In the present study, we provide the first evidence that TRPA1 in DRG neurons mediates the acute phase of oxaliplatin-induced peripheral neuropathy, as supported by the following results. 1) A single administration of oxaliplatin, as well as its metabolite oxalate, produced rapid-onset cold hypersensitivity within 2 h; this response was blocked by a TRPA1 antagonist and by TRPA1 deficiency. 2) Nocifensive behaviors evoked by AITC and menthol, but not by capsaicin, were enhanced 2 h after oxaliplatin administration. 3) Pretreatment of the cultured DRG neurons with oxaliplatin for 2–4 h increased the number of AITC-, but not of menthol- and capsaicin-sensitive neurons.
The peripheral neuropathy caused by chemotherapeutic agents, including oxaliplatin, has been widely evaluated experimentally in rodents as hypersensitivity to mechanical and thermal stimuli in terms of mechanical allodynia and thermal hyperalgesia, respectively. Previous studies in animal models largely focus on the oxaliplatin-induced chronic painful neuropathy that appears several days to several weeks after oxaliplatin administration [19–22, 37], while oxaliplatin-induced acute neuropathy is less well characterized . Our findings in mice, in which cold hypersensitivity was detected as early as 2 h after oxaliplatin administration, is consistent with the clinical observation of a characteristic acute sensory neuropathy triggered by cold that appears during or within hours of oxaliplatin infusion. By contrast, mechanical hypersensitivity in mice was observed 1 day, but not as early as 2 h, after drug administration and persisted for at least 7 days, consistent with previous reports [20, 26, 37]. Moreover, the rapid-onset cold hypersensitivity was not produced by another platinum-based chemotherapeutic agent, cisplatin, or by the non-platinum-containing chemotherapeutic agent, paclitaxel, both of which are known to induce chronic peripheral neuropathy following repeated administration [24, 25, 27, 28]. These findings suggest that the rapid-onset cold hypersensitivity is representative of the acute peripheral sensory neuropathy characteristic to oxaliplatin in mice and that the mouse model is suitable for evaluating the mechanisms of this side effect.
The major finding of this study is that oxaliplatin leads to the selective enhancement of TRPA1-mediated responses within a relatively short time, both in vivo and in vitro. AITC-evoked nocifensive behaviors and Ca2+ influx in DRG neurons are mediated through the activation of TRPA1 [29, 38]. Oxaliplatin increased both of these TRPA1-mediated responses within several hours, suggesting that it rapidly leads to an enhanced TRPA1 responsiveness in sensory neurons. The finding that oxalate enhanced AITC-evoked nocifensive behaviors suggests that the rapid-onset effects characteristic of oxaliplatin are caused by its metabolite, oxalate, or by an oxalate-related structure of oxaliplatin. Consistent with our findings, Sakurai et al. showed that both oxaliplatin and oxalate induce an early-phase (several hours) cold hyperalgesia in the acetone test, whereas a late-phase mechanical allodynia is induced by oxaliplatin or its another metabolite dichloro(1,2-diaminocyclohexane)platinum, but not oxalate, in rats . On the other hand, the oxaliplatin-induced enhancement of AITC-evoked nocifensive behaviors lasted, at least, 3 days after the administration, suggesting that the enhanced TRPA1 responsiveness contributes to not only the acute (several hours), but also, at least, subacute (several days) oxaliplatin-induced cold hypersensitivity.
Despite some controversies, recent evidence points to the involvement of TRPA1 in oxaliplatin-induced subacute and chronic peripheral neuropathy [19–21, 39]. Subacute (several days) mechanical and cold hypersensitivities induced by a single administration of oxaliplatin were shown to be inhibited by either a TRPA1 antagonist or TRPA1 deficiency [19, 20], while the antagonist failed to inhibit the oxaliplatin-enhanced cold-temperature avoidance behavior . Furthermore, subacute (several days) and chronic (several weeks) administration of oxaliplatin increases TRPA1 mRNA levels in DRGs and in the trigeminal ganglion [19, 21]. Controversially, a transient up-regulation of TRPA1 mRNA is observed in DRGs only 6 h after a single administration of oxaliplatin . However, it is unlikely that oxaliplatin is able to increase the expression of functional TRPA1 protein within several hours of its administration. In the Ca2+ imaging experiments of the present study, the AITC concentration was set relatively low (10 μM). Nonetheless, following oxaliplatin treatment, 10 μM AITC produced nearly the same proportion of sensitive neurons (approximately 40%) as obtained with the submaximal concentration of AITC (100 μM) used in the control. Therefore, it is likely that oxaliplatin acutely produces an enhanced TRPA1 responsiveness by increasing the sensitivity to AITC, i.e., through the sensitization of existing TRPA1 and not by an increase in the number of TRPA1-expressing cells. Under inflammatory conditions, TRPA1 sensitization involves its translocation to the plasma membrane via phospholipase C and protein kinase A (PKA) signaling [40, 41]. Acute oxaliplatin may likewise induce the TRPA1 sensitization via PKA signaling  or through other mechanisms specific to oxaliplatin and oxalate.
TRPM8 is expressed in a subpopulation of small-diameter sensory neurons, which correlates with responses to cooling and menthol [18, 42, 43]. Menthol sensation is mainly ascribed to TRPM8, while it also activates TRPA1 in a bimodal manner . In Ca2+ imaging experiments, menthol-sensitive DRG neurons are largely abolished in TRPM8−/− mice, although a small population of menthol-sensitive neurons remains, probably via TRPA1 activation [32, 44]. Nevertheless, we found no change in the number of menthol-sensitive DRG neurons in response to oxaliplatin, suggesting that it has no acute effect on TRPM8-mediated responses. Although menthol-sensitive DRG neurons mediated thorough TRPA1 activation might be increased by acute oxaliplatin, they may be undetectable probably due to too small population or weak activation of TRPA1 by 100 μM menthol in a bimodal phase . Supporting our findings, oxaliplatin enhances Ca2+ responses to icilin (TRPA1/TRPM8 agonist), but not WS12 (TRPM8 selective agonist), in rat DRG neurons . By contrast, our present findings showed that menthol-evoked nocifensive-like behaviors were enhanced after acute oxaliplatin administration, which was inhibited by TRPA1 deficiency. Since it is possible that menthol-evoked nocifensive-like behaviors are mediated through TRPA1 activation , they may be increased by the enhanced responsiveness of TRPA1 after acute oxaliplatin administration in the dose of menthol used in this study. Several studies examine the involvement of TRPM8 in oxaliplatin-induced peripheral neuropathy. The subacute, but not chronic, effects of oxaliplatin administration were shown to include a transient up-regulation of TRPM8 mRNA in DRGs [19, 21, 22]. In TRPM8−/− mice, oxaliplatin-enhanced cold avoidance is abolished, while there is no change in oxaliplatin-induced mechanical hypersensitivity . Pharmacological blockade of TRPM8 has no effect on oxaliplatin-induced subacute cold hypersensitivity . Thus, although TRPM8 involvement in oxaliplatin-induced subacute peripheral neuropathy remains to be clarified, our findings seem to rule out an important role for TRPM8 in oxaliplatin-induced acute peripheral neuropathy.
A body of evidence suggests that TRPV1 plays a role in chemotherapy-induced chronic peripheral neuropathy [21, 24, 25], similar to the neuropathic pain induced by peripheral nerve injury . However, in the present study, neither cisplatin nor paclitaxel altered capsaicin-evoked, TRPV1-mediated nocifensive behaviors. Furthermore, oxaliplatin had no rapid-onset effect on capsaicin-evoked nocifensive behaviors or the number of capsaicin-sensitive DRG neurons, suggesting that oxaliplatin-induced acute peripheral neuropathy is not mediated by TRPV1. Consistent with the present findings, other studies did not find evidence of TRPV1 involvement in oxaliplatin-induced subacute and chronic cold hypersensitivity [20–22].
Accumulating evidence suggests that oxaliplatin, as a platinum-based drug like cisplatin, induces chronic peripheral neuropathy by its direct and indirect neurotoxic effects on peripheral sensory neurons [5, 20]. The painful neurotoxicity may secondarily up-regulate and/or sensitize TRPA1, TRPV1, and TRPM8, as occurs in nerve injury-induced neuropathic pain [46–48]. However, our results indicate that oxaliplatin leads to a rapid, preferentially enhanced responsiveness of TRPA1. Since the rapid effect of the drug is unlikely to be due to its neurotoxicity on sensory neurons, a more likely explanation is an alteration in TRPA1 function, either directly or indirectly, within several hours, although the mechanisms remain unclear.