80 D
ing of injured Ab-fibres [30–32]. Previously, we found animals showed normal behaviour upon recovery from the
NPY acts indirectly via a release of neurotrophin-3 NT-3 anaesthetic. Ethics approval for this study was obtained
from spinal cord to enhance neurite outgrowth of disso- from RNSH UTS Animal Ethics Committee in accordance
ciated dorsal root ganglion DRG cells [30,32]. In vivo with the NHMRC guidelines.
experiments further demonstrate that intrathecal adminis- tration of NT-3 in normal animals induces allodynia and an
2.2. Oligonucleotides expansion of Ab-fibre terminals into lamina II [32]. The
role of endogenous NT-3, however, in nerve injury-in- The oligonucleotides used in this study were NT-3
duced Ab-fibre sprouting and allodynia has not been antisense, 59-CAT CAC CTT GTT CAC-39 and NT-3
determined. sense, 59-GTG AAC AAG GTG ATG-39 Auspep, Aus-
The aim of this transganglionic labelling study is to tralia [26]. The oligonucleotides were fully phosphoro-
examine the influence of intrathecal administration of NT- thioated and HPLC purified. To test the effectiveness of
3 antisense oligonucleotides on the sprouting of Ab-fibres NT-3 antisense to attenuate the synthesis of NT-3, 12.5 ml
following complete sciatic nerve transection. In addition, of 50 mM of either antisense or sense oligonucleotide was
behavioural studies were also conducted to test whether the administered intrathecally and 24 h later animals were
NT-3 antisense oligonucleotide had an influence on al- overdosed with sodium pentobarbitone and samples of
lodynia using animals with partial nerve ligation. spinal cord were removed. The tissue was weighed and
stored at 2808C until processed. The extraction procedure was according to a previous published method [37].
2. Materials and methods Briefly, tissue samples were homogenized, by hand, in 3
ml of ice-cold 100 mM Tris–HCl buffer pH 7.0 con- 2.1. Surgery
taining 2 bovine serum albumin, 1 M NaCl, 0.02 NaN , 4 mM EDTA, 0.2 Triton X-100, 5 mg ml
3
Catheters 18 cm of polyethylene tubing PE-10 were aprotinin, 0.5 mg ml antipain, 157 mg ml benzamide, 0.1
implanted intrathecally by a method similar to that previ- mg ml
pepstatin A,
5.2 mg ml
phenylmethanesul- ously described [4]. Briefly, male Wistar rats 270–300 g;
fonylfluoride. The homogenate was basified to pH 11 with Gore Hill Animal Research Laboratory, Sydney, Australia
1 M NaOH and centrifuged at 13 0003g for 15 min at were anaesthetized sodium pentobarbitone, 50 mg kg;
48C. The supernatant was then acidified to pH 3 with 1 M intraperitoneal and the tissue between the two posterior
acetic acid and centrifuged for 30 min. The resulting articular processes of L5 was cleared and a laminectomy
supernatant was neutralized with 1 M NaOH, centrifuged was performed to expose the spinal cord. The dura was
and the final supernatant was assayed for NT-3 using a pierced using fine forceps and the catheter was gently fed
commercially available ELISA kit Promega. We found cephalad to a length of 2 cm. The catheter was flushed
that the antisense induced a significant decrease in NT-3 with saline, containing 10 U ml heparin, to ensure that
levels in spinal cord 24 h after intrathecal treatment there was no leakage into the surrounding tissue. The
untreated, 24186286 pg g wet tissue wt, n510; sense catheter was secured to the superficial lumbar muscles with
treated, 21296156, n54; antisense treated, 13676340, 4 0 silk sutures and the remainder of the catheter was
n56; untreated vs. antisense, P,0.05 and sense vs. tunnelled subcutaneously to a second skin incision midline
antisense, P,0.05; Student’s t-test. over the upper cervical region. Osmotic pumps 0.5 ml h;
14 days; Alzet, CA were attached to the catheter and 2.3. Transganglionic labelling
implanted subcutaneously either immediately, as for the transganglionic labelling experiments, or a week later in
Transganglionic labelling of Ab-fibres with chol- re-anaesthetized rats for the behavioural experiments. All
eragenoid-horseradish peroxidase C-HRP was examine in solutions delivered by osmotic pumps contained 10 U ml
rats with complete nerve transection. For this, rats were heparin. At the time of attaching the pumps, the left sciatic
re-anaesthetized 2 weeks after injury. The sciatic nerve nerve was either completely transected or partially ligated.
was exposed and 2 ml of a 0.5 solution of C-HRP For the complete transection, the nerve was exposed and
dissolved in saline; List Biological Laboratories, Camp- ligated with 4-0 silk and cut distal to the ligature. In a
bell, CA which labels myelinated fibres [1,35], was separate group of animals, a sham operation was per-
injected into the nerve via a Hamilton syringe with a formed in which the left nerve was exposed. The wounds
33-gauge needle. The wound was closed and the rats were closed with uninterrupted 3 0 silk and rats allowed to
allowed to recover. After 2–3 days the rats were overdosed recover. For the behavioural experiments, the left sciatic
with sodium pentobarbitone and perfused via the left nerve was partially ligated. For this, the dorsal third of the
ventricle with 250 ml of 0.1 M phosphate-buffered saline left sciatic nerve was tightly ligated with 4-0 silk. A sham
PBS followed by 1000 ml of 4 paraformaldehyde in operation was performed on the right sciatic nerve. All
0.1 M phosphate buffer, pH 7.4, over 1 h, then by 500 ml
D .M. White Brain Research 885 2000 79 –86
81
of 30 sucrose in PBS. The lumbar spinal cord was partially ligated and osmotic pumps were attached to the
removed and stored overnight in 30 sucrose. Fifty catheters and implanted subcutaneously. The results are
micron, transverse sections were cut on a freezing mi- expressed as the percentage change in mechanical thres-
crotome. Sections were reacted for HRP using tetra- hold of the injured paw as compared to the sham-operated
methylbenzidine as the chromagen, dehydrated, cleared control and calculated as: hthreshold of injured paw2
and mounted. The density of terminal labelling across the threshold of sham-operated paw3100j threshold of
different laminae was determined by image analysis. Five sham-operated paw.
groups of animals were examined. These were: 1 un- treated controls; 2 sham operated; 3 nerve transection;
2.5. Statistics 4 nerve-transected animals treated intrathecally with NT-
3 antisense oligonucleotide; and 5 nerve-transected ani- Statistical analysis of the data was done using either
mals treated intrathecally with NT-3 sense. The density of one-way analysis of variance ANOVA followed by
labelling was determined by image analysis using a ´
Scheffe test post hoc comparisons, or Student’s t-test. previously published method [1]. The image was captured
directly from the microscope using a JVC digital camera. Four boxes 25325 mm were placed over the image
3. Results
