Prevention of paclitaxel-evoked painful peripheral neuropathy by acetyl-l-carnitine: Effects on axonal mitochondria, sensory nerve fiber terminal arbors, and cutaneous Langerhans cells

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Abstract

Prophylactic treatment with acetyl-l-carnitine (ALCAR) prevents the neuropathic pain syndrome that is evoked by the chemotherapeutic agent, paclitaxel. The paclitaxel-evoked pain syndrome is associated with degeneration of the intraepidermal terminal arbors of primary afferent neurons, with the activation of cutaneous Langerhans cells, and with an increased incidence of swollen and vacuolated axonal mitochondria in A-fibers and C-fibers. Previous work suggests that ALCAR is neuroprotective in other nerve injury models and that it improves mitochondrial dysfunction. Thus, we examined whether the prophylactic efficacy of ALCAR was associated with the prevention of intraepidermal terminal arbor degeneration, the inhibition of Langerhans cell activation, or the inhibition of swelling and vacuolation of axonal mitochondria. In animals with a confirmed ALCAR effect, we found no evidence of a neuroprotective effect on the paclitaxel-evoked degeneration of sensory terminal arbors or an inhibition of the paclitaxel-evoked activation of Langerhans cells. However, ALCAR treatment completely prevented the paclitaxel-evoked increase in the incidence of swollen and vacuolated C-fiber mitochondria, while having no effect on the paclitaxel-evoked changes in A-fiber mitochondria. Our results suggest that the efficacy of prophylactic ALCAR treatment against the paclitaxel-evoked pain may be related to a protective effect on C-fiber mitochondria.

Introduction

Paclitaxel (Taxol®) is one of the most effective and commonly used anti-neoplastic drugs. Its dose-limiting side effect is peripheral neuropathy, which in many patients is accompanied by chronic neuropathic pain (for reviews see Polomano and Bennett, 2001, Dougherty et al., 2004). Painful peripheral neuropathy occurs with other agents in the taxane class, as well as with chemotherapeutics in the vinca alkaloid and platinum-complex classes. The cause of the neuropathy and of the pain syndrome is unknown.

Acetyl l-carnitine (ALCAR) is a naturally occurring amino acid derivative that plays an essential role in transporting long-chain free fatty acids into the mitochondria (reviewed in Friz and Yue, 1963, Rebouche, 2004). Acute administration of ALCAR temporarily reverses the paclitaxel-evoked neuropathic pain state that is seen in experimental animals (Pisano et al., 2003, Ghirardi et al., 2005, Flatters et al., 2006, Xiao and Bennett, 2007). In addition, we have shown that a prophylactic dosing protocol using daily administration of ALCAR during the exposure to paclitaxel and for the period preceding the appearance of the pain syndrome completely prevents the development of allodynia and hyperalgesia (Flatters et al., 2006).

We have shown that rats with paclitaxel-evoked painful peripheral neuropathy have a significantly increased incidence of axonal mitochondria that are swollen and vacuolated (Flatters and Bennett, 2006). ALCAR treatment ameliorates mitochondrial dysfunction caused by a variety of neurotoxic insults (Virmani et al., 1995, Virmani et al., 2005, Bertamini et al., 2002, Hart et al., 2004a, Sayed-Ahmed et al., 2004, Calabrese et al., 2005, Binienda et al., 2006, Mansour, 2006, Wang et al., 2007). Paclitaxel also causes degeneration of the sensory neurons' intraepidermal terminal arbors, without degeneration of the parent axons in the peripheral nerve, and it activates cutaneous Langerhans cells (Polomano et al., 2001, Flatters and Bennett, 2006, Siau et al., 2006). ALCAR has been shown to have neuroprotective and pro-regenerative effects following primary afferent neuron axotomy (Fernandez et al., 1991, Chiechio et al., 2006, Hart et al., 2002, Hart et al., 2004a, Wilson et al., 2003, Wilson et al., 2007), in the peripheral neuropathy produced by antiretroviral chemotherapy (Hart et al., 2004b, Youle and Osio, 2007), and in diabetic peripheral neuropathy (Lowitt et al., 1995, Pacifici et al., 1992, Sima et al., 1996, Sima et al., 2005, DeGrandis and Mirandi, 2002).

Thus, the protective effect of the prophylactic ACLAR treatment protocol might have one or more mechanisms of action: (1) prevention of the degeneration of intraepidermal nerve fibers, (2) inhibition of the activation of cutaneous Langerhans cells, or (3) prevention of paclitaxel's effect on axonal mitochondria. The present study examined each of these possibilities.

Section snippets

Materials and methods

These experiments conformed to the ethics guidelines of the International Association for the Study of Pain (Zimmermann, 1983), the National Institutes of Health (USA), the Canadian Institutes of Health Research, and the Canadian Council on Animal Care. All experimental protocols were approved by the Animal Care Committee of the Faculty of Medicine, McGill University.

Statistics

Data were analyzed using InStat (version 3.0; GraphPad, Inc.; San Diego, CA, USA). Between-groups comparisons for the behavioral data were analyzed with unpaired t-tests. The IENF and LC counts were analyzed with one-way ANOVAs followed by the Bonferroni multiple comparisons test. Between-groups comparisons of the proportion of atypical mitochondria used the Chi-square statistic. Significance was accepted at p < 0.05.

Behavioral assays of the analgesic effects of ALCAR treatment

As shown in Fig. 1, on D25–D27 the expected mechano-allodynia and mechano-hyperalgesia were present in the paclitaxel-treated rats that received vehicle injections. ALCAR treatment completely prevented the paclitaxel-evoked mechano-allodynia and mechano-hyperalgesia.

Intraepidermal nerve fiber counts

IENFs emerged from subepidermal cutaneous nerve fascicles and traveled vertically into the epidermis where they branched into terminal arbors consisting of multiple fine branches bearing terminal and en passant boutons (Fig. 2A). In

Discussion

We found that an ALCAR treatment protocol that completely prevented the appearance of paclitaxel-evoked neuropathic pain also completely prevented the paclitaxel-evoked increase in the incidence of swollen and vacuolated mitochondria in C-fibers, but not in A-fibers. We found no evidence that ALCAR has a neuroprotective effect against paclitaxel-evoked degeneration of IENFs and no evidence of an effect on the paclitaxel-evoked activation of cutaneous LCs.

Acknowledgments

Supported by the National Institute of Neurological Disorders and Stroke (R01-NS36834) and the Canada Foundation for Innovation. H.W.J. was a Ronald Melzack Postdoctoral Research Fellow of The Louise and Alan Edwards Foundation of Montreal. S.J.L.F. was supported by the Brigham and Women's Hospital Anesthesia Foundation. G.J.B. is a Canada Senior Research Chair. We thank Lina Naso and Johanne Ouellette for their assistance.

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