BJA Advance Access originally published online on August 1, 2006
British Journal of Anaesthesia 2006 97(4):553-558; doi:10.1093/bja/ael209
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Alleviation of neuropathic pain by intrathecal injection of antisense oligonucleotides to p65 subunit of NF-
B
1 Department of Anesthesiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology Wuhan 430022, China
2 Department of Pain Management,Shandong Provincial Hospital Jinan 250021, China
3 Department of Anesthesiology, The Second Hospital in Shenzhen Shenzhen 518035, China
*Corresponding author. E-mail: yaoshanglong{at}eyou.com
Accepted for publication May 19, 2006.
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Abstract |
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Background. Treatment of neuropathic pain remains a challenge. The current study investigated the therapeutic effect of intrathecal administration of NF-
B antisense oligodeoxynucleotides (ODNs) on mechanical allodynia and thermal hyperalgesia in a chronic constriction injury (CCI) model of rats.
Methods. Lumbar intrathecal catheters were implanted in male Sprague–Dawley rats and a CCI model was established. Thermal and mechanical nociceptive thresholds were assessed with paw withdrawal latency (PWL) to radiant heat and von Frey filaments. The phosphorothioate-modified antisense ODNs to p65 subunit of NF-B were administered intrathecally on each of five consecutive days post-CCI. Nuclear NF-B p65 expression was assessed by western blot.
Results. CCI induced mechanical allodynia and thermal hyperalgesia and significantly increased NF-B p65 protein expression. Intrathecal injection of antisense ODN markedly suppressed the expression of NF-B p65 protein and significantly attenuated CCI-induced mechanical allodynia and thermal hyperalgesia.
Conclusion. The activation of NF-B pathway may contribute to neuropathic pain in CCI rats. Suppression of NF-B could be a potential new strategy for the treatment of neuropathic pain.
Keywords:
antisense oligodeoxynucleotides;
complications, chronic constriction injury;
NF-B;
spinal cord
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Introduction |
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Neuropathic pain, characterized by hyperalgesia, allodynia and spontaneous pain, often occurs as a result of injuries to the peripheral nerve, dorsal root ganglion (DRG), spinal cord or brain. Treatment of neuropathic pain remains difficult. Recently, the NF-
B pathway has been suggested to be involved in chronic neuropathic pain. It was reported that the percentages of activated NF-B immunoreactive neurons after the partial sciatic nerve injuries in rats in the ipsilateral side of spinal cord to the injury were significantly increased.1 NF-B pathway inhibitors, such as pyrrolidine dithiocarbamate, S1627 (IKK inhibitor) and NF-B decoy, have been used to attenuate chronic pain.2–4 The antisense strategy is a novel gene therapy technique and could be a useful tool for the study of endogenous gene regulation in vivo and in vitro.5 However, few studies have focused on the role of NF-B antisense ODN in neuropathic pain.
In the current study, we administrated NF-B antisense ODN intrathecally in a rat chronic constriction injury (CCI) model to investigate whether the regulation of transcription of NF-B gene is involved in neuropathic pain and to address the feasibility of therapeutic approaches based on specific suppression of the NF-B pathway.
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Materials and methods |
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Animals
A total of 32 male Sprague–Dawley rats (200–250 g, from the Experimental Animal Center of Shandong University) were kept under a 12 h/12 h light–dark cycle regimen, with free access to food and water. Following the IASP guidelines for pain research in animals, all animal studies were approved by the Animal Care and Use Committee at the Shandong University and were in accordance with the University's guidelines for the care and use of laboratory animals.
Experimental groups
Rats were randomly divided into four groups (eight rats in each group): the sham group (Intrathecal normal saline, IT NS), CCI group (CCI+IT NS ), missense ODN group (CCI+IT missense ODN) and antisense ODN group (CCI+IT antisense ODN).
Intrathecal catheter implantation
Intrathecal catheters were implanted as described by Yaksh and Rudy.6 In brief, under anaesthesia with chloral hydrate (300 mg kg–1, i.p.), the occipital muscles were bluntly separated, and then the cisternal membrane was exposed. Polyethylene catheters (PE-10) were inserted via an incision in the cisterna magna and advanced 7.0–7.5 cm caudally to the level of the lumbar enlargement. Correct intrathecal placement was confirmed by the dragging or paralysis of bilateral hind limbs after injection of lidocaine 2%, 10 µl. The incision site was closed in layers and the catheter was fixed firmly under the skin and sealed effectively. The rats were allowed to recover for 3 days before the CCI test and monitored daily after surgery for signs of motor deficiency or infection.
Chronic constriction injury
CCI to the sciatic nerve was performed as previously described by Bennett and Xie.7 Briefly, the rats were anesthetized with chloral hydrate (300 mg kg–1 i.p.) and the left sciatic nerve was exposed at the mid-thigh level. Four chromic gut (4–0) ligatures were tied loosely proximal to the trifurcation of the sciatica's at 1.0 mm intervals. Sham surgery was performed by exposing the left sciatic nerve without ligation. The incision site was closed in layers and penicillin was administered i.m.
Drug administration
Intrathecal drug administration was accomplished using a microinjection syringe connected to the intrathecal catheter in awake, gently restrained rats. The injection was performed manually within 30 s using a single injection volume of ODN (20 µg per 10 µl) or normal saline (10 µl) followed by a flush with 10 µl dd-H2O. Antisense ODN was administered on each of five successive days post-CCI. Normal saline and missense ODN were injected as controls.
Antisense and missense oligonucleotides to the rat p65 subunit were designed according to the cloned 5' end fragment of the rat p65 gene.8 Antisense ODN (5'-GGGGAACAGTTCGTCCATGGC-3') and missense ODN (5'-GGGGCGATGAGGCCTACTATC-3') were synthesized by SBS Genetech Co., Ltd (Beijing, China) and modified with phosphorothioate. Carboxyfluorescein (FAM) was partly labelled to the 5' end of ODN to examine transfection efficacy by fluorescence microscopy. Synthetic ODN was dissolved in nuclease-free dd-H2O to a final concentration of 20 µg per 10 µl.
Evaluation of tactile allodynia and thermal hyperalgesia
Mechanical allodynia was assessed with von Frey filaments. The animals, placed on a wire mesh platform and covered with a transparent plastic dome (20 cm x 25 cm x 15 cm), were allowed to acclimate to their surroundings for 30 min before testing. Each filament was applied perpendicularly to the plantar surface of the hindpaw (ipsilateral to the side of surgery in nerve-injured animals). The paw withdrawal threshold (PWT) was determined by sequentially increasing and decreasing the stimulus strength (the ‘up-and-down’ method), and the data were analysed using the nonparametric method of Dixon, as described by Chaplan and colleagues.9
Thermal hyperalgesia was assessed using the paw withdrawal latency (PWL) to radiant heat according to the protocol of Hargreaves and colleagues.10 Rats were placed in clear plastic cages on an elevated glass plate and allowed to acclimate to their surroundings for 30 min before testing. A high intensity light beam was focused onto the plantar surface of the hindpaw through the glass plate. The nociceptive endpoints in the radiant heat test were the characteristic lifting or licking of the hind paw, while the time to the endpoint was considered the PWL. To avoid tissue damage, a cut-off time of 30 s was used. There were three trials per rat with 5 min intervals between trials. Tests were performed at 1 day before and 1, 3, 5, 7 and 14 days after surgery.
Evaluation of spinal cord ODN uptake11
FAM-labelled ODN was injected intrathecally as a single bolus of 20 µg following the CCI procedure. Eight hours after FAM-labelled ODN administration, rats were deeply anesthetized with chloral hydrate and perfused transcardially with 200 ml of phosphate buffered saline (PBS, pH 7.4) containing heparin (1500 iu litre–1), followed by 500 ml of cold paraformaldehyde 4%. The L4-5 spinal cord was dissected and cryoprotected with sucrose 30% in nuclease-free PBS overnight at 4°C. Frozen sections (20 µm) were thaw-mounted onto glass slides. Fluorescence images were acquired using an inverted fluorescence microscope. As a control, 20 µg of normal saline was injected intrathecally.
Extracts and western blot analysis
Lumbosacral spinal cord samples were extracted and stored in liquid nitrogen. Nuclear extracts were prepared by adaptation of previously described techniques through freeze/thaw cycles between crushed ice and liquid nitrogen.12 Briefly, tissue samples were homogenized in ice-cold buffer A (in mmol litre–1) [HEPES10, MgCl2 1.5, KCl 10, edetic acid 0.1, egtazic acid 0.1, NaF 50, dithiothreitol (DTT) 1, ß-phosphoglycerol 30, Na3VO4 1, benzamidine 1, phenylmethylsulfonyl fluoride (PMSF) 0.5, p-nitrophenyl phosphate (PNPP) 1 and aprotinin 10, leupeptin 10, pepstatin A 10; pH 7.9]. Proteins were left for 10 min, after addition of 10% NP-40 to a final concentration of 1%, the homogenates were vortexed for 30 s and then centrifuged for 15 min at 800 g. The nuclear pellets were resuspended in buffer B (in mmol litre–1) (HEPES 20, NaCl 420, MgCl2 0.5, edetic acid 1, egtazic acid 1, DTT 1, glycerol 20%, and enzyme inhibitors above, pH 7.9) and then were left for 30 min at 4°C with constant agitation. After centrifugation for 15 min at 12 000 g, nuclear extracts were aliquoted and stored at –80°C until use. Protein concentrations were determined using Bradford method. Samples were mixed with loading buffer and boiled for 5 min. Proteins were separated on SDS–PAGE 10% gel, then were electroblotted onto NC membranes (Millipore, Bedford, MA). After blocking for 2 h in PBS with Tween 20 0.1% (PBST) and BSA 3%, membranes were incubated overnight at 4°C with primary rabbit polyclonal anti-p65 (1:1000 Santa Cruz Biotechnology, CA, USA). Membranes were then washed and incubated with secondary antibody (Santa Cruz Biotechnology, California, USA) for 2 h and detected using the NBT/BCIP assay kit.
Statistical analysis
All data are expressed as mean (SD). Statistical analysis was performed using one-way ANOVA or Student's t-test. P<0.05 was considered statistically significant.
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Results |
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Effects of NB-
B p65 antisense ODN on CCI-induced mechanical allodynia and thermal hyperalgesiaCCI, but not sham surgery, produced significant mechanical allodynia and thermal hyperalgesia (P<0.05). The time course of PWT and PWL is presented in Figure 1. Intrathecal injection of NF-
B p65 antisense ODN, not missense ODN, attenuated CCI-induced mechanical allodynia and thermal hyperalgesia (P<0.05). The attenuation of allodynia and hyperalgesia was found 1 day after the procedure and persisted through the observation period of 14 days. There was a significant difference in terms of PWT and PWL between the antisense ODN group and the sham-operated group (P<0.05).
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P<0.05 vs sham group; +P<0.05 vs CCI group.)Uptake of ODN
In order to confirm delivery of ODN into spinal cord cells, antisense labelled with carboxyfluorescein was administrated intrathecally as a single bolus (20 µg) to the lumbar region of the spinal cord. The uptake of labelled ODN by spinal cord cells was evaluated by fluorescence microscopy. No fluorescence was found following intrathecal injection of normal saline (Fig. 2A). Large numbers of cell bodies were highly fluorescent in the group following intrathecal injection of ODN. Fluorescence was observed primarily in the cytoplasm (Fig. 2B). This fluorescence intensity appeared to be variable among the cell bodies irrespective of cell size.
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Western blot of NF-
B p65Effects of the antisense oligonucleotide in inhibiting NF-
B p65 expression were confirmed at the protein level by western blotting (Fig. 3). Compared with the sham group, CCI rats exhibited significantly higher levels of nuclear NF-B p65 expression (P<0.01). The expression of p65 was markedly attenuated after intrathecal injection of antisense ODN (P<0.05). No statistical difference was found between CCI group and missense ODN group for nuclear NF-B p65 expression (P>0.05).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Results of the current study demonstrated that (i) CCI significantly increased nuclear translocation of NF-
B p65 protein in the spinal cord; (ii) ODN was present in the spinal cord after intrathecal injection of FAM labelled antisense ODN; (iii) expression of NF-B p65 protein in the spinal cord was significantly down-regulated by administration of antisense ODNs; (iv) intrathecal injection of NF-B antisense ODN significantly reduced mechanical allodynia and thermal hyperalgesia in CCI animals. Therefore, these results suggest that antisense ODN may alleviate allodynia and hyperalgesia through the NF-B-related pathway.
NF-B pathway plays a pivotal role in cell regulation: proliferation, immune cell activation, apoptosis, stress responses, differentiation and oncogenic transformation. Recently, several studies have demonstrated that activation of NF-B occurs in the DRG and spinal cord, which are both involved in the transmission and processing of nociceptive information. It has been reported that in NF-B knockout mice the analgesic effects of both low and high frequency electroacupuncture were significantly decreased.13 Increased NF-B activation has been observed in rat lumbar DRG neurons after partial sciatic nerve injury1 and in rat lumbar spinal cord after complete Freund's adjuvant (CFA) was injected subcutaneously into the hindpaws.14 NF-B p65 was found to be expressed in a subpopulation (32%) of mixed diameter rat sensory neurons in L4 and L5 dorsal root ganglia, while ipsilateral p65 staining was abolished in a subgroup (60–70%) of these neurons 4 h after crushing the sciatic nerve. The phenomenon was believed because of the failure of retrograde axonal transport of trophic factor(s).15 Together, these results suggest that spinal cord NF-B activation is involved, at least in part, in heightened pain states.
The inhibition of NF-B has been used to attenuate chronic pain states. It has been reported that intrathecal administration of NF-B inhibitors, pyrrolidinedithiocarbamate (PDTC) and SN50, before gp120 partially alleviated gp120-induced allodynia.16 Pretreatment with PDTC reduced the development of allodynia induced by intrathecal administration of dynorphin in mice.17 Also, intrathecal pretreatment with NF-B decoy or PDTC significantly reduced mechanical allodynia and thermal hyperalgesia after unilateral hindpaw inflammation induced by CFA.18 These findings are consistent with our results that NF-B activation is involved, or required, for the induction of allodynia and hyperalgesia.
Several approaches have been described to interfere with the NF-B pathway. Among them, antisense ODN strategy, which hybridizes with the mRNA strand, is highly specific, as its effect is to inhibit RNA transcription and thereby suppress the synthesis of the gene product. Antisense ODN constitutes a potentially important family of therapeutic compounds for the treatment of a range of diseases.19 In our study, we used antisense ODN strategy to inhibit the NF-B pathway. Antisense technologies have been used both in vitro and in vivo to inhibit expression of NF-B proteins in a variety of experimental systems. Antisense to the p65 subunit has been shown to greatly inhibit the expression of cell adhesion molecules in endothelial and smooth muscle cells in vitro.20 In vivo, the administration of antisense p65 ODN inhibited tumour growth in nude mice, prolongs allo- and xenograft survival and alleviates septic shock in LPS-treated animals.21 22 It has been reported that intracolonic administration of NF-B antisense oligonucleotide is effective in ulcerative colitis.23 As the spinal cord is considered to be one of the key relay stations in the transmission and processing of nociceptive information, intrathecal administration of analgesics becomes an effective mode of pain relief. It has been observed that intrathecal administration of antisense ODN against targets such as Na(V)1.8, calcium channel alpha2delta-1, mGluR1 and c-fos could alleviate chronic pain states.24–27 However, little attention has been paid to NF-B antisense ODN, especially in chronic neuropathic pain models. In the current study, we found that our antisense strategy could effectively block NF-B activation and alleviate neuropathic pain.
Inflammatory cytokines play important roles in the modulation of neuropathic pain. For example, overproduction of IL-6 is associated with neuropathic pain. Intrathecal injection of human recombinant IL-6 in the absence of nerve injury was able to induce mechanical allodynia in rats and thermal hyperalgesia in the sciatic cryoneurolysis model.28 Both ipsilateral and mirror-image allodynia in sciatic inflammatory neuropathy (SIN) models of rats were prevented by intrathecal proinflammatory cytokine antagonists specific for interleukin-1, tumour necrosis factor or interleukin-6.29 Interestingly, these cytokines are proteins down-stream of NF-B. It has been demonstrated that single endoneurial injection of NF-B decoy at the site of nerve lesion significantly alleviated thermal hyperalgesia and suppressed the expression of mRNA of the inflammatory cytokines, iNOS, and adhesion molecules at the site of nerve injury.3 As activated NF-B mediates the expression of diverse inflammatory and immune response mediators, the inhibition of NF-B by antisense ODN administration may prevent the onset of thermal hyperalgesia, possibly because of reduced expression of NF-B regulated inflammatory cytokines, such as TNF-
, IL-1ß, IL-6 and IFN-
; reduced expression of adhesion molecules ICAM-1 and VCAM-1; and chemokines, macrophage inflammatory protein, adhesion factors and proinflammatory enzymes (iNOS, COX-2).30 However, further study is needed to confirm the linkage between NF-B and inflammatory cytokines. In addition, the effect of antisense ODN administration on the DRG is still being examined. Further investigations are needed to detail the NF-kB pathway and its relationships between the DRG and the spinal cord.
In summary, the results of the current study suggest that activation of the NF-B pathway in the spinal cord may contribute to the pathogenesis of neuropathic pain induced by CCI and that suppression of NF-B protein expression by antisense ODN could alleviate hyperalgesia and allodynia in CCI model in rats. The effect of antisense ODN to p65 subunit of NF-B is sequence-specific and efficient. It provides not only a potential way for neuropathic pain management but also a method for the study of neuropathic pain.
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Acknowledgments |
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This work was supported by grants from Shandong Provincial Health Bureau (2003HW109).
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