Gene Therapy for Epilepsies - Gene Delivery and Therapy for Neurological Disorders - page 260

4. When loading vectors, draw up slightly more than the volume
required for injection. Before each injection, inject a small
amount to make sure that the needle isn't blocked and the
pump is functional.
5. Wear lab coat, surgical gloves, and mask throughout the surgi-
cal procedure and follow appropriate sterile techniques.
6. The microsyringe pump system from World Precision
Instruments, Inc. can be used with various types of syringes.
Use the manual to find their predefined settings for different
syringe types to ensure that the correct one is used to achieve
the right infusion volume and speed.
7. For rats, make up ketamine/xylazine in a 100 μg/μl ketamine/
20 μg/μl xylazine mixture with sterile water or saline. Then,
inject 1 μl/g of body weight. For mice, make up ketamine/
xylazine in a 10 μg/μl ketamine/2 μg/μl xylazine mixture
with sterile water or saline. Then, inject 10 μl/g of body
weight.
8. Since this is a lengthy procedure due to the slow infusion rate
for vector delivery, we recommend applying some eye oint-
ment to the animal's eyes to prevent drying.
9. A nice trick when determining when the tip just touches the
skull is to check the change in reflection by having a light
source projected on the skull.
10. The DV coordinate can be taken either at bregma or the actual
injection site. Some also take the DV coordinate using the level
of the dura as zero. The key is consistency and to have vali-
dated the coordinates in the same way.

4.4 Seizure Models

1. Usually, one needs to order the internal or injector cannula
with custom length depending on the verified coordinates for
injection. For example, if the DV coordinate is −3.0 mm from
dura, then order the internal cannula that extends 3.0 mm
below the guide cannula.
2. One may also make one's own guide and internal cannula. In
this case, purchase stainless steel tubing of 22-G for the guide
and 28-G for the internal (e.g., from Vita Needle Company,
Needham, MA, USA). Cut them into pieces slightly longer
than desired lengths and then trim and open the hole by pol-
ishing on sandpaper.
3. For the bipolar electrode, scratch the insulation off the very
distal end where the two wires part will enhance signal detec-
tion. However, it is important not to let the non-insulated
ends of the wires of the bipolar electrode meet; otherwise, the
electrode becomes a closed circuit and won't work.