Gene Therapy Approaches to Promoting Axonal Regeneration After Spinal Cord Injury - Gene Delivery and Therapy for Neurological Disorders - page 165

4. Immunostaining. In LV/GFP injected group, the expression
of GFP in the red nucleus and in RST axons could be viewed
directly under a fl uorescence microscope. As the GFP and
DNROCK cDNAs are under the control of human synapsin
promoter, for confi rmation of the neuronal specifi c expression
of the transgenes, sections of midbrain can be immunostained
with a mouse anti-NeuN antibody (1:500; Chemicon,
MAB377). In the DNROCK group, the animals are injected
with a mixture of LV/DNROCK and LV/GFP for easy iden-
tifi cation of the RST axons. For assessment of the co-expres-
sion of DNROCK and GFP in the red nucleus and spinal cord
section, sections of midbrain and cervical spinal cord can be
immunostained with a mouse anti-Flag M2 antibody (1:250,
Sigma, F3165) as a 3 × FLAG tag is fused to the DNROCK
construct ( See Note (1) for viral vectors injected in our study
and Note (2) for the use of an anterograde tracer).
5. Quantifi cation of regenerating axons. GFP-labeled axons in
the white matters are counted in 6-7 horizontal sections at
45 m intervals and in one transverse section 5 mm caudal to

the lesion site. If the DNROCK expression is effective in pro-
moting axonal growth, there should be more GFP-labeled
RST axons growing longer distance toward the lesion border,
crossing the lesion site, and even growing into the spinal cord
caudal to the lesion. Also there may be more GFP-labeled
axons sprouting to the grey matters. Therefore, to accurately
assess the extent of axonal growth, the number of axons needs
to be counted at different regions and at 3-5 distance points,
e.g., 4-5 mm to the lesion center, rostral border of the lesion
site (within 1.5 mm rostral to the lesion center), the lesion
site, and caudal part (within 5 mm caudal to the lesion center)
on horizontal sections, and at 5 mm caudal to the lesion cen-
ter on transverse sections [ 9 ].

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Note (1) : Titers of LV/GFP in the LV/DNROCK and LV/GFP
mixture is adjusted so that it has the same titer as that of LV/GFP
only viral stock. We showed that the co-transduction effi ciency of
red nucleus neurons by LV/DNROCK and LV/GFP is 95.2 ± 0.5 %
[ 9 ]. Therefore, GFP fl uorescence in RST axons in the DNROCK
and GFP co-injected group can be used as an indicator of DNROCK
expression in the same axons.

Note (2) : We injected biotinylated dextran amine (BDA) into the
red nucleus to trace the RST axons in LV/DNROCK injected ani-
mals. However, we found that most LV/DNROCK transduced
neurons were not labeled by BDA for some unknown reasons.
Therefore, BDA may not be suitable as an anterograde tracer for
LV transduced RST axons.