Biomedical Engineering Reference
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6. In order to delimitate brain nuclei and spinal cord laminae,
immunoreact an additional set of sections, as described above,
and counterstain with formol-thionin [
15
] (
see
Note 2
).
To confi rm the noradrenergic nature of transduced neurons upon
THZ injection into the DRt, a double immunodetection of
Double Immunodetection
of ʲ-Galactosidase
and Tyrosine Hydroxylase
-gal
expressed by THZ and endogenous TH was performed with the
animals being stereotaxically injected following the procedure in
Sect.
3.2.1
and then sacrifi ced at 22 days p.i., time of maximal
behavioral effect (Fig.
4a, b
) [
7
]. The double immunoreaction is
performed as follows:
ʲ
1. After incubation in PBS-T containing 0.1 M glycine and 10 %
normal swine serum for 2 h to decrease background staining,
incubate the sections overnight with a monoclonal anti-
ʲ
-gal
Fig. 4
HSV-1 (THZ vector) injected at the DRt transduces noradrenergic afferents of the nucleus (
a
,
b
).
Photomicrographs representing double-labeled neurons for
ʲ
-gal and TH (
yellowish
) in the locus ceruleus (
a
)
and the A5 noradrenergic cell group (
b
).
-gal-positive neurons are shown in
red,
and TH-positive neurons are
shown in
green
(
a
). Scale bar in
b
: 40 µm (
a
is at the same magnifi cation). The insertion of TH in antisense
orientation into HSV-1 (THa vector) induced analgesia in the spared nerve injury (SNI) model of neuropathic
pain (
c
,
d
). THa induced a sustained attenuation of mechanical hyperalgesia evaluated by the pin-prick test (
c
)
and cold allodynia evaluated by the acetone test (
d
). THa and the control vectors (THZ and THTH) were injected
at time 0, i.e., 2 weeks after SNI induction. Data are presented as mean ± SEM (
n
= 6 for each group); *
P
<0.05,
**
P
<0.01, ***
P
<0.001 THa− vs. THZ
ʲ
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