Gene Therapy for Huntington’s Disease - Gene Delivery and Therapy for Neurological Disorders - page 145

3. If a titering control has been used, results can be normalized to
the known value of the titering control. Therefore, enabling
comparisons to be made across different titering runs.

3.4 Stereotaxic
Delivery of Vectors
into the Rodent Brain

This section describes our procedures for infusion of viral vectors
into the striatum. We typically use male Wistar or Sprague-Dawley
rats in the 230-300 g weight range:

1. Animals are anesthetized according to institutional guidelines
and the animal positioned in a stereotaxic frame (David Kopf
Instruments, Tujunga, CA).
2. Make an incision through the scalp, expose the skull surface,
and identify bregma and lambda points. Adjust the incisor bar
height such that bregma and lambda are at equal dorsoventral
height (flat skull).
3. Coordinates for intrastriatal infusion of viral vector into the rat
brain are determined with reference to the atlas of Paxinos and
Watson [ 95 ] and are as follows: anterior-posterior (AP)
+0.4 mm, mediolateral (ML) −3.0 mm, dorsoventral (DV)
−5.5 mm, and bregma =0.
4. Make a small burr hole in the skull using a high-speed rotary
drill at the appropriate AP-ML coordinates, and lower the tip of
a 10 μL Hamilton syringe (Hamilton, Reno, NV) with a 25 G
needle under control of a microinfusion pump controller (World
Precision Instruments, Sarasota, FL) into the infusion site.
5. AAV vector (up to 3 μL) is infused into the striatum at an infu-
sion rate of 70-100 nL/min.
6. After completion of the infusion, the syringe is left in place for
a further 5 min to allow diffusion of the vector before it is
slowly withdrawn over a 5 min period and the scalp sutured.
7. Follow institutional guidelines for the application of analgesia
and monitoring recovery from surgery as required.

Spontaneous locomotor activity can be assessed by placing rats in a
circular enclosure, 1.8 m in diameter divided into segments of
equal area. Locomotion under dim lighting condition is video-
recorded for 5 min, and the number of lines (the boundary between
each segment) crossed is manually quantified by the investigator.
Monitoring of this type of activity can also be conducted by utiliz-
ing automated video-tracking systems such as the Noldus
EthoVision XT system.

3.5 Behavioral
Testing

3.5.1 Spontaneous
Locomotor Activity
in an Open Field

The gait of rats can be analyzed to detect deficits in locomotion.
Rats are trained to walk along an enclosed 2 m long, 20 cm wide
runway into an escape box. On the day of the trial, place the rats'
hind- and forepaws in red and blue non-toxic dye, respectively
(20 % food coloring in glycerol), before releasing the rat onto a

3.5.2 Footprint Test