Gene Therapy Approaches to Promoting Axonal Regeneration After Spinal Cord Injury - Gene Delivery and Therapy for Neurological Disorders - page 164

exploration the rat independently places its left, right, or both fore-
paws against the side of the cylinder. However, after unilateral RST
transection, use of the forelimb on the lesioned side will reduce
signifi cantly. To examine the forelimb use during spontaneous ver-
tical exploration the rat is placed in the cylinder for 10 min and the
testing session is recorded with a video camera. The usage of the
non-impaired forelimb, and the impaired forelimb, and both fore-
limbs for contacting the wall of the cylinder during vertical explora-
tion is scored blindly by another researcher at a later date.
The results are expressed as percentage of usage of the left, right, or
both forelimbs relative to the total number of forelimb usage.

Note (1): The rats may not rear up so frequently after they have
got used to the glass cylinder. If not enough rear-ups are recorded,
the rat is placed back into its home cage and repeat the session
again after a while.

Note (2): Place a mirror on the opposite side of the video camera
at a suitable angle so that the camera can capture the positions of
the forelimbs when the rat turns its back to the camera.

Modifi ed rope crossing : In this test, rats are to cross a 125 cm long
horizontally oriented rope (4 cm in diameter) between two plat-
forms. This task requires hind-limb weight support, precise paw
placement, posture and coordinated balance of the body. However,
as rats with RST lesion fall easily from the rope due to the swinging
of the rope, which may mask small improvement in limb motor
function, we modifi ed the device by wrapping a thinner rope
around a wood pole to make it rigid (Fig. 1c, d ). Unlesioned rats
run across the rope very quickly and rarely slip or fall during the
run, while lesioned rats cross slowly with foot slips and sometimes
fall from the rope. During each run the number of slips and falls is
counted and the ratio of slips versus total steps in two runs at each
time point for each animal is calculated and presented as error
index. The ratio is defi ned as 1 whenever the rat falls.

1. Dissecting the tissues. Under deep anesthesia, perfuse the ani-
mals with 0.9 % saline fi rst and then with 4 % paraformalde-
hyde in phosphate buffer. Dissect the midbrain and spinal cord
segments containing the lesion site.
2. Processing the tissues. The dissected tissues are post-fi xed in
4 % paraformaldehyde for overnight, and cryoprotected in
30 % sucrose solution until they sink to the bottom. Divide
each spinal cord into three blocks, a 10 mm lesion block cen-
tered at the lesion site ('lesion block'), and two 5 mm blocks
at either ends of the lesion block (rostral and caudal blocks).
3. Tissue sectioning. Cut horizontal sections at 15

3.2.5 Immunohistoche-
mistry and Quantifi cation
of RST Axons

m in thickness
for the lesion block, transverse sections for both the rostral and
caudal blocks of the spinal cord as well as the midbrains.

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