Gene Therapy for Chronic Pain: How to Manipulate and Unravel Pain Control Circuits from the Brain? - Gene Delivery and Therapy for Neurological Disorders - page 333

antibody raised in mouse (Roche, USA) diluted at 1:1,000 in
PBS-T containing 2 % normal swine serum.
2. Wash with PBS-T, and then incubate for 1 h with a rabbit
biotinylated anti-mouse serum at 1:200 (Dako, Denmark) diluted
in PBS-T containing 2 % normal swine serum. After several
washes in PBS-T, incubate with an Alexa streptavidin 594
(Molecular Probes, Netherlands) at 1:1,000 in PBS-T for 1 h.
3. After washing, incubate again with PBS-T containing 0.1 M
glycine and 10 % normal swine serum for 2 h, and then incu-
bate overnight, with rabbit antisera against TH (Affi niti, UK),
at 1:1,000 in PBS-T containing 2 % normal swine serum.
4. Wash in PBS-T and incubate with an Alexa 488 goat anti-rab-
bit antibody (Molecular Probes, Netherlands) at 1:1,000 in
PBS-T for 1 h ( see Note 3 ).
5. Mount sections on gelatin-coated slides and add a drop of
glycerol/phosphate [3:1 (vol/vol) mixture of glycerol and
0.4 M PBS] to the coverslip.

3.3 Effects of HSV-1-
Mediated Gene
Delivery

The effects of the THa vector on nociceptive behaviors were tested
on a neuropathic pain model, the spared nerve injury (SNI) model
developed by Decosterd and Woolf [ 16 ]. The animals were fi rst
subjected to a surgery to induce the SNI model and 14 days later
were stereotaxically injected into the DRt following the procedure
in Sect. 3.2.1 with the THa vector and/or the control vectors
THTH and THZ. The surgery for SNI induction is performed on
Wistar rats (Charles River, Spain) weighing 210-220 g as follows:

3.3.1 Nociceptive
Behavioral Effects

Neuropathic Pain Induction
and Behavioral
Assessment

1. Anesthetize the animals with an intraperitoneal (i.p.) injection
of a mixture of ketamine hydrochloride (0.06 g/kg) and
medetomidine (0.25 g/kg).
2. Clean the left thigh with Betadine ® solution. Make an incision
in the skin on the lateral surface, and then section directly
through the biceps femoris muscle exposing the sciatic nerve
and its three terminal branches: the common peroneal, tibial,
and sural nerves.
3. Proceed with the ligation of the common peroneal and tibial
nerves with a 5.0 silk and the sectioning of the nerves distal to
the ligation removing 2-4 mm of the distal nerve stump. Avoid
any contact with or stretching of the intact sural nerve.
4. Suture the muscle with the 5.0 silk, and then close the skin
with surgical suture clips.

After SNI induction, animals develop a sustained and robust
mechanical hyperalgesia and cold allodynia [ 16 ]. Mechanical
hyperalgesia and cold allodynia were assessed by the pin-prick and
acetone tests, respectively, before SNI induction and several days