Targeted Gene Therapy for Ischemic Stroke - Gene Delivery and Therapy for Neurological Disorders - page 198

3.4 Behavioral Tests
(See Note 6)

Mice are placed between two boards with identical dimensions
(30 × 20 cm). When mice are near the corner, both sides of their
vibrissae will be stimulated. They will rear forward and upward and
then turn back to face the open end. Normal mice will turn to the left
or right side with equal frequency, whereas stroke mice will turn more
often to the ipsilateral side of the lesion (left). The percentage of left
turns is recorded in three different sets of ten trials. Turning move-
ments not incorporated in a rearing movement are not recorded.

3.4.1 Corner Test

Adhesive tape (0.3 × 0.3 cm) is applied on each paw. The time it
takes for the mice to remove the tape from each paw is recorded
with a maximum testing time of 120 s. Mice will be trained three
times daily for 4 days before the surgery to obtain an optimal level
of performance.

3.4.2 Adhesive
Removal Test

1. Cut 20 m coronal sections of the brain and fi x in 0.5 % glu-

taraldehyde for 10 min.
2. Incubate the sections in X-gal staining solution for 2 h and
photograph.
3. Calculate the transduction volumes by multiplying the trans-
duction areas by the thickness of the sections using NIH
ImageJ 1.63 software.

ʼ 3.5 Tissue Stains

and Morphometry

3.5.1 Assay Transgene
Expression (X-gal Staining,
Fig. 3b )

1. Cut brain into 20 m serial sections.

2. Fix one of every 10 s in 4 % paraformaldehyde solution.
3. Incubate the sections in cresyl violet solution for 5-10 min.
4. Differentiate sections in 70, 90, and 100 % ethanol sequen-
tially, for 2 × 3 min each. Incubation time can vary slightly
according to staining density and section thickness.
5. Transfer sections to xylene for 2 × 5 min each.
6. Mount sections with Permount.
7. Outline the infarct/atrophic (pear blue) and the ipsilateral
hemisphere areas using ImageJ software (Fig. 4 ).
8. Estimate the infarct/atrophic volumes by each area's sum
multiplied by 200

ʼ 3.5.2 Assay Infarct/

Atrophic Volume (Cresyl
Violet Staining)

ʼ m ( see Note 7 ).

Fig. 4 Nissl staining illustrates the infarct area in the pMCAO model. The infarcted
brain ( arrow ) is lighter than the normal brain