Gene Therapy Approaches to Promoting Axonal Regeneration After Spinal Cord Injury - Gene Delivery and Therapy for Neurological Disorders - page 166

3.3 Gene Delivery
to the Spinal Cord
in a Dorsal
Hemisection Model

For assessing the effects of treatments in promoting sensory axon
regeneration, a commonly used model is the dorsal column transec-
tion, which cuts the ascending sensory axons that are responsible
for transmitting fi ne touch, vibration and conscious proprioceptive
information to the cerebral cortex. We used this model to assess the
effect of LV mediated expression of NGF-sNgR1 fusion protein in
the spinal cord to promote sensory axonal regeneration [ 44 ].

1. Anesthetize female rats (200-250 g) as described above with
Fluothane (ICI).
2. Shave the operated areas around the eighth thoracic vertebra
(about 4 cm × 3 cm) using veterinary hair clippers and clean
the exposed skin with iodine and 70 % alcohol.
3. Place the rat on a heated pad and cover with a sterile drape.
4. Make an incision about 3 cm in the skin at the level of the
eighth thoracic vertebra (T8) rostrocaudally. Incise the under-
lying musculature using a scalpel blade along both sides of the
spine to expose the lamina of T8. Perform blunt dissection to
split the deeper muscle layers.
5. Perform a laminectomy of T8 using a Malleus Nipper (Fine
Science Tools, #16140-11) under an operating microscope.
Open the dura with a pair of microsurgical scissors.
6. Crush the dorsal column of T8 spinal cord with a pair of
watchmakers' forceps at a depth of 1.5 mm to prevent heavy
bleeding of the posterior spinal vein, and then cut the dorsal
column using microsurgical scissors at the same depth. Place a
small piece of fi brinogen fl ake from rat plasma (Sigma-Aldrich,
#6755) on the surfaces of the spinal cord to cover the transec-
tion site and prevent further bleeding.

3.3.1 Surgical Procedure
for Dorsal Hemisection

To minimize the injection damage caused by breathing the stereo-
tactic frame used for injection is modifi ed with a frame connected
with two forceps to immobilize the spinal cord by holding the
spines of the T7 and T9 ( See Fig. 2 ).

3.3.2 Injection
of the Viral Vector into
Spinal Cord

1. After the spinal cord transection transfer the rat to the above
device.
2. Load 3-4 l Hamilton syringe.

3. Stop the bleeding of injury before the viral vector injection.
4. Under the operation microscope, using a 30 gauge needle to
make a small puncture through the arachnoid mater and pia
mater at the ipsilateral of rostral margin of the lesioned spinal
cord. The insertion point should be away from blood vessels
to avoid bleeding.
5. Lower the injecting needle (33 gauge) through the puncture.
When the needle reaches the surface of the spinal cord, take a

ʼ l of viral vectors into a 10 ʼ