Brain Research 885 2000 79–86 www.elsevier.com locate bres
Research report
Neurotrophin-3 antisense oligonucleotide attenuates nerve injury-induced Ab-fibre sprouting
Deborah M. White
Department of Anaesthesia and Pain Management , University of Sydney, Royal North Shore Hospital, St. Leonards, N.S.W. 2065, Australia
Accepted 29 August 2000
Abstract
It is proposed that following peripheral nerve injury abnormal sprouting of Ab-fibre primary afferent neurons in the spinal cord contributes to the allodynia that often occurs with such injury. Allodynia is characterized as pain due to a stimulus which is normally
non-noxious. Our recent in vivo experiments show that intrathecal administration of neurotrophin-3 NT-3, in normal animals, induces allodynia and sprouting of Ab-fibres. In this study, we examine whether intrathecal administration of NT-3 antisense oligonucleotides 50
mM, via an osmotic pump for 14 days, attenuates nerve injury-induced sprouting and allodynia. The oligonucleotides used in this study were phosphorothioate modified and control experiments, using an ELISA, confirm that intrathecal administration of the antisense induces
a significant decrease in NT-3 levels in the spinal cord. All surgery was conducted on anaesthetized Wistar rats sodium pentobarbitone, i.p. 50 mg kg. Consistent with previous studies, transganglionic labelling of Ab-fibres with choleragenoid-horseradish peroxidase
C-HRP shows that complete transection of the sciatic nerve induces an expansion of C-HRP label into lamina II of the spinal dorsal horn. Using image analysis, we find that intrathecal administration of NT-3 antisense attenuates the density of C-HRP labelling in lamina
II in nerve injured animals. A NT-3 sense oligonucleotide 50 mM has no effect. To test the effect of NT-3 antisense on allodynia, the nociceptive flexion reflex is examined, using an Ugo Basile Analgesymeter, in animals with partial sciatic nerve ligation. Intrathecal
administration of 50 mM NT-3 antisense significantly attenuates nerve injury-induced allodynia, whereas the sense oligonucleotide has no effect. These results provide further evidence that endogenous NT-3 contributes to both nerve injury-induced Ab-fibre sprouting and
allodynia and demonstrates the potential of neurotrophin-3 antisense oligonucleotides as therapeutic agents for neuropathic pain.
2000
Elsevier Science B.V. All rights reserved.
Theme : Sensory systems
Topic : Pain: pathways
Keywords : Neurotrophin-3; Antisense oligonucleotide; Ab-fiber; Allodynia; Nerve injury
1. Introduction central terminals of sensory neurons within the dorsal horn
of the spinal cord following nerve injury, which is Neuropathic pain, commonly associated with peripheral
proposed to contribute to nerve injury-induced allodynia nerve injury, is characterized by spontaneous pain, in-
[25,34]. In particular, Ab-fibres, which respond to non- creased responsiveness to painful stimuli hyperalgesia
noxious stimuli, sprout from lamina III into lamina II, a and by the perception of normally non-noxious stimuli as
region where
small diameter
unmyelinated C-fibre
painful allodynia. Multiple mechanisms presumably con- nociceptors normally terminate [33]. Ultrastructual studies
tribute to the development and maintenance of neuropathic show that the sprouting terminals of Ab-fibre in lamina II
pain and extensive research has been done to examine the form predominantly axodendritic synapses [3,35]. Electro-
injury-induced plasticity of sensory neurons in an attempt physiological experiments indicate that these are functional
to ascertain
the mechanisms
underlying such
pain synapses as stimulation of injured Ab-fibres activates
[6,29,34]. Of recent interest, is the reorganization of the lamina II neurons via either mono- or polysynaptic inputs
[13,36]. Recent studies from this laboratory have aimed to
Tel.: 161-2-9926-8420; fax: 161-2-9906-4079. E-mail address
: debbeauozemail.com.au D.M. White.
characterize the trophic factors contributing to the sprout-
0006-8993 00 – see front matter
2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 9 4 0 - 1
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
