The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

Summary

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.

Abstract

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.

Protocol

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

1. Inject mice subcutaneously with buprenorphine (0.05mg/kg) 2 h before surgery, immediately after the surgery and then every 3-5 h during the first 24 h post-operative period.
2. Sterilize all the surgical instruments, gauze spo....

Discussion

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 establi.......