Erythropoietin as a Neuroprotectant for Neonatal Brain Injury: Animal Models - Tissue-Protective Cytokines: Methods and Protocols - page 115

1. Prepare a sterile field in the area adjacent to the dissecting
microscope. Tools are autoclaved prior to use, and a hot bead
sterilizer is used between animals. Surgeons wear sterile gloves,
face masks, and clean lab coats.
2. Lay out sterilized surgical instruments, warming pad and ani-
mal cradle (see Fig. 1 , top panel).
3. Prepare dissecting microscope by turning on light source, and
checking focal range.
4. Prepare anesthesia delivery system by connecting regulated
oxygen supply to the anesthetic vaporizer. Connect the vapor-
izer to the induction chamber. The induction chamber con-
nects to the anesthesia outlet (Fig. 1 , bottom panel, arrow)
which is connected to an exhaust or trap. A rubber dam can be
secured to the anesthesia outlet to decrease the escape of anes-
thetic gas into the environment. To do this, cut a 3 in. square
from a laboratory glove, secure to the outlet with a rubber
band, and cut a small hole in the taut surface. The pup's nose
will be inserted into this hole.

3.1.2 Surgical Station

(Fig. 1 )

1. Obtain an 8% O 2 tank and regulator, then connect tubing to
channel the gas supply through a humidifier followed by a
water trap then run the line to the hypoxia chamber. The
humidifier and gas trap are readily made using two Erlenmeyer
flasks, the humidifier contains dH 2 O and is immersed in the
water bath to warm and humidify the gas supply.
2. The hypoxia chamber must have an input and one-way exhaust
port to prevent pressure build up. The optimal chamber is
transparent acrylic or plastic with an air-tight opening. In
advance, the minimal flow rate needed to quickly flush the
chamber with 8% oxygen should be determined using an oxy-
gen meter.
3. Prior to gas exposure, heat the water bath/chamber apparatus
so that the humidifier and chamber will be at the desired
temperatures.

3.1.3 Hypoxia Station

Select, identify, weigh, and assign animals to treatment groups as
appropriate for the experimental protocol.

3.2 Hypoxic-
Ischemic Brain Injury

1. Fill vaporizer with anesthetic.
2. Open gas supply and adjust gas flow to 2-3 LPM, depending
on size of induction chamber.
3. Turn on vaporizer to 2.5-3%.
4. Place animal in chamber. Before the start of any surgical steps,
ensure adequate sedation by checking for loss of spontaneous
and reflexive movements (including lack of postural/geotaxis

3.2.1 Anesthesia