Brain Ischemic Injury in Rodents: The Protective Effect of EPO - Tissue-Protective Cytokines: Methods and Protocols - page 82

3. The respirator is set to provide artificial ventilation with a respi-
ratory volume of 2 mL at 80 breath rates per minutes. Those
parameters should be adjusted regarding the End-tidal CO 2 ,
and the blood gazes measurements (paO 2 , paCO 2 ) and pH.
4. Body temperature is monitored continuously during anesthe-
sia with a rectal probe and maintained around 37.5°C with a
thermostatically controlled heating pad. Rectal temperature
could be monitored after ischemia to evaluate if the animals
experience fever.

The rat is subjected to MCAO by insertion of the 0.380 mm diam-
eter coated nylon monofilament previously described. The MCA
occlusion is confirmed by the reduction of CBF as measured by
LDF probe (Fig. 2 ).

3.1.4 Induction of MCAO

1. Under anesthesia, the rat is placed on its back on the heat-
ing pad. Rectal temperature is maintained at 37.5 ± 0.5°C.
2. Apply artificial tear on the eyes.
3. Shave the fur on the ventral neck region and disinfect the sur-
gical site with betadine.
4. Under the dissecting microscope, a ventral right median inci-
sion of about 2 cm long is performed on the neck.
5. Using forceps, the external and internal carotid arteries (ECA-
ICA) and the common carotid artery (CCA) are carefully
exposed under the operating microscope. The CCA and the
ICA are carefully separated from the vagus nerve. The ptery-
gopalatine artery needs to be exposed too.
6. Once the CCA, the ECA and the ICA are isolated and are free
from surrounding tissue, a permanent ligature is placed on the
distal portion of the ECA. Furthermore, a knot is prepared on
the proximal portion of the ECA near the permanent suture
but is not tight. All knots are performed using 5.0 silk suture.
7. Transient ligatures are placed on the CCA, and the ICA to
temporarily block blood flow coming through when the inci-
sion in the ECA is performed.
8. A small incision is performed in the ECA between the previ-
ously installed knots.
9. The filament is introduced into the ECA, with the coated
extremity directed towards the CCA. Then the proximal knot
is tightened to avoid bleeding from the ECA but allowing the
filament to move easily.
10. The ECA is cut and tilted to be oriented in the direction of the
ICA.
11. The knot previously placed on the ICA is removed.