Gene Therapy for Chronic Pain: How to Manipulate and Unravel Pain Control Circuits from the Brain? - Gene Delivery and Therapy for Neurological Disorders - page 334

after vector injection for 2 months (Fig. 4c, d ) [ 7 ]. The pin-prick
and acetone tests are performed as follows:

1. Before performing the tests, habituate the animals to the
experimenter and the testing room 1 h per day for a week.
2. Place the animal on a transparent cage with a mesh wire bot-
tom allowing the stimulation of the lateral plantar surface of
the injured hind paw during the pin-prick and acetone tests
(steps 3 and 4, below).
3. To perform the pin-prick test, apply a brief stimulation with a
safety pin at an intensity suffi cient to indent the skin, and
record the duration of the paw withdrawal with an arbitrary
minimal time of 0.5 s [ 17 ].
4. To perform the acetone test, apply a drop of acetone and
record the duration of the withdrawal response, with a mini-
mal value of 0.5 s [ 18 ].

In order to confi rm that the behavioral effects of the THa vector
were mediated through an action on

Reversal of Behavioral
Effects ʱ -adrenoreceptors, the

ʱ 1-adrenoreceptor agonist phenylephrine was administered into

the DRt of THa-injected animals (Fig. 5a, b ) [ 7 ]. Fourteen days
after SNI induction, the animals were placed on a stereotaxic frame
for THa injection and the implantation of a guide cannula into the
left DRt following the procedure in Sect. 3.2.1 with the exception
that the guide cannula was implanted 3 mm above the DRt. At 22
days after the stereotaxic surgery (timing of maximal behavioral
effects), the animals were injected with 0.5 µl of saline or phenyl-
ephrine (10 µg; Sigma-Aldrich, St Louis, MO, USA) with a needle
protruding 3 mm beyond the ventral tip of the guide cannula. The
pin-prick and acetone tests were performed following the proce-
dure in section “Neuropathic Pain Induction and Behavioral
Assessment” before and 15, 30, 45, and 60 min after injection.

In order to verify the extracellular levels of noradrenaline at the
DRt upon injection of the THa vector, microdialysis experiments
were conducted upon injection of the THa vector or the control
vectors THTH and THZ into the DRt (Fig. 6 ) [ 7 ]. Fourteen days
after SNI induction, the animals were placed on a stereotaxic frame
for vector injection and a guide cannula implantation into the left
DRt following the procedure in Sect. 3.2.1 with the exception that
the guide cannula was implanted 2 mm above the DRt. Microdialysis
was performed 22 days (timing of maximal behavioral effects) after
injection and guide cannula implantation by using a microdialysis
probe extending 2 mm beyond the ventral tip of the guide cannula.
Microdialysis procedures are as follows:

3.3.2 Neurochemical
Effects

1. Prior to microdialysis, habituate the animals to the experi-
menter and the testing room 2 h per day for a week.