AAV Gene Therapy Strategies for Lysosomal Storage Disorders with Central Nervous System Involvement - Gene Delivery and Therapy for Neurological Disorders - page 283

ICV Injection
14. A custom-constructed positioning device is used to mount the
ultrasound probe and biopsy guide to the micromanipulator of
the stereotaxic apparatus for controlled injection of the lateral
ventricle.
15. An incision is made in the skin above the sagittal suture to
expose the skull.
16. The left lateral ventricle is visualized using an 8-5 MHz ultra-
sound probe. Because the sheep skull is thick and fontanelles
have closed by the time of surgery, craniotomy is performed
with a Hall air drill to create an ultrasound window for the
visualization of the lateral ventricle.
17. A 20 G spinal needle is inserted through the biopsy guide of
the ultrasound probe and into the brain parenchyma to serve
as a guide and anchor for the injection needle.
18. A 22 G, 2 L Hamilton

syringe is affi xed to a micromanipulator for insertion through
the guide needle. The injection needle is advanced into the
lateral ventricle manually, with visualization of ventricular
placement by ultrasound.
19. A total of 350

″ injection needle attached to a 500 ʼ

L i of vector is delivered (with or without 4 %
gadoteridol in the injectate g ) in 50

ʼ

ʼ L boluses at a rate of

L/min.
20. The injection needle remains in place for 2 min postinjection
and is then removed, followed by the removal of the guide
needle e .

50 ʼ

Postoperative Care

21. All skin incisions are closed with a Ford interlocking pattern
using 0 monofi lament suture.
22. Morphine (0.05 mg/kg) delivered intravenously is used for
analgesia after the surgery j .
23. Florfenicol (20 mg/kg) delivered intramuscularly is used as a
postoperative antibiotic.
24. Most sheep recover fully from anesthesia within 4 h. Animals
are observed carefully for several days after surgeries.

3.4 Notes

(a) MRI, cerebrospinal fl uid analysis, and complete blood count
(CBC) and chemistry are performed prior to surgery to ensure
adequate health of the animal.
(b) If restrained properly, most kittens do not require sedation for
catheter placement.
(c) If desired, an arterial line can be placed for blood gas analysis
and/or invasive blood pressure monitoring.