Gene Therapy for Epilepsies - Gene Delivery and Therapy for Neurological Disorders - page 253

syringe in the process. Set the infusion setting on the Micro4
microsyringe pump controller to inject at 1 μl over 10 min.
7. Weigh the animal and anesthetize the animal with ketamine/
xylazine (100 and 20 mg/kg; i.p.). Place the animal individu-
ally in a new cage after anesthetic injection and check for the
absence of paw reflex (by pinching the hind paws) as an indica-
tion of adequate anesthesia.
8. Once the animal is in a deep anesthetic state, shave the head of
the animal with an electric hair shaver.
9. Secure the animal via ear bars and incisor bar on the stereotaxic
frame. When sliding the ear bars into each ear canal, be careful
not to rupture the eardrums. Once firmly and accurately posi-
tioned, the head of the animal should not wobble. The two ear
bars should be equal in distance from the incisor bar for correct
alignment.
10. Clean the skin with a cotton-tipped applicator soaked with
70 % ethanol or Betadine.
11. Make a midline incision with a scalpel from the front to the
back of the skull. Use cotton-tipped applicators to part the
scalp and clean the underlying fascia and pericranium and allow
the skull to dry.
12. Identify the bregma, which is the intersection of the coronal
and the sagittal sutures. Mark the bregma with a marker.
13. To achieve accurate injection, ensure the animal's head is in a
flat skull position. This can be done by measuring the dorso-
ventral coordinate of the bregma and the lambda, which is the
intersection of sagittal and lambdoid sutures. The two coordi-
nates should be very close, if not identical. Adjust the height of
the toothbar if the head is not in a flat skull position.
14. Move the tip of the needle to the bregma position. Lower the
needle so the tip is just touching the skull. Take the bregma
coordinates (AP, anteroposterior; ML, mediolateral; DV, dor-
soventral) and use these to calculate the injection coordinates.
15. Move the needle to the calculated injection coordinates and
mark the position on the skull with a marker. Use an electric
dental drill to carefully drill a burr hole slightly bigger than the
size of the injection needle at the target site. Clean the skull
and burr hole with cotton applicators.
16. Carefully open the dura with the tip of a disposable 26-G nee-
dle. Clean any blood and fluid with a cotton applicator, and
then slowly lower the syringe needle to the target position.
Start infusion of the vector using the micropump controller.
17. At the end of the infusion, leave the needle in place for an addi-
tional 3 min before slowly withdrawing the needle. This is to
minimize backflow of the vector into the needle tract.