Neurotrophin-3 antisense oligonucleotide attenuates nerve injury-induced Aβ-fibre sprouting

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 Neuropathic pain, commonly associated with peripheral nerve injury, is characterized by spontaneous pain, increased responsiveness to painful stimuli (hyperalgesia) and by the perception of normally non-noxious stimuli as painful (allodynia). Multiple mechanisms presumably contribute to the development and maintenance of neuropathic pain and extensive research has been done to examine the injury-induced plasticity of sensory neurons in an attempt to ascertain the mechanisms underlying such pain [6,29,34]. Of recent interest, is the reorganization of the *Tel.: 161-2-9926-8420; fax: 161-2-9906-4079. E-mail address: [email protected] (D.M. White).

central terminals of sensory neurons within the dorsal horn of the spinal cord following nerve injury, which is proposed to contribute to nerve injury-induced allodynia [25,34]. In particular, Ab-fibres, which respond to nonnoxious stimuli, sprout from lamina III into lamina II, a region where small diameter unmyelinated C-fibre nociceptors normally terminate [33]. Ultrastructual studies show that the sprouting terminals of Ab-fibre in lamina II form predominantly axodendritic synapses [3,35]. Electrophysiological experiments indicate that these are functional synapses as stimulation of injured Ab-fibres activates lamina II neurons via either mono- or polysynaptic inputs [13,36]. Recent studies from this laboratory have aimed to characterize the trophic factors contributing to the sprout-

0006-8993 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )02940-1

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ing of injured Ab-fibres [30–32]. Previously, we found NPY acts indirectly via a release of neurotrophin-3 (NT-3) from spinal cord to enhance neurite outgrowth of dissociated dorsal root ganglion (DRG) cells [30,32]. In vivo experiments further demonstrate that intrathecal administration of NT-3 in normal animals induces allodynia and an expansion of Ab-fibre terminals into lamina II [32]. The role of endogenous NT-3, however, in nerve injury-induced Ab-fibre sprouting and allodynia has not been determined. The aim of this transganglionic labelling study is to examine the influence of intrathecal administration of NT3 antisense oligonucleotides on the sprouting of Ab-fibres following complete sciatic nerve transection. In addition, behavioural studies were also conducted to test whether the NT-3 antisense oligonucleotide had an influence on allodynia using animals with partial nerve ligation.

2. Materials and methods

2.1. Surgery Catheters (18 cm of polyethylene tubing (PE-10)) were implanted intrathecally by a method similar to that previously described [4]. Briefly, male Wistar rats (270–300 g; Gore Hill Animal Research Laboratory, Sydney, Australia) were anaesthetized (sodium pentobarbitone, 50 mg / kg; intraperitoneal) and the tissue between the two posterior articular processes of L5 was cleared and a laminectomy was performed to expose the spinal cord. The dura was pierced using fine forceps and the catheter was gently fed cephalad to a length of 2 cm. The catheter was flushed with saline, containing 10 U / ml heparin, to ensure that there was no leakage into the surrounding tissue. The catheter was secured to the superficial lumbar muscles with 4 / 0 silk sutures and the remainder of the catheter was tunnelled subcutaneously to a second skin incision midline over the upper cervical region. Osmotic pumps (0.5 ml / h; 14 days; Alzet, CA) were attached to the catheter and implanted subcutaneously either immediately, as for the transganglionic labelling experiments, or a week later in re-anaesthetized rats for the behavioural experiments. All solutions delivered by osmotic pumps contained 10 U / ml heparin. At the time of attaching the pumps, the left sciatic nerve was either completely transected or partially ligated. For the complete transection, the nerve was exposed and ligated with 4-0 silk and cut distal to the ligature. In a separate group of animals, a sham operation was performed in which the left nerve was exposed. The wounds were closed with uninterrupted 3 / 0 silk and rats allowed to recover. For the behavioural experiments, the left sciatic nerve was partially ligated. For this, the dorsal third of the left sciatic nerve was tightly ligated with 4-0 silk. A sham operation was performed on the right sciatic nerve. All

animals showed normal behaviour upon recovery from the anaesthetic. Ethics approval for this study was obtained from RNSH / UTS Animal Ethics Committee in accordance with the NH&MRC guidelines.

2.2. Oligonucleotides The oligonucleotides used in this study were NT-3 antisense, 59-CAT CAC CTT GTT CAC-39 and NT-3 sense, 59-GTG AAC AAG GTG ATG-39 (Auspep, Australia) [26]. The oligonucleotides were fully phosphorothioated and HPLC purified. To test the effectiveness of NT-3 antisense to attenuate the synthesis of NT-3, 12.5 ml of 50 mM of either antisense or sense oligonucleotide was administered intrathecally and 24 h later animals were overdosed with sodium pentobarbitone and samples of 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]. Briefly, tissue samples were homogenized, by hand, in 3 ml of ice-cold 100 mM Tris–HCl buffer (pH 7.0) containing 2% bovine serum albumin, 1 M NaCl, 0.02% NaN 3 , 4 mM EDTA, 0.2% Triton X-100, 5 mg / ml aprotinin, 0.5 mg / ml antipain, 157 mg / ml benzamide, 0.1 mg / ml pepstatin A, 5.2 mg / ml phenylmethanesulfonylfluoride. The homogenate was basified to pH 11 with 1 M NaOH and centrifuged at 13 0003g for 15 min at 48C. The supernatant was then acidified to pH 3 with 1 M acetic acid and centrifuged for 30 min. The resulting supernatant was neutralized with 1 M NaOH, centrifuged and the final supernatant was assayed for NT-3 using a commercially available ELISA kit (Promega). We found that the antisense induced a significant decrease in NT-3 levels in spinal cord 24 h after intrathecal treatment (untreated, 24186286 pg / g wet tissue wt, n510; sense treated, 21296156, n54; antisense treated, 13676340, n56; untreated vs. antisense, P,0.05 and sense vs. antisense, P,0.05; Student’s t-test).

2.3. Transganglionic labelling Transganglionic labelling of Ab-fibres with choleragenoid-horseradish peroxidase (C-HRP) was examine in rats with complete nerve transection. For this, rats were re-anaesthetized 2 weeks after injury. The sciatic nerve was exposed and 2 ml of a 0.5% solution of C-HRP (dissolved in saline; List Biological Laboratories, Campbell, CA) which labels myelinated fibres [1,35], was injected into the nerve via a Hamilton syringe with a 33-gauge needle. The wound was closed and the rats allowed to recover. After 2–3 days the rats were overdosed with sodium pentobarbitone and perfused via the left ventricle with 250 ml of 0.1 M phosphate-buffered saline (PBS) followed by 1000 ml of 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, over 1 h, then by 500 ml

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of 30% sucrose in PBS. The lumbar spinal cord was removed and stored overnight in 30% sucrose. Fifty micron, transverse sections were cut on a freezing microtome. Sections were reacted for HRP using tetramethylbenzidine as the chromagen, dehydrated, cleared and mounted. The density of terminal labelling across the different laminae was determined by image analysis. Five groups of animals were examined. These were: (1) untreated controls; (2) sham operated; (3) nerve transection; (4) nerve-transected animals treated intrathecally with NT3 antisense oligonucleotide; and (5) nerve-transected animals treated intrathecally with NT-3 sense. The density of labelling was determined by image analysis using a 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 within lamina II and in the adjacent lamina III area. The area of C-HRP label within these boxes was computed using Scion Image (Windows version of NIH Image; Scion Corp., MA). An average of the four measurements was calculated and the results are expressed as the ratio of lamina II / lamina III. Transganglionic labelling of Abfibres was also done on two groups of animals with partial nerve ligation. In one group the C-HRP was injected proximal to the ligature to label all Ab-fibres and, in the second group, the conjugated was injected distal to the ligature to label the intact fibres. In control experiments, the cross-sectional area of cells labelled with C-HRP was examined in L5 dorsal root ganglion cells ipsilateral and contralateral to complete transection of the sciatic nerve (n54). Twenty-micron serial sections were cut, mounted and processed as described above. Cross-sectional area of individually labelled cells was determined in every 20th section. The circumference of labelled cells, in which the nucleus could be seen, was manually drawn using a computer mouse and from this the cross-sectional area was computed.

2.4. Behavioural test The nociceptive flexion reflex was quantified using an Ugo Basile Analgesymeter (Comerio-Varese, Italy). A mechanical force increasing linearly with time was applied to the dorsum of the rat’s hindpaw by a dome-shaped plunger (diameter 1.4 mm, radius of curvature 368). The nociceptive threshold was defined as the force, in grams, at which the rat withdrew its paw. The chronic intrathecal catheters were inserted 1 week before commencing behavioural studies to allow rats sufficient time for recovery. Nociceptive thresholds were then determined on a daily basis over a 3-week period. Each day, thresholds were determined at 10-min intervals for a period of 2 h. The mean of the last six measurements represented the nociceptive threshold for that day. After the first 5 days of testing, the rats were re-anaesthetized, the sciatic nerve was

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partially ligated and osmotic pumps were attached to the catheters and implanted subcutaneously. The results are expressed as the percentage change in mechanical threshold of the injured paw as compared to the sham-operated control and calculated as: h(threshold of injured paw)2 (threshold of sham-operated paw)3100j /(threshold of sham-operated paw).

2.5. Statistics Statistical analysis of the data was done using either one-way analysis of variance (ANOVA) followed by Scheffe´ test post hoc comparisons, or Student’s t-test.

3. Results

3.1. Effect of NT-3 antisense oligonucleotide on nerve injury-induced Ab -fibre sprouting In sham-operated controls (n55), C-HRP label is present in laminae I, III and deeper lamina, but not present in lamina II (Fig. 1A). This distribution of C-HRP is consistent with previous transganglionic labelling studies using normal animals [1,35]. The image analysis data also shows that the density of C-HRP label in lamina II of sham-operated animals is not different compared to untreated animals (Fig. 2; n54). Two weeks following complete transection of the sciatic nerve (n55), C-HRP label expands into lamina II (Fig. 1B) and the density is significantly different from both untreated and sham rats (Fig. 2; ANOVA, P,0.05). Intrathecal administration of NT-3 antisense oligonucleotide for 14 days via an osmotic minipump significantly attenuated the density of C-HRP label in lamina II of injured rats (n54) compared to the control group of animals with complete nerve transection (Figs. 1C and 2; nerve injury vs. nerve injury1antisense, P,0.05). Control experiments show that intrathecal administration of sense oligonucleotide (n54) had no effect on the nerve injuryinduced expansion of C-HRP into lamina II (Figs. 1D and 2). A comparison of the cross-sectional area of cells labelled with C-HRP reveals there is no significant difference in size of injured DRG cells compared to control cells of the contralateral DRG (injured, 1062619 mm 2 , 528 profiles, n54; control, 1086624 mm 2 , 404 profiles, n54).

3.2. Effect of NT-3 antisense oligonucleotide on nerve injury-induced allodynia The influence of NT-3 antisense and sense oligonucleotides on nerve injury-induced allodynia was also examined in animals with partial nerve ligation. Partial nerve ligation

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Fig. 1. Photomicrographs of dorsal horn of lumbar spinal cord showing area of termination of Ab-fibres transganglionically labelled with C-HRP injected into the left sciatic nerve. (A) In sham-operated animals (n55), C-HRP label is present in lamina I, III and deeper laminae. No C-HRP label is present in lamina II (*). (B) Two weeks following complete sciatic nerve transection (n55), C-HRP label expands into lamina II. (C) Intrathecal administration of NT-3 antisense oligonucleotides via osmotic pumps for 14 days (n54) attenuated the intensity of C-HRP label in lamina II in nerve injured animals. (D) Intrathecal administration of NT-3 sense (n54) had no influence on intensity of C-HRP label in lamina II of rats with nerve injury. Calibration bar550 mm.

induces a significant decrease in threshold to mechanical stimulation compared to measurements determined prior to ligation in week 1 (Fig. 3; P,0.0002, n58). Animals treated intrathecally with NT-3 antisense significantly attenuated the nerve injury-induced allodynia compared to the untreated control group of animals (Fig. 3A; P,0.05, n510). The analgesic effect of the NT-3 antisense became significant 5 days after nerve injury and persisted for the remainder of the treatment period. In control experiments, intrathecal administration of NT-3 sense oligonucleotides had no significant effect on nerve injury-induced allodynia compared to untreated controls (Fig. 3B; n510). Transganglionic labelling of Ab-fibres in animals with partial nerve ligation shows an uneven spread of C-HRP label into lamina II (Fig. 4B; n54). As there was a large variation in C-HRP density in lamina II between animals with partial ligation, image analysis of animals treated with oligonucleotides was not examined. Of note, injection of

C-HRP distal to the ligature also results in expansion of label in lamina II, suggesting sprouting occurs in intact Ab-fibres (Fig. 4C; n53).

4. Discussion The use of NT-3 antisense oligonucleotides to attenuate nerve injury-induced sprouting and allodynia implicates a contribution of endogenous NT-3. The findings are consistent with our previous study showing that exogenous NT-3, administered intrathecally, induces an expansion of the area of termination of Ab-fibres in the spinal cord and allodynia in normal animals [32]. Little is actually known about the actions of endogenous NT-3 in the adult spinal cord. The focus of many studies has been the effect of exogenous NT-3 which, in general, appears to enhance

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Fig. 2. Image analysis of the density of C-HRP in lamina II of the spinal cord. Two weeks after complete nerve transection (n55) there is a significant increase in the density of C-HRP in lamina II compared to both untreated (n54) and sham-operated animals (n55; P,0.05, ANOVA). Intrathecal administration of NT-3 sense oligonucleotide (50 mM; n54) had no effect on the C-HRP density in lamina II. The density of C-HRP in lamina II was significantly attenuated in nerve injured animals treated intrathecally with NT-3 antisense oligonucleotide (50 mM; n54; P,0.05). The results are expressed as a ratio of the area occupied by C-HRP-labelled nerve terminals in lamina II compared to lamina III.

repair and survival of injured neurons [5,7,17,20,22,27]. With respect to sensory neurons, exogenous NT-3 enhances neurite outgrowth of dissociated DRG cells [2,18,32]; enhances regrowth of injured dorsal roots into the spinal cord in adult animals [20]; and accelerates the regeneration of injured sciatic nerve [22]. Consistently, exogenous NT-3 also up-regulates the expression of cytoskeleton proteins such as MAP5 [21] and tubulin [18] in dissociated sensory neurons. It has been suggested that NT-3 may be clinically useful for neuronal regeneration after injury. Such an approach would be ill-advised in view of the present study showing that NT-3 contributes to aberrant neuronal growth and neuropathic pain. Intrathecal administration of NT-3 antisense oligonucleotides only partially attenuates nerve injury-induced Ab-fibre sprouting and allodynia. One explanation for this was the incomplete inhibition of NT-3 synthesis induced by the antisense treatment. Another possible explanation is that the transganglionic labelling experiments are likely to show sprouting of only cutaneous Ab-fibres sprouting from lamina III into II [34]. In normal animals, the NT-3 receptor, trk C, is localized on a small subpopulation of cutaneous DRGs whereas it is predominantly found on muscle afferents [16], which terminate in lamina V and deeper laminae [19]. NT-3 antisense may, therefore, be attenuating the sprouting of a small population of DRGs and multiple mediators are likely to be involved in nerve injury-induced sprouting of sensory neurons. Recent data cast doubt on the selectivity of C-HRP for Ab-fibres after nerve injury, suggesting that small diameter DRGs are also labelled [28]. In the present study, control

Fig. 3. Effect of intrathecally administered NT-3 antisense on nerve injury-induced allodynia. Partial nerve ligation induces a significant decrease in threshold to mechanical stimuli compared to the thresholds measured prior to nerve injury (n58; P,0.0002, ANOVA). (A) Intrathecal administration of NT-3 antisense significantly attenuates the nerve injury-induced allodynia (n510; P,0.05). The effect of NT-3 antisense becomes significant 5 days after nerve injury and osmotic pump implantation. (B) Nerve injury-induced allodynia was not influenced by NT-3 sense oligonucleotide (n510).

experiments show no change in cell size of DRGs labelled by C-HRP following nerve injury compared to normal DRGs, which is in agreement with two other studies [1,35]. Furthermore, it has been demonstrated that the size of axons in the dorsal roots containing C-HRP is not different after nerve injury [35]. In the present study, it is likely that the NT-3 antisense-induced changes in C-HRP density in lamina II are due to an action on Ab-fibres and not C-fibres as it is the large diameter DRGs that express the trk C receptor. The reason for the discrepancy over the specificity of C-HRP in nerve injured animals is unclear. In the partial nerve ligation animal model of neuropathic pain it has been previously reported that the allodynia to mechanical stimuli is mediated, in part, by myelinated primary afferents [23] and biochemical changes, consistent with complete nerve transection, also occurs [8,14]. In the

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Fig. 4. Photomicrographs of C-HRP labelled nerve terminals in spinal cord of rats with partial nerve ligation. (A) C-HRP injected into the left sciatic nerve of a sham-operated animals (same as Fig. 1A). C-HRP is present in lamina I, III and deeper lamina. There is no label in lamina II (*). (B) C-HRP was injected into the left sciatic nerve proximal to the ligature. There is an uneven density of C-HRP label across lamina II (n54). Calibration is same as in A. (C) Following injection of C-HRP distal to the ligature, there is a small area of label in lamina II, suggesting intact neurons may also sprout into lamina II (n54). Calibration bar550 mm.

current study, the transganglionic labelling experiments also show that rearrangement of the central terminals of Ab-fibres occurs in both intact and ligated sensory neurons. In view of the similarities between partial ligation and complete nerve transection it is tempting to speculate that the attenuation of the nerve injury-induced allodynia by NT-3 antisense is consistent with the suggestion that sprouting of Ab-fibres contributes to the allodynia. As there is no direct evidence showing sprouting of Ab-fibres induces allodynia other factors need to be considered. For example, NT-3 has been shown to enhance synaptic transmission of hippocampus neurons, which possibly involves a pre-synaptic mechanism and local protein synthesis [9,10]. NT-3 also weakly excites central neurons [11] and enhances spontaneous activity of cortical neurons putatively by attenuating GABA inhibitory input [12]. Although the actions of NT-3 on membrane potentials has not been examined in the spinal cord, increased spontaneous activity, enhanced synaptic transmission and attenuation of inhibitory inputs are all plausible mechanisms that one would expect to contribute to neuropathic pain. It was also observed that the NT-3 antisense significantly attenuated the allodynia 5 days after injury, which is before sprouting of Ab-fibres into lamina II is fully established [34,35]. A study by Shortland and Molander [24] provides evidence that Ab-fibres influence activity of lamina II neurons before sprouting occurs. Following nerve injury, stimulation of Ab-fibres induces the expression of c-fos in lamina II as early as 3 days after injury [24]. How Ab-fibres influence the biology of neurons in lamina II at such an early time point is unknown. A possible explanation, is that mediators released from injured Ab-fibres diffuse into lamina II. This is exemplified by the extensive diffusion throughout the dorsal horn of NPY following stimulation of Ab-fibres of an injured sciatic nerve [15]. Whether NT-3 contributes to allodynia via early changes in Ab-fibres that influence lamina II neurons needs to be examined. Many studies have shown that exogenous NT-3 enhances regrowth and survival of injured neurons in both the peripheral and central nervous systems. This study provides evidence that endogenous NT-3 contributes to the aberrant sprouting of Ab-fibres primary afferents and allodynia that occurs following peripheral nerve injury. The data further suggests that intrathecal administration of agents that attenuate the biological actions of NT-3 may potentially have therapeutic benefits in neuropathic pain.

Acknowledgements This project was funded by the Australian and New Zealand College of Anaesthetists and Northern Sydney Area Health Service. Thanks go to Dr Suellen Walker for her support.

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