Gene Therapy for Epilepsies - Gene Delivery and Therapy for Neurological Disorders - page 256

cannula dummy in the cannula, with edge just protruding
from the cannula, to prevent cannula blockage by tissue entry.
16. Place the animal in a clean cage. Monitor the animal until it
recovers from the anesthesia and return it to the holding room.
17. Conduct the intrahippocampal KA injection at least 7 days
postsurgery. Set up the EEG apparatus according to the manu-
facturer's directions. Connect one end of an appropriate con-
nector cable to the EEG (e.g., Product 363-340/6, Plastics
One).
18. Securely hold the animal with one hand. Remove the dust cap
from the electrode pedestal. Attach the plug end of the con-
nector cable to the electrode pedestal. Place the animal in a
transparent Plexiglas circular enclosure with an open top. Set
up video for recording seizure behavior.
19. Allow 10 min for the animal to acclimatize and then record
EEG for 15-30 min until an adequate baseline is obtained.
20. Dissolve 1 mg of KA in 12.5 ml saline to give a dilution of
80 ng/μl. Store 10 μl aliquots at -20 °C.
21. Prepare the needle for KA injection. The length of the 28-G
stainless needle is the length of the cannula (12 mm) plus the
desired depth to reach the target injection site (DG). For exam-
ple, if the DV coordinate is −3.0 mm from dura, then the nee-
dle is 12 mm + 3 mm =15 mm. Connect the needle to a plastic
tubing that is connected to a 10-μl Hamilton syringe. Clean the
syringe and tubing with sterile water and then fill the tubing
with sterile water. Draw up a little air, followed by 5 μl of KA,
so that there is a small air bubble between the water and KA.
22. After baseline EEG is established, take the animal out from the
Plexiglas enclosure and remove the stopper from the cannula.
Carefully insert the needle into the cannula and inject 0.5 μl of
KA slowly over approximately 1 min. Mark on the EEG trace
to indicate time of injection. Let the needle stay in place for
another 3 min before taking it out and replace the stopper.
23. Continue EEG and video recording for up to 180 min after
KA infusion.
24. At the end of 180 min, stop EEG and video recording and give
the animal 40 mg/kg pentobarbital i.p. injection to stop any
further seizure before returning it to its cage.
25. Score behavioral seizure according to Racine [ 68 ]: Stage 1,
staring, mastication; Stage 2, head nodding, wet dog shakes
(WDS); Stage 3, unilateral forelimb clonus; Stage 4, rearing
with bilateral forelimb clonus; Stage 5, rearing with bilateral
forelimb clonus and falling.
26. Analyze EEG traces and note any activity different from
the baseline. EEG seizures are defined by the simultaneous