Modeling Stroke in Mice – Middle Cerebral Artery Occlusion with the Filament Model

To guarantee high quality and reproducibility, we recommend the use of Standard Operating Procedures (SOP). In this video, published SOPs as developed and used in our laboratory are applied. ¹

1. Middle Cerebral Artery Occlusion

1. Mice are anaesthetized with an appropriate anaesthetic regime in consult with veterinary staff. (E.g. induction with 1.5 – 2 % Isoflurane and maintainance with 1.0 – 1.5 % Isoflurane in 2/3 N2O and 1/3 O2 using a vaporizer).
   1. Body temperature of the mice is maintained at 36.5°C ± 0.5°C during surgery with a heating plate. A feedback controlled heating pad, which warms according to the rectal temperature of the mouse, is highly recommended.
   2. Disinfect the skin and surrounding fur with an appropriate agent (e.g. 70% ethyl alcohol) and dry it afterwards.
2. A midline neck incision is made and the soft tissues are pulled apart.
3. The left common carotid artery (LCCA) is carefully dissected free from the surrounding nerves (without harming the vagal nerve) and a ligature is made using 6.0/7.0 string. 5.0 string can also be used.
4. The left external carotid artery (LECA) is then separated and a second knot is made.
5. Next, the left internal carotid artery (LICA) is isolated and a knot is prepared with a 6.0 filament.
6. After obtaining good view of the left internal carotid artery (LICA) and the left pterygopalatine artery (LPA), both arteries are clipped, using a microvascular clip.
7. A small hole is cut in the LCCA before it bifurcates to the LECA and the LICA. A monofilament made of 8.0 nylon coated with silicon hardener mixture (see below) is then introduced into the LICA, until it stops at the clip. Attention has to be paid not to enter the occipital artery. (Figure 1)
8. The clipped arteries are opened while the filament is inserted into the LICA to occlude the origin of the LMCA in the circle of Willis.
9. The third knot on the LICA is closed to fix the filament in position.
10. The mice receive saline 0.5 mL subcutaneously as volume replenishment. For pain relief, Lidocaine gel is topically applied in the wound.
11. If reperfusion is intended, mice stay for 30 – 90 min occlusion in a heated cage, the wound could be closed with a small suture clip. Afterwards, a second anaesthesia is performed, the third knot on the ICA is momentary opened and the filament withdrawn.
12. The remaining sutures are shortened and the skin is adapted with a surgical suture.
13. All animals receive a second volume replenishment as described above.
14. Animals are put in a heated cage for two hours to control for body temperature.
    1. The animals must be checked daily after surgery for signs of discomfort. The mice could show some post surgical weight loss. They receive mashed food in a petri-dish placed on the floor to encourage eating. The food is replaced daily for seven days.

2. Sham Operation

1. For sham operations the filament is inserted to occlude LMCA and withdrawn immediately to allow instant reperfusion (1.8). The subsequent operation is identical to that performed on the animals undergoing cerebral ischaemia (1.9 – 1.14) including a second anaesthesia.

3. Preparation of the Filament

1. Sterility of the filament should be considered. The use of sterilized equipment as well as an appropriate handling of the filament afterwards is a prerequisite for a sterile surgery. Disinfection of the filament is difficult, since many of the common sterilization methods may worsen the quality of the filament. However methods such as radiation, for example with UV light or γ rays, or chemical sterilization, for example with highly reactive gases such as ethylene oxide, are applicable.
2. 8.0 nylon filament is cut into lengths of 11 mm under the microscope
3. The filament tip must be coated completely and evenly over a length of 8 mm with a hardener mixture of Xantopren M Mucosa and Activator NF Optosil

4. Representative Results

Depending on the duration of blood flow restriction, different motor and behavioural deficits result. Both after 30 and 60 min of cerebral ischaemia, animals in most cases show decreased resistance to lateral push and circling due to disturbance in locomotion. Milder lesions manifest as a flexor position in the front limbs. These easily observable signs can be used as a basic score for the success of the operation.²

Morphologically the lesion can be assessed using either histology or Magnetic Resonance Imaging (MRI). Sixty minutes occlusion of the middle cerebral artery produces tissue pannecrosis in an area including both the striatum and the neocortex, whereas 30 minutes of ischaemia cause mainly neuronal cell death limited to the striatum. ³ ( Figure 2) In terms of infarct volume, we expect a standard deviation lower than 30% in a set of operations. Mortality depends on the occlusion time with around 5 % after 30 min of ischaemia and 10 – 20 % after 60 min.

Another minimal invasive possibility is the use of Laser Doppler flowmetry (LDF) during the operation, which allows a direct control of its success. In an individual animal, reduction to 10 – 20 % of preocclusion values clearly indicates successful induction of focal cerebral ischemia. ⁴ However, LDF cannot be used as a method for interindividual comparisons, since LDF can only measure quantitative changes (in percentages) of blood flow within a small and limited tissue sample volume. It does not give information about the spatial extent of blood flow reduction. ⁵

There are several tests to assess behavioural aspects after stroke, including gait analysis ^6,7, Rotarod ⁸, Pole test ^9,10, adhesive removal test ^11,12, staircase test ^13,14, ladder rung test^15,16 and Morris water maze¹⁷. In all these tests, mice subjected to MCAo perform less successfully than control animals.

Figure 1. Scheme of the vessel architecture supplying the brain (depicted in background) in the mouse. Different strains may show variations, for example the occipital artery sometimes leaves from the internal carotid artery.

Figure 2. Schematic illustration of typical lesion sizes after different time points of reperfusion in the proximal MCAo model. In the middle, the typical course of functional activity and cerebral blood flow after MCAo are depicted. (MCAo: Middle cerebral artery occlusion, LDF: Laser Doppler flow measurement)