tape | Scienceware, Bel-art Products F13463-2075 |
Gauze Sponges | Kendall Curity 2252, Johnson and Johnson 6415 |
Cotton Tip Applicators | Puritan 806-WCL |
C. Surgery Tools | |
Surgical Spring Scissors | Fine Science Tools 15003-08 |
Microdissecting forceps angled | Fine Science Tools 5/45 |
Microdissecting forceps curved | Fine Science Tools 5 Ti |
Fine straight scissors | Fine Science Tools 14568-12 |
Curved bone rongeur | Fine Science Tools 16021-14 |
Hartman Hemostat Curved | Fine Science Tools 13003-10 |
Table 1. The list of materials, tools and chemicals used for the MCA ligation procedure. A. Solutions and Chemicals, B. Surgical Materials and Equipment, C. Surgery Tools.