Gene Therapy in Spinal Muscular Atrophy (SMA) Models Using Intracerebroventricular Injection into Neonatal Mice - Gene Delivery and Therapy for Neurological Disorders - page 316

3. Grasp the immobilized mouse fi rmly by the skin behind the
head using the free hand. Place the animal's head on the fi ber-
optic light to illuminate relevant anatomical structures that
can serve as useful guides (Fig. 8b ).
4. The next step is to insert the needle 2 mm deep, perpendicular
to the skull surface, at a location approximately 0.25 mm lat-
eral to the sagittal suture, and 0.50-0.75 mm rostral to the
neonatal coronary suture (Fig. 8c ). Each person can choose
either the left ventricle (Fig. 8c ) or right ventricle (Fig. 8d ) for
injection based on the fl exibility of the hand motion.
5. Following the insertion of the needle, the plunger is pushed
using the palm of the injecting hand. This is done very slowly
and carefully until all the content of the needle is emptied into
the brain (see Notes 1 and 2). Monitor for ruptured vessels or
facial swelling.
6. Remove the needle 10 s after the injection to prevent back-
fl ow. For recovery, mice should be placed in a warm container
for 5-10 min until movement and general responsiveness are
restored.

To verify the success of ICV injection, the dissemination pattern of
the dye in the brain should be monitored (see Note 3). Successful
injection into one of the ventricles will allow visualization of the
dye on the injected side and the distribution of the dye to the adja-
cent ventricle within a few minutes postinjection (Fig. 9a ). A uni-
form distribution of the dye in both right and left cerebral
hemispheres and olfactory bulbs will be visible approximately
60 min postinjection [ 96 ]. Accurate injection can also be visual-
ized by the dispersal of the dye in the rostral central spinal canal
within a few hours postinjection (Fig. 9b ). Injections can be
repeated if the fi rst injection was not satisfactory (see Note 4). The
injection can also be repeated on P3 and P4 if more materials need
to be administered into the mouse brain.

3.1.5 Verifying
a Successful ICV Injection

4 Notes

1. During the injection process, some tissues may enter the nee-
dle and block the tip. In this case, change the needle or slightly
break the tip.
2. While holding the needle, the plunger may be unintentionally
pushed by the palm before the needle is penetrated into the
brain. Some practice is required to avoid emptying the content
of the needle outside of the brain.
3. Inaccurate ICV injections can be distinguished by lack of color
in cerebral hemispheres. In this case, the needle may be pene-
trated too deep, and the injected solution is probably distrib-
uted underneath the cerebral ventricles. Widespread and