AAV Gene Therapy Strategies for Lysosomal Storage Disorders with Central Nervous System Involvement - Gene Delivery and Therapy for Neurological Disorders - page 279

catheter and maintain using isofl urane (0.5-1.5 %) in oxygen
delivered through an endotracheal tube. Intravenous fl uids are
administered at a rate of 10 mL/kg/h.
4. Monitor continuously physical parameters and anesthetic
depth using a rectal temperature probe, electrocardiogram,
capnograph (end tidal CO 2 ), pulse oximeter (O 2 saturation)
and Doppler peripheral pulse monitor, and sphygmomanom-
eter (blood pressure) c .
5. Provide supplemental heat (Hot Dog veterinary patient warm-
ing system) to prevent hypothermia.

Craniotomy
6. Place animal in a sternal position, and fi x the head in a Horsley-
Clarke stereotaxic apparatus.
7. Clip the hair off of the dorsal cranium, and prepare it asepti-
cally. Make a single longitudinal incision through the skin and
periosteum to expose the skull.
8. Create craniotomy entry sites through the skull and dura with
a 20 G hypodermic needle (which is more than suffi cient to
penetrate the soft skull in the 4-12.5 weeks old cat).

Thalamic Injection

9. Stereotaxic coordinates (in cm) for the thalamus relative to
bregma are given in three planes, anterior-posterior (AP),
mediolateral (ML), and dorsoventral (DV): AP −0.7, ML ±0.4,
DV −1.6. The DV zero point is marked when the needle tip
touches meninges d .
10. Make a single craniotomy directly above each thalamic target.
11. Use a sterile 100 L Hamilton syringe fi tted with a non-coring

needle (22-25 G) to inject the AAV vector.
12. Advance the needle through the skull and into the target struc-
ture (thalamus) manually at a rate of 1 mm/s.
13. Inject a total of 70

ʼ

L/
min. Raise the needle by 0.15 cm between each injection bolus,
so that the fi nal needle position is 1.15 cm ventral to meninges
(~2.5 mm ventral to the lateral ventricle).
14. Leave the needle in the thalamus for 1 min after the fi nal injec-
tion, and then raise it halfway. After another minute, withdraw
the needle completely over 30 s e .

ʼ L in 10-20 ʼ L boluses at a rate of 2 ʼ

DCN Injection

15. Stereotaxic coordinates (in cm) for the deep cerebellar nuclei
(DCN) relative to lambda are AP 0.0, ML ±0.4, DV −1.25.
The DV zero point is marked when the needle tip touches
meninges d .