Gene Delivery and Gene Therapy for Alzheimer’s Disease - Gene Delivery and Therapy for Neurological Disorders - page 110

3. During recovery from anesthesia, maintain the body temperature
at 37 °C using an electric heating pad with a sterile drape
between the animal and the heating pad to avoid thermal
injury. Operated animals should be monitored until they are
fully recovered from anesthesia. The recovery criteria include
anesthetic depth, respiratory rate and pattern, and mucus
membrane color. Thereafter, examine the animals' health sta-
tus at least once daily for any signs of ill health, such as distress,
pain, loss of appetite, or dehydration. Postoperative analgesia
(carprofen, 5 mg/kg, SC) should be administered once a day
for the fi rst 2-3 days after surgery.
4. In order to minimize the incidence of infection, topical triple
antibiotic ointment treatments may be applied to the incised
region after completion of surgical procedures.

1. Once anesthetized, place the mouse in a stereotaxic apparatus.
Secure the animal's head with earbars and mouthpiece so that
the skull is fi xed, horizontal, and symmetrical, and administer
a steady fl ow of isofl urane (1-3 %) to sustain anesthesia.
2. Clean the skin and make a longitudinal incision on the skull
with a small blade.
3. Identify and mark bregma. Using bregma as a guiding point,
mark the left and right hemisphere target areas with a sterilized
pencil. To deliver AAV to the hippocampus, we use a site in the
dorsal hippocampus in the apical dendritic zones of the CA1
region near the hippocampal fi ssure. For delivery to this site,
mark the skull bilaterally at the following stereotaxic coordi-
nates of bregma: anterioposterior (AP) −1.8 mm, mediolateral
(ML) ±1.8 mm, dorsoventral (DV) −1.8 mm.
4. Next, make holes approximately 0.5 mm in diameter using a
0.5 mm drill bit with a handheld electric drill at the marked
points. AAV will be directly injected through the holes. Inject
a volume of 0.5-1 µl AAV per site over 1 min using an auto-
mated microinjector connected to a 10 µl Hamilton syringe
and a 30-gauge needle. Remove the microinjector 5 min after
the completion of the injection to assure suffi cient diffusion of
AAV. Inject a total of 1-2 µl AAV (0.5-1 µl × 2 sites) per mouse
( see Fig. 2 for example of protein expression after AAV-GFP or
AAV-7ND injected at 2 × 10 10 VP/2 µl/site into the hippo-
campus (b) or cortex (c)).
5. After the injection, close the 0.5 mm hole with a very small
wax ball and close the skin incision using a 4-0 nylon suture in
a simple interrupted pattern. The suture can be removed 7-10
days after the operation. Animals may be administered an S.C.
injection of 500 µl saline to prevent dehydration and 5 mg/kg

3.3.3 Intracranial
Injection