Hartman Behavioral Neuroscience Lab:Notebook/Neonatal hypoxic-ischemia and hydrogen therapy/2008/05/08

--Richard E. Hartman 00:50, 9 May 2008 (EDT)

• P10 RVM.... The following are Melissa's notes:

Pilot study for cerebral palsy and hydrogen treatment begun

Setup

1. Pups were marked (#s1-11) with a permanent marker to the back; 9 pups from 1 litter and 2 pups from another litter
2. Pups weighed and returned to mothers (who were kept in the dark by a black tarp covering). Separating the mother from the pups with a fitted cut-out cardboard helps avoid being bitten
3. Surgeries started. Things noted: surgery start and end time, surgeon, pup #, and any notes

Surgery protocol for Unilateral Carotid Artery Occlusion on the neonate rat pup (p10)

1. Anesthetize the P10 rats with 3% isoflourine via gas tube
2. Check pain response (tail pinch) to ensure the animal is sufficiently under for surgery
3. Secure the rat with face to the tube (duct tape cover) to keep pup under anesthesia
4. Once rat is secure, use small surgical scissors to make a caudal to rostral incision slightly lateral of the medial line. (to the your left).
5. Use tweezers in both hands to slowly deepen the incision into the issue. As long as you sweep caudal to rostral damage along arteries and nerves is minimized if not avoided completely.
6. Search for the carotid artery: landmarks: sternocledomastoid muscle is caudal to carotid artery. Vegas nerve is dorsal to the carotid artery.
7. Be sure to discriminate between the jugular and the carotid. The jugular will not be pulsating, the carotid artery will be pulsating in the live animal.
8. Once the carotid is located, use the hooked tweezers to weave a looped (pre-cut) suture string under the carotid: the closed end of the string was my right and the open end to the left so that “two” string sections were passed under the carotid.
9. Cut the “looped” section of the string with scissors
10. Using the instrument, tie the ends of the now two separate suture lengths together leaving a length of the carotid exposed between the tied sutures. For each suture tie at least twice. BE SURE SUTURE IS TIGHT ENOUGH TO CUT OFF BLOOD FLOW OR WHEN YOU CUT CAROTID ANIMAL WILL BLEED OUT AND DIE.
11. Sever completely the carotid artery between the tied sutures, and tuck suture ends below skin
12. Suture skin together. Be sure skin is slightly raised where skin pushes together at the incision. If you have a clean incision, one suture tied three times is sufficient. More sutures may be needed.
13. Return pup to its mother

Surgery should take no longer than 10 to 20 minutes.  The less time the animal spends under anesthesia the better.  Get used to raising and lowering (focusing) the microscope before you begin surgery.  All surgeries should be done by same person to minimize differences.

Two hours after last rat pup was returned to its mother, rats were subjected to treatment. Pups were randomly assigned to the hypoxic treatment or the hypoxic + hydrogen treatment

(Don’t have full protocol for this)

1. Large beakers were placed in water maintained at 37 °C (?). One tube fed 8% oxygen to one beaker and a separate tube fed 8% oxygen + ___% hydrogen. Gas flow was tested prior to treatment. Be sure to pay attention to safety valves and the proper order of opening and closing compressed tanks!. Note: both tubes had pathways that were emerged partially under the water, so gas was cooled briefly before entering the beaker.
2. Once gas flow was at appropriate levels, pups were placed in the beaker corresponding to their randomly assigned treatment type.
3. Beakers were closed snugly
4. pups were left in this treatment for 2 hours for both conditions

Post treatment, pups were returned to mothers (with the exception of two pups which died during treatment)