Gene Therapy Approaches to Promoting Axonal Regeneration After Spinal Cord Injury - Gene Delivery and Therapy for Neurological Disorders - page 167

Fig. 2 Set-up for microinjection of viral vectors into rat thoracic spinal cord. ( a ) A photo showing the whole setup
for microinjection of viral vector to thoracic spinal cord. Note how the two pairs of forces are fi xed to the ste-
reotaxic frame and hold the rat spine to reduce the movement of spine due to animal breathing. ( b ) A close- up
photo showing how the forceps hold the rat spine

reading on the stereotactic frame, and then slowly insert the
needle into the spinal cord. When the needle reaches 1 mm
into the spinal cord, withdraw the needle slightly and wait for
approximately 2 min to allow the tissue to seal around the
needle and help to prevent leakage ( See Note (1) and (2) for
more options of injection).
6. Inject 1

ʼ

l of LV at a speed of 200 nl/min as described above.
7. After the injection leave the needle in situ for 5 min and then
gradually withdraw over 2-3 min to allow the diffusion of viral
vector from the injection site and to prevent leakage from the
needle track.
8. Make a second injection at the caudal margin of the lesion site
of the ipsilateral spinal cord as described above.
9. Suture the muscles and skin with a 3-0 absorbable suture.

Note (1): A glass pipette needle can be pulled to a much fi ner tip
than that of a 33 gauge stainless steel needle, therefore, glass nee-
dles cause less damage to the tissue. However, glass needles may
clog or break easily, which prolongs the surgery time and causes
wastage of viral vectors as the loaded needle has to be discarded
and a new needle is loaded.

Note (2): Instead of inserting the needle vertically into the spinal
cord, the needle may be inserted to the site of injection at an angle
to the spinal cord. The advantage is that the longer needle track
will reduce the leakage and increase the spread of the viral vector.
The disadvantage is that it will cause more tissue damage.