Gene Therapy for Parkinson’s Disease: AAV5-Mediated Delivery of Glial Cell Line-Derived Neurotrophic Factor (GDNF) - Gene Delivery and Therapy for Neurological Disorders - page 71

2.3 Injection of rAAV

In this section, the procedure of stereotactic infusion into the brains
of test animals is described. A good description of the procedure
has also been described by Dr. Ruitenberg and coworkers [ 53 ].

1. Preparing the infusion device. Infusions into the brain paren-
chyma can be performed using stainless steel 30-G beveled
injection needle (outer diameter ~300 l

Hamilton syringe. They are attached using polyethylene tubing
with a 0.28 mm diameter. The device is fi rst fi lled with water
from the back of the Hamilton detaching the plunger and insert-
ing it back. At this point, testing for leakages is performed. After
the system is fi lled with water, an air bubble of 1

ʼ m), attached to a 10- ʼ

l is introduced
before the actual viral vector is sucked up from a vector solution.
This can be done most easily from the cap of an Eppendorf reac-
tion tube. The needle can be attached to the stereotactic frame,
and the Hamilton syringe laced into a Harvard micropump.

ʼ

2. Anesthesia. For testing in rodents, procedures are relatively
similar for mice and rats, except differences in injection coor-
dinates and dosing of the anesthetic. Animals are fi rst anesthe-
tized using an FFM mix (Hypnorm/Dormicum/water;
1:1:10) at a dose of 70 ʼ l/10 g bodyweight per mice and of

ʼ

l/100 g per rat, intramuscular. Sedation is further
enhanced using the same dose of Dormicum. This dose should
be suffi cient to perform the procedure. The animals will be
fully sedated for approximately 45 min and further sleep for
three more hours. These numbers are estimates and vary
among animals. Depth of anesthesia is tested by pinching the
toes with a pair of tweezers. Eye refl ex should also be absent.
Please note that hearing is still intact. Post-surgery, a single
dose of Fynadine (2.4 mg/kg body wt; Schering-Plough,
Belgium) is administered for pain relief.
3. Placement in stereotactic frame. The placement into the ste-
reotactic frame allows procedures to be carried out without
actual movement of the head while performing the surgery.
The animal is placed into the frame, fi rst with an ear bar into
one of the earshafts. Some pressure may be applied. When the
animal blinks with his eye, the bar is placed correctly, and the
other bar can be applied at the same mirrored position. When
the bars are fi xed, the head of the animal cannot move; other-
wise, the procedure has to be repeated. With both bars inserted,
the head of the animal needs to be positioned at the midline of
the frame by eye. The mouthpiece is now also applied. It is
important that the skull is positioned horizontally.

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4. Opening the skin. The head of the animal is shaved using elec-
tric razors and cleaned with 70 % ethanol. Using a scalpel, a
small incision is made at the midline. Depending on the loca-
tion of the injection, the incision is roughly 5-10 mm. When
the striatum is targeted, the incision is approximately around
the position of bregma.