Oxaliplatin Causes Transient Changes in TRPM8 Channel Activity

Oxaliplatin is a third-generation platinum-based anticancer drug that is widely used as first-line treatment for colorectal carcinoma. Patients treated with oxaliplatin develop an acute peripheral pain several hours after treatment, mostly characterized by cold allodynia as well as a long-term chronic neuropathy. These two phenomena seem to be causally connected. However, the underlying mechanisms that trigger the acute peripheral pain are still poorly understood. Here we show that the activity of the transient receptor potential melastatin 8 (TRPM8) channel but not the activity of any other member of the TRP channel family is transiently increased 1 h after oxaliplatin treatment and decreased 24 h after oxaliplatin treatment. Mechanistically, this is connected with activation of the phospholipase C (PLC) pathway and depletion of phosphatidylinositol 4,5-bisphosphate (PIP2) after oxaliplatin treatment. Inhibition of the PLC pathway can reverse the decreased TRPM8 activity as well as the decreased PIP2-concentrations after oxaliplatin treatment. In summary, these results point out transient changes in TRPM8 activity early after oxaliplatin treatment and a later occurring TRPM8 channel desensitization in primary sensory neurons. These mechanisms may explain the transient cold allodynia after oxaliplatin treatment and highlight an important role of TRPM8 in oxaliplatin-induced acute and neuropathic pain.


Introduction
Oxaliplatin is a first-line cytostatic and the major component of the widely used FOLFOX regimen for the treatment of advanced colorectal cancer [1]. However, in up to 90% of the treated patients oxaliplatin causes neurotoxicity and acute pain that already begins several hours after treatment and that is characterized by cold allodynia [2,3]. Apart from these transient effects, in up to 80% of the patients oxaliplatin also causes a long-term distal sensory neuropathy that can persist lifelong [4,5]. The acute pain after oxaliplatin treatment usually subsides within 2-3 days after treatment; however, clinical studies suggest a causal connection of the oxaliplatin-induced acute pain and the later occurring chronic neuropathy [6]. Although many substances were investigated in clinical trials, currently, there is no preventative treatment available for oxaliplatin-induced neuropathic pain [7].
The cellular and molecular mechanisms of the acute response to oxaliplatin are still unclear. Several studies suggest that oxaliplatin treatment rapidly causes damage to the peripheral nervous system by inducing synthesis of reactive oxygen species (ROS), mitochondrial dysfunction and altered activity of neuronal ion channels [8][9][10][11].
The formation of ROS had already been suggested as target for chemotherapy-induced neuropathic pain (CINP) before. However, clinical studies using antioxidants or neuroprotective substances to prevent or treat chemotherapy-induced neuropathic pain, such as vitamin E, glutathione or α-lipoic acid, all failed to ameliorate CINP in patients [12]. Altered activity of ion channels has been reported to contribute to oxaliplatin-induced neuropathic pain. In this context, the activity of the voltage-gated sodium channel Na (v) 1.6, the potassium channels KCNK2 and KCNK4 as well as the activity of transient receptor potential (TRP) channels was found to be altered by oxaliplatin [11,13,14].
TRP channels are ligand-gated ion channels. Some members of this ion channel family can detect external noxious stimuli, such as heat, cold and chemicals. They respond to these stimuli by opening their pores and allowing calcium to enter the cell [15]. Several TRP channels are highly expressed in sensory neurons. Upon their activation and subsequent calcium influx, depolarization and action potentials can be triggered, causing acute or persistent pain [16]. The most important TRP channels for pain are TRPV1 (vanilloid), TRPA1 (ankyrin) and TRPM8 (melastatin). TRPV1 can be activated by noxious heat or capsaicin, TRPA1 by pungent environmental substances and TRPM8 by noxious cold or menthol [16]. More recently, TRPM3 was identified as an additional receptor for noxious heat [17,18].
Altered activity of TRPA1, TRPV1 and TRMP8 have been attributed to oxaliplatininduced neuropathic pain in previous preclinical studies [13]. In these studies, the TRPchannel activity was assessed several days to weeks after initial oxaliplatin-treatment, thus investigating its contribution to oxaliplatin-induced long-term chronic neuropathic pain. However, as shown recently, the early oxaliplatin-induced acute pain, which starts several hours after treatment in patients, is causally linked to the chronic neuropathy, and patients with strong oxaliplatin-induced acute pain have a higher risk of developing a more severe chronic neuropathy [6]. It is therefore necessary to investigate TRP-channel activity at early timepoints after oxaliplatin treatment to understand its contribution to oxaliplatin-induced neuropathy. Unfortunately, this has not yet been addressed experimentally.
Here, we investigate the activity of the ligand-gated calcium channels TRPV1, TRPA1, TRM8, TRPM3 and the purinergic receptor P2X3, a nucleotide-gated calcium-channel that is expressed in sensory neurons and that is known to contribute to chronic pain [19]. We found that the activity of TRPM8 but not of the other calcium channels was transiently increased 1 h after oxaliplatin treatment and decreased 24 h after oxaliplatin treatment, a timepoint at which mice show mechanical hypersensitivity [20]. This altered TRPM8 activity was not related to changes in the channel's gene expression. Instead, we found that the decreased TRPM8 activity 24 h after oxaliplatin treatment was mediated by phospholipase C (PLC) and phosphatidylinositol 4,5-bisphosphate (PIP 2 )-depletion. In summary, we found that aberrant TRPM8 activity in sensory neurons was a crucial and a very early effect of oxaliplatin-treatment, an effect that seems to be linked to oxaliplatin-induced neuropathy and that may be targeted pharmacologically to alleviate oxaliplatin-induced pain.

The TRPM8 Channel Activity Is Affected after Oxaliplatin-Induced Acute Peripheral Pain
Previously, we showed that oxaliplatin induced mechanical hypersensitivity 24 h after treatment [20]. For the current study, we first investigated whether the activity of the TRPM8 channel was altered in response to oxaliplatin treatment. Using the TRPM8 specific agonist menthol (100 µM, 30 s), we observed that the TRPM8 channel activity was significantly reduced in oxaliplatin-treated animals after 24 h when compared with vehicle-treated animals (p = 0.0006, Figure 1A,B). Similar to the other investigated channels, the number of neurons responding to the selective TRPM8 agonist menthol was not affected ( Figure 1C). the number of neurons responding to the selective TRPM8 agonist menthol was not affected ( Figure 1C). Δratio F340/F380 of the amplitude after a transient Ca 2+ influx after TRPM8 channel stimulation (B) 24 h or (E) 1 h after oxaliplatin treatment. Data represent the means ± SEM from n = 24-64 primary sensory neurons per condition from n = 2-5 mice; ** p < 0.01; *** p < 0.001; unpaired two-tailed t-test with Welch's correction. (C,F) Percentage of neurons responding to TRPM8 (C) 24 h or (F) 1 h after oxaliplatin treatment. Data represent the means ± SEM from n = 20-42 independent measurements per condition; unpaired two-tailed t-test with Welch's correction. Primary sensory neurons were identified by stimulating the DRG cultures with 50 mM KCl for 60 s.
To investigate the impact of oxaliplatin on the TRPM8 channel more transiently, we next analyzed TRPM8 activity 1 h after oxaliplatin treatment. Interestingly, oxaliplatin treatment led to significantly increased TRPM8 channel activity when compared with vehicle-treated animals (p = 0.0089, Figure 1D,E). Likewise, we analyzed the number of neurons responding to the TRPM8 channel agonist menthol 1 h after oxaliplatin treatment ( Figure 1F). As already observed for the later timepoint, no alterations in the number of responding neurons could be observed 1 h after oxaliplatin treatment ( Figure 1C,F).
We next investigated the activity as well as the number of responding neurons of four additional ligand-gated calcium channels that have previously been suggested to contribute to persistent pain states and to hypersensitivity after oxaliplatin treatment (Figure 2). We first started to investigate the TRPV1 and the TRPA1 channel activity as well as the number of neurons responding to their agonists capsaicin (200 nM, 20 s) and AITC To investigate the impact of oxaliplatin on the TRPM8 channel more transiently, we next analyzed TRPM8 activity 1 h after oxaliplatin treatment. Interestingly, oxaliplatin treatment led to significantly increased TRPM8 channel activity when compared with vehicle-treated animals (p = 0.0089, Figure 1D,E). Likewise, we analyzed the number of neurons responding to the TRPM8 channel agonist menthol 1 h after oxaliplatin treatment ( Figure 1F). As already observed for the later timepoint, no alterations in the number of responding neurons could be observed 1 h after oxaliplatin treatment ( Figure 1C,F).
We next investigated the activity as well as the number of responding neurons of four additional ligand-gated calcium channels that have previously been suggested to contribute to persistent pain states and to hypersensitivity after oxaliplatin treatment ( Figure 2). We first started to investigate the TRPV1 and the TRPA1 channel activity as well as the number of neurons responding to their agonists capsaicin (200 nM, 20 s) and AITC (allyl isocyanate; 100 µM, 30 s), as several studies described an involvement of both channels in the induction of oxaliplatin-induced acute peripheral pain [14,21,22]. However, neither TRPV1 nor TRPA1 channel activity nor the number of responding neurons was affected 24 h after oxaliplatin treatment (Figure 2A-F). Viable neurons were identified by a short KCl stimulation at the end of each measurement, causing depolarization and activation of voltage-gated calcium channels. Previously, we could observe that the mRNA expressions of TRPV1 and TRPA1 were unchanged after oxaliplatin treatment [20]. We next investigated the mRNA expression levels of several other ion channels that have previously been connected with oxaliplatin-induced hypersensitivity [14]. For this experiment, DRGs were harvested from mice 24 h after oxaliplatin treatment (3 mg kg −1 ), and qPCR analysis was performed. However, we could not observe any alterations in mRNA expression levels for KCNK2, KCNK4, HCN1, piezo1 and P2X3 24 h after oxaliplatin treatment ( Figure S1). Interestingly, the TRPM3 transcript was significantly increased in oxaliplatin-treated mice when compared with vehicle-treated animals after 24 h (p = 0.0348, Figure S1). We next investigated the activity of the TRPM3 channel but could not detect any differences in the activity and number of neurons responding to the TRPM3-specific agonist pregnenolone sulfate (PS, 40 µM, 45 s, Figure 2G-I) [23]. We additionally analyzed the activity of the nucleotide-gated P2X3 channel and the number of neurons responding to the P2X3specific agonist α,β-methylene-ATP (50 µM, 20 s) [24] because ATP-dysregulation after oxaliplatin treatment was described as a mechanism of oxaliplatin-induced pain by several studies [9,25]. However, when compared with vehicle-treated mice, no differences in Ca 2+ influx in primary sensory neurons nor in the number of neurons responding to α,β-methylene-ATP could be detected after stimulating the P2X3 ion channel of oxaliplatintreated animals ( Figure 2J-L).
In summary, measuring the ligand-gated and calcium-permeable ion-channels revealed transient changes of TRPM8 activity in sensory neurons 1 h and 24 h after oxaliplatin treatment.

The TRPM8 Channel Activity Can Be Reconstituted after PLC and PKC Pathway Inhibiton
Since we saw transient changes in the TRPM8 channel activity after oxaliplatin treatment and a decrease of TRPM8 activity at the later timepoint, we next tested the hypothesis that the TRPM8 channel is desensitized after oxaliplatin treatment. It was shown previously that TRPM8 channel desensitization can occur after activation of the phospholipase C (PLC) pathway followed by depletion of PIP 2 (phosphatidylinositol 4,5-bisphosphate) to IP 3 (inositol 1,4,5-triphosphate) and DAG (1,2-diacylglycerol) [26]. To investigate the influence of oxaliplatin, we used a heterologous expression system with TRPM8 transfected HEK293 cells since the TRPM8 channel is only expressed in 5-10% of primary sensory neurons, and the downstream signaling of TRPM8 activation is triggered only in this fraction of neurons ( Figure 3A). It was important to check whether the heterologous expression system with TRPM8-transfected HEK cells reflects the findings of the Ca 2+ imaging measurements with DRG neurons. We therefore treated TRPM8-transfected HEK cells with two different oxaliplatin concentrations (5 µM and 10 µM) for 24 h prior to Ca 2+ imaging measurements. Treating the cells with 5 µM showed no effect ( Figure S2). However, treatment with 10 µM oxaliplatin for 24 h showed a significantly reduced TRPM8 channel activity similar to the effect in DRG neurons (p < 0.0001, Figure 3B,C). To ensure that the heterologous expression system was comparable to the ex vivo system with primary sensory neurons, we also investigated the activity of the TRPV1 channel in TRPV1-transfected HEK cells ( Figure S3). TRPV1 activity was not affected by oxaliplatin treatment in the heterologous expression system ( Figure S3), which is in accordance with our previous results in primary sensory neurons 24 h after oxaliplatin treatment (Figure 2A,B). We conclude that the heterologous expression system can appropriately reflect the activity of the TRPV1 and the TRPM8 channel and can do so in a comparable manner to its activation in primary sensory neurons 24 h after oxaliplatin treatment.    Next, we analyzed the underlying mechanisms that lead to TRPM8 channel desensitization after oxaliplatin treatment. We therefore performed Ca 2+ imaging with TRPM8transfected HEK cells that were treated with a PLC inhibitor (U 73122 [27], 1 µM for 24 h) in addition to oxaliplatin. Interestingly, the decreased TRPM8 channel activity in transfected HEK cells could be reversed after PLC inhibition (p = 0.0073, Figure 3B).
It is known that PLC pathway activation causes hydrolyzation of PIP 2 into IP 3 and DAG. This can also lead to the activation of the protein kinase C (PKC). As previously shown, PKC activation may lead to TRPM8 channel desensitization as well [28]. We analyzed TRPM8 channel activity after inhibiting PKC ( Figure 3B,C) and, similar to PLC pathway inhibition, we could observe significantly increased TRPM8 channel activity in transfected HEK cells after PKC inhibition (GF 109203X [29], 1 µM for 24 h, p < 0.0001). These results suggest that acute oxaliplatin treatment can induce PLC and PKC pathway activation.
To determine the effect of the PLC-PKC pathway in modulating TRPM8 activity, we next used a specific PLC agonist ( Figure 3D). While we saw a tendency of decreased TRPM8 activity with the PLC activator alone (m-3M3FBS 25 µM), we could reproduce the reduction of TRPM8 activity by oxaliplatin (oxaliplatin: p = 0.003; oxaliplatin ±10 µM PLC agonist: p < 0.0001; oxaliplatin ±25 µM PLC agonist: p < 0.0019). Additional treatment with the PLC activator (m-3M3FBS [30], 10 µM and 25 µM for 24 h) did not cause additional reduction of TRPM8 activity, indicating that the desensitizing effect of oxaliplatin on TRPM8 activity may already be at maximum and cannot be further potentiated by additional PLC activation ( Figure 3D,E).

TRPM8 Channel Desensitization Occurs after TRPM8 Channel Activation and PIP 2 Depletion upon PLC Pathway Activation
In the previous experiments, we observed that PLC pathway inhibition can reverse TRPM8 channel desensitization ( Figure 3B,C). Since PLC activation leads to a depletion of its substrate PIP 2 , we next analyzed the PIP 2 concentrations of untransfected, emptyvector-transfected and TRPM8-transfected HEK cells in the presence of oxaliplatin using a PIP 2 ELISA. Comparing the relative PIP 2 concentrations of vehicle-and oxaliplatintreated HEK cells, we saw a significantly reduced PIP 2 concentration, especially in TPM8transfected HEK cells (p < 0.0001, Figure 4A). Interestingly, the relative PIP 2 concentrations in TRPM8-transfected HEK cells were significantly decreased after oxaliplatin treatment in comparison with the empty-vector-transfected HEK cells and untransfected HEK cells (TRPM8-transfected HEK cells vs. empty-vector-transfected HEK cells after oxaliplatin treatment: p = 0.0001; TRPM8-transfected HEK cells vs. untransfected HEK cells after oxaliplatin treatment: p = 0.0499, Figure 4A). These results show that oxaliplatin can lead to PIP 2 depletion. TRPM8 channel activation causes PLC pathway activation. In turn, PLC hydrolyzes PIP2 to IP3 (inositol 1,4,5-triphosphate) and DAG (1,2-diacylglycerol). PIP2-depletion is responsible for TRPM8 channel desensitization after oxaliplatin treatment.

Discussion
In summary, we could observe that among the different investigated calcium channels, only TRPM8 was affected by oxaliplatin treatment for 24 h. The decreased TRPM8 channel activity is likely to occur due to an early transient activation and subsequent PLC (phospholipase C) pathway activation. Oxaliplatin caused decreases in PIP2 (phosphatidylinositol 4,5-bisphosphate) concentrations, which were observed mainly in TRPM8transfected HEK cells, and this effect could be reversed with a specific PLC inhibitor. (C) Schematic overview of the proposed mechanisms leading to TRPM8 channel desensitization after acute oxaliplatin treatment. TRPM8 channel activation causes PLC pathway activation. In turn, PLC hydrolyzes PIP 2 to IP 3 (inositol 1,4,5-triphosphate) and DAG (1,2-diacylglycerol). PIP 2 -depletion is responsible for TRPM8 channel desensitization after oxaliplatin treatment.
To verify the effect of oxaliplatin activation of the PLC pathway leading to PIP 2 depletion, we next performed a PIP 2 ELISA with HEK cells that were treated with vehicle, oxaliplatin and a PLC inhibitor (D609 [31], 10 µM for 5 h, Figure 4B). The ELISA measurements revealed a significantly increased PIP 2 concentration in TRPM8-transfected HEK cells after treatment with the PLC inhibitor in addition to oxaliplatin, when compared with TRPM8-transfected HEK cells treated with oxaliplatin alone (p < 0.0001, Figure 4B). The results obtained from the PIP 2 ELISA strengthen the presumption that oxaliplatin can affect TRPM8 channel activity by PLC pathway activation.
As shown in Figure 4C, based on the results of this study, we propose that TRPM8 channel desensitization after oxaliplatin treatment (24 h) occurs due to early TRPM8 channel activation and subsequent PLC pathway activation. In turn, PLC can degrade PIP 2 , which positively regulates TRPM8 channel activity, into IP 3 and DAG. Eventually, activation of the PLC pathway can cause TRPM8 channel desensitization after oxaliplatin treatment ( Figure 4C).

Discussion
In summary, we could observe that among the different investigated calcium channels, only TRPM8 was affected by oxaliplatin treatment for 24 h. The decreased TRPM8 channel activity is likely to occur due to an early transient activation and subsequent PLC (phospholipase C) pathway activation. Oxaliplatin caused decreases in PIP 2 (phosphatidylinositol 4,5-bisphosphate) concentrations, which were observed mainly in TRPM8-transfected HEK cells, and this effect could be reversed with a specific PLC inhibitor. These findings suggest a transient and dynamic change of TRPM8 activity after oxaliplatin treatment.
Oxaliplatin does not directly activate the TRPM8 channel [14]. This leads to the conclusion that modulation of TRPM8 by oxaliplatin is mediated in an indirect manner. It was previously described that oxaliplatin increases the expression of TRPM8 in mediumsized neurons of the dorsal root ganglia four days after treatment [32]. In another study, the authors did not see any differences in TRPM8 expression 90 h after treatment [14].
The effects of PLC are crucial for the activity of multiple ligand-gated calcium channels, among them several thermo-sensitive TRP channels. The substrate of PLC, PIP 2 , was previously shown to modulate TRP channel activity with contrasting effects. For example, PIP 2 can increase the activity of TRPM5, is required for osmotic activation of TRPV4, and can inhibit desensitization of TRPA1, while it can decrease the activity of TRPV3 [33]. For TRPM8, it was shown that PIP 2 seems to bind to a C-terminal region, causing positive modulation of the channel [26]. Among the PLC isoforms, the family of PLCds and the isoform PLCd4 seem to be responsible for modulating the activity of TRPM8 [34,35]. The crucial role of PIP 2 for normal TRPM8 activity was confirmed by incorporation of TRPM8 in lipid bilayers. In this system, both menthol and cold activation of TRPM8 depended on PIP 2 [36]. Likewise, it was shown that PIP 2 depletion can lead to tachyphylaxis of TRPM8 and that PIP 2 is required for maintaining the threshold temperature of TRPM8 [37,38], which is in line with our data.
For TRPA1, another supposed cold receptor, the activity modulation of PIP 2 seems to be different and more complex. For example, PIP 2 was shown to inhibit mustard oilinduced desensitization of TRPA1 and TRPV1-dependent cross-desensitization of TRPA1 in cells expressing both channels [39,40]. However, there are contrasting reports indicating negligible effects of PIP 2 on TRPA1 activity [41,42].
According to our results, TRPM8 activity is increased after one hour of oxaliplatin treatment. Although it is known that oxaliplatin does not directly activate TRPM8 [14], it has been shown before that oxaliplatin can rapidly enter sensory neurons and gilal cells after treatment, probably via organic cation transporters (OCT), and can remain in these cells for a long time [43,44]. The accumulation of oxaliplatin in neurons may lead to increased TRPM8 activity, which is in accordance with the acute cold pain that is observed rapidly after treatment in patients [6].
In contrast, after 24 h, TRPM8 activity was decreased, which is probably mediated by previous calcium influx, leading to PLC activation and PIP 2 depletion. It remains to be investigated whether the oxaliplatin levels within the neurons remain the same after initial uptake or if they increase slowly. Moreover, the metabolization of oxaliplatin into oxalate and platinum may additionally modulate TRPM8 activity by accumulation of oxalate and calcium depletion in the cytosol [45]. While the initial increase in TRPM8 activity fits to the acute cold pain response in patients, the decreased TRPM8 activity after 24 h seems to diverge.
However, we suggest that TRPM8 is the first responder to oxaliplatin accumulation in sensory neurons. At later timepoints, this system may be exhausted and other stress responses may take over to maintain the neuron in a sensitized state. After three to four days, the expressions of TRPM8, KCNK2, KCNK4, TRPA1 and Na v 1.8 increase in sensory neurons to initiate a new phase of increased neuronal activity and oxaliplatin-induced hypersensitivity [14,32].
In this regard, it would be interesting to investigate whether an early blockade of excessive TRPM8 activity can also prevent subsequent events that lead to chronic neuropathic pain.
While we see an increased expression of the TRPM3 transcript after oxaliplatin treatment, we do not see any detectable increase of TRPM3 activity in our calcium imaging experiments. We assume that oxaliplatin causes several transcriptional stress responses in sensory neurons, among them the early increase of TRPM3 expression. However, due to posttranscriptional or posttranslational modifications, increased expression does not necessarily correlate with increased protein synthesis or activity. Moreover, several alternative splice variants of TRPM3 have been described that may alter TRPM3 activity and make it more difficult to compare the expression of the channel with its activity [46]. In summary, while TRPM3 represents an important calcium channel that is relevant for pathological pain states, from our observations in this manuscript, we do not have any evidence for its contribution to oxaliplatin-induced acute pain.
Transient changes in TRPM8 channel activity may play an important role in the development of cold allodynia in oxaliplatin-induced acute peripheral pain as shown by oxaliplatin studies focusing on acutely induced pain. Even though we could not observe any changes in the activity of the other TRP channels, PIP 2 is known to modulate the activity of several other TRP channels [47]. For example, it was shown that PIP 2 can lead to TRPV1 channel sensitization [48]. Moreover, Anand et al., reported an increased responsiveness of the TRPV1 channel after acute exposure of oxaliplatin to DRG neurons [49]. According to our findings, apart from PIP 2 depletion, PKC activation can also cause TRPM8 desensitization. A similar mechanism has been observed before by Premkumar et al., who showed that PKC activation can result in dephosphorylation of TRPM8 after bradykinin stimulation, causing decreased TRPM8 activity [50]. Interestingly, PKC seems to have contrasting effects on the activity of different TRP channels. It can mediate dephosphorylation of TRPM8 and reduce its activity, but for example, in TRPV1-positive neurons, it causes phosphorylation of TRPV1 and increased activity of the channel [51]. These contrasting mechanisms of both PLC and PKC strengthen the presumption that heat and cold nociceptors are regulated in a reciprocal manner and that the activation of one of the two neuronal populations leads to decreased activity of the other population, allowing it to focus on relevant external stimuli.
Nevertheless, we could show that particularly the activity of the TRPM8 channel is affected during oxaliplatin-induced acute peripheral pain. This is in line with the findings of previous studies that showed an involvement of the TRPM8 channel during oxaliplatin-induced neuropathic pain but which investigated later timepoints after oxaliplatin treatment. We conclude that targeting TRPM8 early after oxaliplatin treatment may ameliorate oxaliplatin-induced acute pain and may thus reduce the risk of patients developing a strong long-term neuropathy.

Animals
All animal experiments were approved by the local Ethics Committees for Animal Research (Darmstadt, Germany) under the approval code FK/1113, approved on 29 March 2019. In addition, all animal experiments were performed according to the Working Group PPRECISE (Preclinical Pain Research Consortium for Investigating Safety and Efficacy) [52] and the recommendation of the Guide of the Care and Use of Laboratory Animals of the National Institute of Health and the ARRIVE guidelines [53].
All experimental C57BL/6NRj animals were purchased from the commercial breeding company Janvier (Le Genest-Saint-Isle, France). They were housed in a day night cycle of a 12 h rhythm and food and water were available ad libitum. In addition, all animal experiments were performed with 8-to 12-week-old C57BL/6NRj male mice.

Oxaliplatin-Induced Acute Peripheral Pain Model
First, an oxaliplatin stock (Cayman Chemical, Ann Arbor, MI, USA) solution of 3 mg/mL in autoclaved deionized water was prepared. Then, the oxaliplatin stock solution of 3 mg/mL was diluted 1:4 in saline (sodium chloride 0.9% (v/v); Fresenius Kabi, Bad Homburg, Germany). Finally, a dose of 3 mg/kg of the 1:4 diluted oxaliplatin stock solution or vehicle (saline; 0.9% sodium chloride) was injected intraperitoneally (i.p.) in 8-to 12-week-old C57BL/6NRj male animals as described previously [22]. Thus, the application of the described oxaliplatin-induced neuropathic pain model was in accordance with the suggestions of the PPRECISE Working Group [52].

Animal Tissue Isolation
For qPCR analysis, dorsal root ganglia (DRGs) of oxaliplatin-and vehicle-treated mice after 24 h were used. For Ca 2+ imaging experiments, DRGs of oxaliplatin-and vehicle-treated animals after 1 h and 24 h were used.
DRG tissue dissection from oxaliplatin-and vehicle-treated male mice was performed as previously described [54].
DRG tissue for qPCR analysis was immediately frozen in liquid nitrogen and stored until RNA-isolation at −80 • C. For Ca 2+ imaging experiments, dissected DRGs were put in ice-cold HBSS containing CaCl 2 and MgCl 2 (Gibco Life Technologies, Carlsbad, CA, USA).

HEK293 Cell Transfection
Ca 2+ imaging in vitro experiments and PI(4,5)P2 ELISA measurement analysis were performed with transfected HEK cells as a heterologous expression system.

HEK293 Cell Treatment
Prior to Ca 2+ imaging measurement (r)TRPM8-transfected or empty-vector-transfected HEK cells were treated with 5 µM or 10 µM oxaliplatin or saline for 24 h. HEK cells transfected with (h)TRPV1 or empty-vector plasmid DNA were treated with 10 µM oxaliplatin prior to Ca 2+ imaging experiments.
For PI (4,5) P2 ELISA measurement analysis, (r)TRPM8-transfected or empty-vectortransfected and untransfected HEK cells were treated with 10 µM oxaliplatin for 24 h. For investigating the contribution of the PLC pathway to the PIP 2 amount, (r)TRPM8transfected or empty-vector-transfected and untransfected HEK cells were treated with 10 µM oxaliplatin ±10 µM of the PLC inhibitor D609 (Tocris) for 5 h.

PI(4,5)P2 ELISA
To analyze the PI(4,5)P2 concentration, the (r)TRPM8-transfected, empty-vectortransfected and untransfected HEK cells, which were treated as previously described, were grown to 80% confluency in a 6-well plate. Afterward, the lipid extraction of PIP 2 was performed according to the manufacturer s instructions of the PIP 2 ELISA kit (Echelon Biosciences, Salt Lake City, UT, USA) and as previously described [57]. Briefly, after carefully aspirating the old cell media, 1 mL of ice-cold 0.5 M TCA (Sigma-Aldrich, St. Louis, MO, USA) solution was added to the HEK cells. Next, HEK cells were immediately scraped from the 6-well plate. After centrifugation of the HEK cells for 7 min at 1000× g at 4 • C, the cell pellet was washed twice with 1 mL of a 5% TCA solution containing 1 mM EDTA (Honeywell Fluka™, Morristown, NJ, USA). Between each washing step, the HEK cells were first vortexed for 30 s and then centrifuged for 5 min at 1000× g at RT. Afterward, the supernatant was carefully aspirated, and the HEK cell pellet was resolved in 1 mL of MeOH:CHCl 3 (2:1) (MeOH: Sigma-Aldrich; CHCl 3 : Fisher Scientific, Pittsburgh, PA, USA), then the mixture was vortexed for 10 min and centrifuged for 5 min at 1000× g at RT. This step was repeated one more time. Thereafter, 750 µL of MeOH:CHCl 3 :HCl (80:40:1) (HCl: Sigma-Aldrich) was added to the pellet. The mixture was vortexed for a further 25 min prior to centrifuging the mixture for a further 5 min at 1000× g at RT. Next, the supernatant was carefully transferred to a fresh 2 mL Eppendorf tube. Afterward, 250 µL of CHCl 3 and then 450 µL of 0.1 N HCl were added to the supernatant. The mixture was vortexed for 30 s and then centrifuged for 5 min at 1000× g at RT. At least 500 µL of the lower organic phase was collected in a new 1.5 mL Eppendorf tube. Afterward, the lower organic phase was evaporated under a gentle stream of nitrogen at 45 • C. Extracted PIP 2 lipid samples from HEK cells were stored at −20 • C until usage.
After PIP 2 lipid extraction from the treated HEK cells, the PIP 2 concentration was detected by performing a PIP 2 ELISA according to manufacturer s instructions and as previously described [58,59].

Quantitative Real-Time PCR
First, RNA from DRGs of oxaliplatin-and vehicle-treated male mice was isolated (24 h after treatment) by using the mirVana mRNA Isolation kit (Invitrogen by Thermo Fisher Scientific, Carlsbad, CA, USA). For cDNA syntheses, 400 ng of isolated RNA from DRGs was used. The cDNA syntheses were performed with the First Strand cDNA syntheses kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer s instructions. Finally, the following qPCR analysis of the TRPM3, TRPV4, kcnk2, kcnk4, hcn1, piezo1 and P2X3 transcript was performed by the use of assay primers of the TaqMan ® Gene Expression system (Thermo Fisher Scientific) on a QuantStudio 5 Real-Time PCR system (Thermo Fisher Scientific) according to manufacturer s instructions and as previously described [60,61]. For relative mRNA expression quantification, the software QuantStudio™ Design and Analysis Software v1.4.3 (Thermo Fisher Scientific) was used. In addition, expression analysis was evaluated according to the ∆∆C(T) method, as described previously [62].

Data Analysis and Statitics
All data are represented as means ± SEM. For in vitro experiments comparing only two groups, the unpaired two-tailed t-test with Welch s correction was used. For in vitro experiments comparing more than two groups, one-way analysis of variance (ANOVA) was used. Dunnett s post hoc test was used for Ca 2+ imaging and PI(4,5)P2 Elisa experiments; Sidak s post hoc test was employed for qPCR analysis. Data with a p-value smaller than 0.05 were considered as statistically significant. Statistical analysis for all experiments was done by using GraphPad Prism 7 software (GraphPad, San Diego, CA, USA).

Conclusions
In summary, we showed that oxaliplatin leads to TRPM8 channel desensitization after transient TRPM8 channel activation and subsequent PLC pathway activation. Hence, transient changes in TRPM8 channel activity contribute to the induction of oxaliplatininduced acute peripheral pain and may be targeted pharmacologically to reduce oxaliplatininduced neuropathic pain.  Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.