Gene Therapy Approaches to Promoting Axonal Regeneration After Spinal Cord Injury - Gene Delivery and Therapy for Neurological Disorders - page 168

To study the regeneration of sensory axons after spinal cord injury
cholera toxin

3.3.3 Injection
of Retrograde Tracers
to the Sciatic Nerve

subunit (CTB) can be used to transganglionically
label the ascending sensory axons. The animals are maintained for a
designed period (normally for about 6-10 weeks after surgery). CTB
can be injected 3 days before the termination of the experiment.

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1. Anesthetize the rat as before and place the rat in a prone posi-
tion on a heated mat.
2. Shave and sterilize the mid-thigh area of the ipsilateral leg.
3. Make an incision of the skin and expose sciatic nerve.
4. Load 5 l 1 % CTB (List Biological Laboratories, Campbell,

CA) into a 10

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l Hamilton syringe attached with 30-gauge
needle (bevelled 30°).
5. Make a small nick in the perineurium of the sciatic nerve using
a pair of microscissors, through which the needle is inserted
and pushed along sciatic nerve for a further 1 cm.
6. Inject the tracer slowly into the sciatic nerve. At the end of the
injection leave the needle in situ for 2-3 min and then gradu-
ally withdraw the needle.
7. Close the skin incision with 3-0 absorbable sutures.

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Perfuse the animals with 0.9 % saline and then 4 % paraformalde-
hyde 3 days after CTB injection. Remove segments of spinal cord
containing the lesion site and brainstem for morphological exami-
nation. Cut sagittal cryostat sections (15

3.3.4 Immunohistoche-
mistry and Quantifi cation
of Regenerating Axons

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m thick) of spinal cord
containing the lesion site and transverse sections of brainstem. To
view the glial scar around the lesion border, stain the sections with
an antibody against glial fi brillary acidic protein (GFAP), an astro-
cyte marker. CTB-labeled sensory axons can be identifi ed by
immunostaining with a goat anti-CTB IgG antibody (1:4,000, List
Biological Laboratories, Campbell, CA, USA. # 703).
Quantifi cation of CTB-labeled axonal profi les within and ros-
tral to the lesion cavity can be done by counting the number of
CTB + axonal profi les in several regions including various distance
points to the caudal border of the lesion, at caudal border of lesion,
in the lesion cavity, at the rostral border, and at various distance
points from the rostral border of the lesion. CTB + axonal profi les
in 3-5 sections from each animal may be counted.

As it has been described in Sect. 1 , cell transplantation is consid-
ered as an important component in combinatorial treatments for
spinal cord injury. Schwann cells (SCs) have been used for grafting
into injured spinal cord in animal experiments for many years as
they can be obtained easily and can myelinate CNS axons [ 75 , 76 ],
which is crucial for functional recovery. However, because they are
the myelinating cells for peripheral nerves, Schwann cells do not
mingle well with CNS glial cells and do not migrate into the host
tissue. Since PSA is a highly hydrated sugar chain attached to

3.4 Transplantation
of Genetically Modifi ed
Schwann Cells
in a Corticospinal
Tract Injury Model