Intrathecal interleukin-10 gene therapy attenuates paclitaxel-induced mechanical allodynia and proinflammatory cytokine expression in dorsal root ganglia in rats

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Abstract

Paclitaxel is a commonly used cancer chemotherapy drug that frequently causes painful peripheral neuropathies. The mechanisms underlying this dose-limiting side effect are poorly understood. Growing evidence supports that proinflammatory cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor (TNF), released by activated spinal glial cells and within the dorsal root ganglia (DRG) are critical in enhancing pain in various animal models of neuropathic pain. Whether these cytokines are involved in paclitaxel-induced neuropathy is unknown. Here, using a rat neuropathic pain model induced by repeated systemic paclitaxel injections, we examined whether paclitaxel upregulates proinflammatory cytokine gene expression, and whether these changes and paclitaxel-induced mechanical allodynia can be attenuated by intrathecal IL-1 receptor antagonist (IL-1ra) or intrathecal delivery of plasmid DNA encoding the anti-inflammatory cytokine, interleukin-10 (IL-10). The data show that paclitaxel treatment induces mRNA expression of IL-1, TNF, and immune cell markers in lumbar DRG. Intrathecal IL-1ra reversed paclitaxel-induced allodynia and intrathecal IL-10 gene therapy both prevented, and progressively reversed, this allodynic state. Moreover, IL-10 gene therapy resulted in increased IL-10 mRNA levels in lumbar DRG and meninges, measured 2 weeks after initiation of therapy, whereas paclitaxel-induced expression of IL-1, TNF, and CD11b mRNA in lumbar DRG was markedly decreased. Taken together, these data support that paclitaxel-induced neuropathic pain is mediated by proinflammatory cytokines, possibly released by activated immune cells in the DRG. We propose that targeting the production of proinflammatory cytokines by intrathecal IL-10 gene therapy may be a promising therapeutic strategy for the relief of paclitaxel-induced neuropathic pain.

Introduction

The chemotherapeutic drug paclitaxel (Taxol®) frequently induces painful peripheral neuropathies (Dougherty et al., 2004). The mechanisms underlying this dose-limiting side effect are unclear and few drugs prevent or control it. Available drug therapies primarily target neurons. However, in the past decade, data strongly support that proinflammatory cytokines, in particular interleukin-1 (IL-1) and tumor necrosis factor (TNF), produced by activated glia (microglia and astrocytes), are critical in the creation and maintenance of neuropathic pain in animal models (Marchand et al., 2005, Moalem and Tracey, 2006, Watkins and Maier, 2003).

Targeting glial proinflammatory cytokines may therefore provide an effective approach for the control of neuropathic pain. A promising candidate therapeutic is the anti-inflammatory cytokine interleukin-10 (IL-10), a powerful suppressor of the production and activity of several proinflammatory mediators (Moore et al., 2001, Strle et al., 2001). Indeed, IL-10 has been reported to be effective in the control of acute and chronic pain. Intrathecal IL-10 protein blocks the development of IL-1-mediated pain changes induced by intrathecal dynorphin (Laughlin et al., 2000) and by peri-sciatic snake venom phospholipase A2 (Chacur et al., 2004). Intrathecal IL-10 protein also reverses sciatic nerve chronic constriction injury (CCI)-induced mechanical allodynia and thermal hyperalgesia (Milligan et al., 2005a). In addition, intrathecal administration of adenoviral or adeno-associated viral vectors containing cDNA encoding IL-10 prevented and/or reversed mechanical allodynia induced by intrathecal. HIV-1 gp120, sciatic nerve inflammation (sciatic inflammatory neuropathy, SIN), and CCI (Milligan et al., 2005a, Milligan et al., 2005). More recently, we have been pursuing intrathecal delivery of ‘naked’ plasmid DNA encoding rat IL-10, which results in a far more prolonged reversal of CCI-induced allodynia (Milligan et al., 2006).

Recently, a rat model has been described wherein repeated intraperitoneal injections of low doses of paclitaxel induce stable mechanical allodynia, but no significant systemic toxicity or motor impairment (Polomano et al., 2001). The behavioral responses last for several weeks to months (Flatters and Bennett, 2004, Polomano et al., 2001), thus modeling painful neuropathies in patients that can persist for months after termination of chemotherapy. Pain enhancement in this model is relatively resistant to opioid treatment and does not appear to involve activation of NMDA receptors (Flatters and Bennett, 2004). In contrast, ethosuximide, an anti-epileptic and calcium channel blocker, was effective in reversing mechanical allodynia/hyperalgesia in this model (Flatters and Bennett, 2004). Whether proinflammatory cytokines contribute to paclitaxel-induced neuropathic pain is as yet unknown.

Thus, the purpose of the present study is to assess whether paclitaxel-induced neuropathic pain in rats is mediated by proinflammatory cytokines, and is associated with spinal cord glial activation. We therefore examined whether paclitaxel-induced low-threshold mechanical allodynia is associated with increases in the expression of spinal glial activation markers and whether it can be reversed by intrathecal IL-1 receptor antagonist (IL-1ra) and/or intrathecal IL-10 gene therapy. In addition, the present study explores whether these putative pain modulatory effects are associated with changes in proinflammatory cytokine expression in spinal cord, meninges, and/or dorsal root ganglia.

Section snippets

Subjects

Pathogen-free adult male Sprague–Dawley rats (325–375 g; Harlan Laboratories, Madison, WI) were used in all experiments. Rats were housed in temperature (23 ± 3 °C) and light (12 h:12 h light:dark cycle; lights on at 7 AM) controlled rooms with standard rodent chow and water available ad libitum. Behavioral testing and injections were performed during the light cycle. All procedures were approved by the Institutional Animal Care and Use Committee of the University of Colorado at Boulder.

Drugs and plasmid DNA

Paclitaxel

Experiment 1. Time course of paclitaxel-induced mechanical allodynia

To establish the relative magnitude and duration of effects using different paclitaxel doses, mechanical allodynia was assessed using the von Frey test in rats following i.p. vehicle or paclitaxel (separate groups receiving a cumulative dose of 4 or 8 mg/kg). Both paclitaxel-treated groups developed reliable bilateral allodynia between Days 14–21 (Fig. 1). Mechanical allodynia lasted for approximately 6 weeks in the 4 mg/kg group (p < 0.05 vs. vehicle for Days 24–63, 78), and about 16 weeks in the 8

Discussion

These studies evaluate the efficacy of IL-10 gene therapy in attenuating painful neuropathy in rats produced by the chemotherapeutic paclitaxel. The data demonstrate that intrathecal pDNA-IL-10 reduced paclitaxel-induced bilateral mechanical allodynia (von Frey test) for several weeks. In contrast, control plasmid was only transiently effective. pDNA-IL-10 administered shortly after the last paclitaxel administration delayed subsequent onset and progression of allodynia. This study also

Acknowledgment

This work was supported by Avigen and NIH Grants DA018156 and DA015642. We thank Amgen (Thousand Oaks, CA, USA) for the gift of IL-1ra and vehicle.

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    Disclosure statement. A.L., K.W.J., and R.A.C. are currently employees of and have stock options in Avigen, Inc. D.M. is currently an employee of Amgen. L.R.W. has received research support by Avigen, Inc.

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