Mu-Opioid Receptor (MOR) Dependence of Pain in Chemotherapy-Induced Peripheral Neuropathy

Abstract

We recently demonstrated that transient attenuation of Toll-like receptor 4 (TLR4) in dorsal root ganglion (DRG) neurons, can both prevent and reverse pain associated with chemotherapy-induced peripheral neuropathy (CIPN), a severe side effect of cancer chemotherapy, for which treatment options are limited. Given the reduced efficacy of opioid analgesics to treat neuropathic, compared with inflammatory pain, the cross talk between nociceptor TLR4 and mu-opioid receptors (MORs), and that MOR and TLR4 agonists induce hyperalgesic priming (priming), which also occurs in CIPN, we determined, using male rats, whether (1) antisense knockdown of nociceptor MOR attenuates CIPN, (2) and attenuates the priming associated with CIPN, and (3) CIPN also produces opioid-induced hyperalgesia (OIH). We found that intrathecal MOR antisense prevents and reverses hyperalgesia induced by oxaliplatin and paclitaxel, two common clinical chemotherapy agents. Oxaliplatin-induced priming was also markedly attenuated by MOR antisense. Additionally, intradermal morphine, at a dose that does not affect nociceptive threshold in controls, exacerbates mechanical hyperalgesia (OIH) in rats with CIPN, suggesting the presence of OIH. This OIH associated with CIPN is inhibited by interventions that reverse Type II priming [the combination of an inhibitor of Src and mitogen-activated protein kinase (MAPK)], an MOR antagonist, as well as a TLR4 antagonist. Our findings support a role of nociceptor MOR in oxaliplatin-induced pain and priming. We propose that priming and OIH are central to the symptom burden in CIPN, contributing to its chronicity and the limited efficacy of opioid analgesics to treat neuropathic pain.

Significance Statement

It remains unknown why opioid analgesics are less effective against neuropathic pain, including that induced by cancer chemotherapy. Here we demonstrate a crucial role of nociceptor mu-opioid receptors (MORs) in chemotherapy-induced peripheral neuropathy (CIPN) pain and hyperalgesic priming (priming), the latter a mechanism involved in the transition from acute to chronic pain. In addition to neuropathic pain and priming, opioid-induced hyperalgesia (OIH) also develops in chemotherapy-treated rats, which is reversed by inhibitors of second messengers that also reverse Type II priming, as well as by MOR and TLR4 antagonists. Taken together, our results support the suggestion that priming and OIH contribute to CIPN and its limited responsiveness to opioid analgesics.

Introduction

The pain associated with chemotherapy-induced peripheral neuropathy (CIPN), which develops hours to days following their administration, and persists in ∼68% of patients, when assessed 1 month following chemotherapy, and 30% when assessed at 6 months (Seretny et al., 2014; Pachman et al., 2015), responds poorly to available treatments. Despite being used by up to 97% of patients with CIPN pain (Chou et al., 2015; Shah et al., 2018), opioid analgesics are far from an optimal treatment for these patients due to side effects, including the development of tolerance and opioid-induced hyperalgesia (OIH), and to loss of efficacy overtime (Chou et al., 2015; Shah et al., 2018), relegating opioids to third-line therapies for neuropathic pain (Finnerup et al., 2015).

While most chemotherapy agents have relatively poor CNS penetrance (Sandler et al., 1969; Weiden and Wright, 1972; Casey et al., 1973; Alberts and Noel, 1995), secondary to a weaker blood nerve barrier, they have greater access to the peripheral nervous system (PNS; Devor, 1999; Jimenez-Andrade et al., 2008). Dorsal root ganglion (DRG) neurons have been suggested to be key targets for the side effects of many cancer chemotherapies (Negri and Ibison, 1979; Cavaletti et al., 1992; McDonald and Windebank, 2002; Ta et al., 2006; Zhang et al., 2007). One such mechanism, which has been implicated in paclitaxel and oxaliplatin CIPN, is the activation of pattern recognition receptors (PRRs), such as Toll-like receptor 4 (TLR4) and the receptor for advanced glycation end products (RAGE; Araldi et al., 2024), both of which are receptors for damage-associated molecular patterns (DAMPs), and the consequent engagement of innate immune mechanisms within DRG (Han and Smith, 2013; Boyette-Davis et al., 2015; Illias et al., 2022; Araldi et al., 2024). Importantly, nociceptor TLR4 has also been implicated in OIH (Lewis et al., 2010; Hutchinson et al., 2011; Due et al., 2012; Bai et al., 2014; Johnson et al., 2014; Ellis et al., 2016), and we have recently demonstrated that antisense knockdown of TLR4 in DRG neurons eliminated hyperalgesia and hyperalgesic priming (priming) induced by the systemic administration of low-dose morphine (Araldi et al., 2019). We have also recently demonstrated that hyperalgesic priming, another side effect, developed in rats with CIPN (Staurengo-Ferrari et al., 2023).

One DAMP, high mobility group box 1 (HMGB1), a nuclear protein that functions as a pronociceptive mediator when released to the extracellular space, plays a key role in CIPN (Sekiguchi and Kawabata, 2020), activating several PRRs, including TLR4, to accelerate inflammation and pain (Yanai et al., 2012; Yamasoba et al., 2016; Sekiguchi and Kawabata, 2020; Tsujita et al., 2021). Of note in this regard, chronic intrathecal administration of morphine increases the expression of HMGB1 and TLR4 in the rat spinal dorsal horn (Qian et al., 2020). Since dorsal rhizotomy eliminates most of the MOR in the spinal dorsal horn (Stevens and Seybold, 1995), it has been suggested that spinal MOR is located on the central terminals of sensory neurons (Kline and Wiley, 2008). In another study, in a preclinical model of neuropathic pain, systemic administration of morphine produced a persistent increase in HMGB1 expression in the spinal cord (Grace et al., 2018). Taken together, these findings support the suggestion that morphine, acting at MORs on the central terminals of DRG neurons, chronically increases the expression of HMGB1 and TLR4 (Grace et al., 2018; Qian et al., 2020). Given the established cross talk between TLR4 and MOR (Hutchinson et al., 2011; Meng et al., 2013; Araldi et al., 2019; Zhang et al., 2020), and the role of TLR4 in oxaliplatin and paclitaxel CIPN (Han and Smith, 2013; Boyette-Davis et al., 2015; Illias et al., 2022; Araldi et al., 2024), in the present experiments we have explored the impact of attenuating DRG neuron MOR on CIPN pain, as well as the role of nociceptor MOR in OIH and hyperalgesic priming induced by neurotoxic cancer chemotherapy drugs.

Material and Methods

Animals

Experiments were performed on 260–460 g adult male Sprague Dawley rats, supplied by Charles River Laboratories. Experimental animals were housed three per cage, under a 12 h light/dark cycle, in a temperature- and humidity-controlled animal care facility at the University of California, San Francisco. Rats had food and water available ad libitum, in their home cage. Experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of California at San Francisco and adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Efforts were made to minimize the number of animals used and their suffering.

Nociceptive threshold testing

Mechanical nociceptive threshold was quantified using an Ugo Basile Analgesymeter (Randall-Selitto paw-withdrawal device, Stoelting), which applies a linearly increasing mechanical force to the dorsum of a rat's hindpaw (Taiwo et al., 1989; Taiwo and Levine, 1989; Araldi et al., 2015, 2017, 2018b, c, 2019). To minimize restraint stress, rats were placed in cylindrical acrylic restrainers designed to provide ventilation and allow hind leg extension from lateral ports, for the testing of mechanical nociceptive threshold using the Randall-Selitto paw-withdrawal test. To acclimatize rats to the experimental testing procedure, they were placed in restrainers for 1 h prior to starting training sessions, daily, for 3 consecutive days, and for 40 min prior to experimental manipulations. Nociceptive threshold was defined as the force applied by the analgesymeter, in grams, at which a rat withdrew its paw. Baseline threshold was defined as the mean of three readings taken before test agents were injected. To minimize experimenter bias, individuals conducting the behavioral experiments (D.A., L.S.-F., and I.J.M.B.) were blinded to experimental interventions.

Drugs

The following compounds were used in this study: oxaliplatin, paclitaxel, morphine sulfate salt pentahydrate (a nonselective opioid receptor agonist), a selective mu-opioid receptor (MOR) antagonist (CTOP, D-Pen-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2), cordycepin 5′-triphosphate sodium salt (a protein translation inhibitor), prostaglandin E₂ (PGE₂, a hyperalgesic agent that acts directly to sensitize nociceptors), SU6656 (a Src family kinase inhibitor), and U0126 (a MAPK/ERK inhibitor), all of which were purchased from Sigma-Aldrich and the TLR4 antagonist [LPS-RS (lipopolysaccharide from Rhodobacter sphaeroides) Ultrapure], purchased from InvivoGen.

The stock solution of PGE₂ (1 μg/μl) was prepared in 10% ethanol with additional dilutions made with physiological saline (0.9% NaCl), yielding a final ethanol concentration of <1%. Cordycepin, CTOP, LPS-RS Ultrapure, and morphine were dissolved in saline. SU6656 and U0126 were dissolved in 100% DMSO (Sigma-Aldrich) and further diluted in saline containing 2% Tween 80 (Sigma-Aldrich). The final concentration of DMSO and Tween 80 was ∼2%.

Intradermal drug administration was performed at the site of nociceptive threshold testing, on the dorsum of the hindpaw, using a 30-gauge hypodermic needle adapted to a 50 μl Hamilton syringe by a segment of PE-10 polyethylene tubing (Becton Dickinson). The combination of SU6656 and U0126 was diluted to a concentration of 1 μg/2 μl each and the combination injected by adding 2 μl of the test agent, into a syringe separated by an air bubble to prevent mixing in the syringe. The intradermal administration of cordycepin, or the combination of SU6656 plus U0126, were preceded by a hypotonic shock to transiently enhance the permeability of the cell membrane to these agents (1 μl of distilled water separated from the drug by an air bubble, to avoid mixing in the syringe) to transiently enhance entry of molecules into nerve terminals (Borle and Snowdowne, 1982; Burch and Axelrod, 1987). Importantly, control experiments, in vivo, showed that the final concentration of ethanol (2%) used to prepare the solution of PGE₂ had no effect on the mechanical threshold per se (Table 1); 2% DMSO and Tween 80, used to dissolve SU6656 and U0126, also did not affect nociceptive threshold (Table 1).

Table 1.

Mechanical nociceptive threshold, in grams, before and 30 min after intradermal treatment with control/vehicle agents in naive rats

Treatment (intradermal, i.d.)	Mechanical nociceptive threshold (g) before treatment	Mechanical nociceptive threshold (g) 30 min after treatment	Statistics (paired Student's t test)	n of paws
adH2O	129.717 ± 4.83 g	130.833 ± 4.55 g	p = 0.4065, t(5) = 0.9059	6
b2% ethanol	133.100 ± 5.40 g	132.917 ± 6.03 g	p = 0.8338, t(5) = 0.2211	6
cdH20, cordycepin	131.833 ± 4.99 g	130.000 ± 4.79 g	p = 0.6109, t(5) = 0.5423	6
ddH2O, 2% DMSO, 2% Tween 80	131.667 ± 2.30g	128.833 ± 4.18 g	p = 0.3460, t(5) = 1.040	6
edH2O, SU6656, U0126	125.417 ± 3.84	125.700 ± 2.80	p = 0.8562, t(5) = 0.1908	6
fLPS-RS Ultrapure	130.417 ± 3.50 g	131.667 ± 3.85 g	p = 0.5177, t(5) = 0.6956	6
gMorphine	126.670 ± 4.50 g	126.667 ± 5.16 g	p = 0.3774, t(5) = 0.9682	6

Intradermal drug administration was performed at the site of nociceptive threshold testing, on the dorsum of the hindpaw, using a 30-gauge hypodermic needle adapted to a 50 μl Hamilton syringe by a segment of PE-10 polyethylene tubing. The following control experiments were performed in naive male rats:

^a1 μl of distilled water (dH₂O) separated from the saline (5 μl) by an air bubble, to avoid mixing in the syringe.

^b5 μl of saline containing 2% ethanol (PGE₂ control).

^c1 μl of dH₂O separated by an air bubble from the 1 μg of cordycepin dissolved in 5 μl of saline.

^d1 μl of dH₂O separated, by an air bubble, from 5 μl of saline containing 2% DMSO and 2% Tween 80.

^e1 μl of dH₂O separated by an air bubble, from SU6656 (1 μg/2 μl) separated by an air bubble from U0126 (1 μg/2 μl). SU6656 and U0126 were dissolved in saline containing 2% DMSO and 2% Tween 80.

^f1 μg of LPS-RS Ultrapure dissolved in 5 μl of saline.

^g1 μg of morphine dissolved in 5 μl of saline.

Data is presented as mechanical nociceptive threshold (g), mean ± SEM, n = 6 paws per group. As demonstrated in the table, none of the vehicle/control treatment agents produced statistically significant changes in mechanical nociceptive threshold 30 min after their intradermal administration (paired Student's t test).

Preclinical models of CIPN

Oxaliplatin chemotherapy-induced neuropathic pain (CIPN)

Oxaliplatin, dissolved in saline was injected intravenously as a single dose (2 mg/kg), producing a reproducible preclinical model of CIPN in which distinct early and late phases are present (Staurengo-Ferrari et al., 2021; Staurengo-Ferrari et al., 2022; Araldi et al., 2024).

Paclitaxel chemotherapy-induced neuropathic pain (CIPN)

Paclitaxel was prepared in a mixture of absolute ethanol and polyethoxylated castor oil (Cremophor EL; 1:1; Sigma-Aldrich) and further diluted in saline to a final concentration of 1 mg/ml, immediately prior to administration. To produce CIPN, paclitaxel (1 mg/kg) was injected intraperitoneally every other day for a total of four doses (Ferrari et al., 2020; Staurengo-Ferrari et al., 2022; Araldi et al., 2024).

Oligodeoxynucleotide antisense to MOR mRNA

To investigate the role of MOR in the decrease in mechanical nociceptive threshold (hyperalgesia) associated with oxaliplatin and paclitaxel CIPN, and in oxaliplatin-induced hyperalgesic priming, our previously validated oligodeoxynucleotide (ODN) antisense (AS) for MOR mRNA (Ferrari et al., 2019) was employed. The AS-ODN sequence for MOR, 5′-CGC-CCC-AGC-CTC-TTC-CTC-T-3′, is directed against a unique region of rat MOR [UniProtKB database entry P33535 (OPRM_RAT)], to block translation and downregulate gene expression of all eight known MOR isoforms. The ODN mismatch (MM) sequence, 5′-CGC-CCC-GAC-CTC-TTC-CCT-T-3′, is a scrambled version of the antisense sequence with four mismatched bases (denoted by bold letters) that has the same base pairs and GC ratio, with little or no homology to any mRNA sequences posted at GenBank. A nucleotide BLAST search was performed to confirm that the mRNA sequence targeted by the AS-ODN, or its MM-ODN control, were not homologous to any other sequences in the rat database. Oligodeoxynucleotides were synthesized by Thermo Fisher Scientific. Before use, lyophilized ODNs were reconstituted in nuclease-free 0.9% NaCl and then administered intrathecally (i.t.) at a dose of 6 µg/µl in a volume of 20 µl (120 µg/20 µl). As described previously (Alessandri-Haber et al., 2003), after rats were anesthetized with isoflurane (2.5% in O₂), ODN was injected, using a 0.3 ml syringe (300 units/µl; Walgreens), with a 29-gauge hypodermic needle, inserted between the L4 and L5 vertebrae, into the subarachnoid space. The intrathecal site of injection was confirmed by the elicitation of a sudden flick of the rat's tail, a reflex evoked by accessing the subarachnoid space and bolus injection of ODN (Mestre et al., 1994). A total of 120 μg of ODN, in a volume of 20 μl, was then injected. Rats regained consciousness ∼2 min after stopping anesthesia. The use of intrathecal AS-ODN to attenuate the expression of proteins essential for their role in nociceptor sensitization is well supported by previous studies (Song et al., 2009; Su et al., 2011; Bogen et al., 2012; Quanhong et al., 2012; Sun et al., 2013; Oliveira-Fusaro et al., 2017; Ferrari et al., 2019; Pagliusi et al., 2020; Araldi et al., 2024).

Hyperalgesic priming and OIH produced by oxaliplatin

Recently we demonstrated that oxaliplatin induces hyperalgesic priming, a latent state of nociceptor hyper-responsiveness to pro-algesic mediators (Staurengo-Ferrari et al., 2023). This model of nociceptor neuroplasticity is characterized by the prolongation of mechanical hyperalgesia induced by PGE₂, lasting >4 h, as opposed to PGE₂-induced hyperalgesia in naive control rats, which is fully resolved by 2 h (Aley and Levine, 1999).

To evaluate whether nociceptor MOR plays a role in hyperalgesic priming produced by oxaliplatin, ODN antisense to MOR was administered intrathecally for 4 consecutive days and then three more doses administered, one every other day (total of seven doses). Approximately 17 h after the third dose of ODN, oxaliplatin was administered, to induce CIPN. Mechanical nociceptive threshold was measured on days −3 (before the first intrathecal administration of ODN) and 14. Then, on Day 14, PGE₂ (100 ng) was injected intradermally, and nociceptive threshold evaluated 30 min and 4 h after PGE₂. The continued presence of hyperalgesia at the fourth hour characterizes the presence of priming (Aley et al., 2000; Ferrari et al., 2014; Araldi et al., 2019; Staurengo-Ferrari et al., 2023).

To investigate whether oxaliplatin produces OIH, morphine was injected intradermally (i.d., 1 μg/5 μl) at the site of nociceptive threshold testing, in rats with oxaliplatin CIPN, and mechanical nociceptive threshold evaluated 30 min later. To investigate the second messengers involved in OIH, their inhibitors were administered 10 min before the injection of morphine in the hindpaw of rats treated with oxaliplatin, 14 d prior.

In rats with oxaliplatin CIPN, PGE₂- and morphine-induced hyperalgesia (OIH) were measured, on Day 14, as a further decrease in mechanical nociceptive threshold, in addition to the pre-existing hyperalgesia induced by oxaliplatin. This data is shown as change in mechanical paw-withdrawal threshold, expressed as percentage change from threshold on Day 14 after oxaliplatin, at which time rats still express CIPN hyperalgesia, or in absolute values in grams (g).

Data analysis

Data are presented as mean ± SEM of n independent observations. Statistical comparisons were made using GraphPad Prism 9.0 statistical software (GraphPad Software). A p value of <0.05 is considered statistically significant. In the behavioral experiments, the dependent variable is the change in mechanical paw-withdrawal threshold, expressed in grams (g) and/or as percentage change from baseline. As specified in the figure legends, Student's t test or two-way repeated-measures ANOVA followed by Bonferroni's post hoc test was performed to compare the magnitude of hyperalgesia induced by chemotherapeutic agents, PGE₂, or morphine.

Data availability

Upon a reasonable request, the data generated during the current study are available from the corresponding author.

Results

ODN antisense for MOR mRNA both prevents and reverses oxaliplatin CIPN

To determine the role of the MOR in the onset of oxaliplatin-induced hyperalgesia, male rats received intrathecal antisense (AS)-ODN to MOR mRNA or mismatch (MM)-ODN (120 μg/20 μl, i.t.), once daily for 4 consecutive days and then three more doses, one every other day for a total of seven doses. Approximately 17 h after the 3rd dose of ODN, oxaliplatin was administered (2 mg/kg, i.v.) and mechanical nociceptive threshold measured, from days −3 (before the first intrathecal administration of ODNs) to 28. In the group treated with AS-ODN, oxaliplatin-induced hyperalgesia was prevented (Fig. 1A,B). Thus, knockdown of MOR in nociceptors, prior to administration of oxaliplatin, prevents CIPN.

Figure 1.

Intrathecal administration of an oligodeoxynucleotide (ODN) antisense (AS) to MOR mRNA prevents and reverses oxaliplatin CIPN. A, B, Separate groups of male rats were treated intrathecally (i.t.) with AS-ODN against MOR mRNA or a mismatch (MM)-ODN (both 120 μg in 20 μl/day), once daily, starting 3 d before treatment with oxaliplatin (i.e., 7 doses over 10 d). On Day 0, rats received oxaliplatin (2 mg/kg, i.v., administered on Day 0), and mechanical nociceptive threshold was evaluated before the first intrathecal administration of ODN (Day −3) and then from Days 0 to 28 after oxaliplatin. Compared with the MOR MM-ODN-treated group, in the AS-ODN-treated group, oxaliplatin CIPN hyperalgesia was almost completely prevented, an effect of AS-ODN that was undiminished for the entire 28 d testing period. A. Data is presented as percentage change from baseline, mean ± SEM. Two-way repeated-measures ANOVA, time × MOR AS-ODN interaction, F_(6,60)= 19.93, p < 0.0001; MOR AS-ODN treatment, F_(1,10)= 118.4, p < 0.0001; Bonferroni's multiple post hoc comparisons test: ****p < 0.0001, ***p = 0.0002, and ^###p = 0.0006 (MOR MM-ODN vs AS-ODN). B, Data is presented in mechanical threshold (grams), mean ± SEM. Two-way repeated-measures ANOVA, time × MOR AS-ODN interaction, F_(7,70)= 15.55, p < 0.0001; MOR AS-ODN treatment, F_(1,10)= 89.81, p < 0.0001; Bonferroni's multiple post hoc comparisons test: ****p < 0.0001 (MOR MM-ODN vs AS-ODN). N = 6 rats per group. C, D, Two additional groups of male rats received oxaliplatin (2 mg/kg, i.v., on Day 0). After developing CIPN, one group was treated intrathecally with AS-ODN against MOR mRNA and the other with MM-ODN (both 120 μg in 20 μl/day), once a day starting 3 d after administration of oxaliplatin, for seven doses, over 10 d. Mechanical nociceptive threshold was measured from Day 0 before to 28 after oxaliplatin. In the MOR AS-ODN-treated group, CIPN hyperalgesia was markedly attenuated in oxaliplatin-treated rats, attenuation that was undiminished for the 28 d testing period. C, Data is presented as percentage change from baseline, mean ± SEM. Two-way repeated-measures ANOVA, time × MOR AS-ODN interaction, F_(5,50)= 28.95, p < 0.0001; MOR AS-ODN treatment, F_(1,10)= 107.5, p < 0.0001; Bonferroni's multiple post hoc comparisons test: ****p < 0.0001 (MOR MM-ODN vs AS-ODN). D, Data is presented in mechanical threshold (grams), mean ± SEM. Two-way repeated-measures ANOVA, time × MOR AS-ODN interaction, F_(6,60)= 29.5, p < 0.0001; MOR AS-ODN treatment, F_(1,10)= 16.38, p = 0.0023; Bonferroni's multiple post hoc comparisons test: **p = 0.0022, ***p = 0.0004, ****p < 0.0001, and ^##p = 0.0092 (MOR MM-ODN vs AS-ODN). N = 6 rats per group.

To determine if the maintenance of oxaliplatin CIPN is also MOR dependent, we evaluated whether MOR antisense can reverse CIPN once it is established. In these experiments rats received oxaliplatin (2 mg/kg, i.v., on Day 0) and 3 d later, at which time CIPN hyperalgesia is fully established, MOR AS-ODN or MM-ODN was administered intrathecally (120 μg/20 μl, i.t.), for 4 consecutive days, and then every other day for three additional doses (total of seven doses). In this protocol, AS-ODN for MOR reversed oxaliplatin-induced hyperalgesia by (Fig. 1C,D) a reversal that persists even at time points following AS-ODN when the level of MOR in DRG would have been expected to recover. These findings support the suggestion that the presence of MOR in nociceptors is necessary for the maintenance of oxaliplatin-induced CIPN.

MOR antisense prevents and reverses paclitaxel CIPN

While oxaliplatin causes neuronal apoptosis via nuclear DNA and mitochondrial damage (Quasthoff and Hartung, 2002; Argyriou et al., 2008), paclitaxel hyperstabilizes microtubules and alters mitochondrial dynamics, producing a “dying back axonopathy” (Argyriou et al., 2008). Since there are differences between the mechanisms mediating the neurotoxic effects of different classes of chemotherapies, we evaluated if MOR also plays a role in paclitaxel CIPN. Rats received intrathecal MOR AS-ODN or MM-ODN (120 μg/20 μl, i.t.) daily for 4 consecutive days and then three more doses, one every other day for a total of seven doses. Starting ∼17 h after the third ODN dose, to induce CIPN, paclitaxel was administered (1 mg/kg, i.p.) every other day for a total of four doses. Mechanical nociceptive threshold was measured from Day −3 (before the first intrathecal administration of AS- and MM-ODNs) to Day 28. In the rats treated with MOR AS-ODN, paclitaxel-induced hyperalgesia was attenuated (Fig. 2A,B), indicating that knockdown of nociceptor MOR prevents the development of CIPN induced by chemotherapy drugs, whose neurotoxic effects are produced by different mechanisms.

Figure 2.

MOR antisense both prevents and reverses paclitaxel CIPN. A, B, Male rats were treated intrathecally (i.t.) with antisense (AS)-ODN against MOR mRNA or mismatch (MM)-ODN (both 120 μg in 20 μl/day), once daily starting 3 d before (day −3) the first dose of paclitaxel (i.e., 7 doses, over 10 d). On Day 0, rats received paclitaxel (1 mg/kg, i.p., on Days 0, 2, 4, and 6). Mechanical nociceptive threshold was evaluated before the first intrathecal administration of ODN (Day −3) and then from Day 0 before the first dose of paclitaxel to 28 d after. In the MOR antisense-treated rats, paclitaxel-induced hyperalgesia was almost completely prevented, an attenuation that was undiminished over the 28 d testing period. A, Data is presented as percentage change from baseline, mean ± SEM. Two-way repeated-measures ANOVA, time × MOR AS-ODN interaction, F_(7,70)= 54.23, p < 0.0001; MOR AS-ODN treatment, F_(1,10)= 261.5, p < 0.0001; Bonferroni's multiple post hoc comparisons test: ***p = 0.0007, ****p < 0.0001 (MOR MM-ODN vs AS-ODN). B, Data is presented in mechanical threshold (grams), mean ± SEM. Two-way repeated-measures ANOVA, time × MOR AS-ODN interaction, F_(8,80)= 54.52, p < 0.0001; MOR AS-ODN treatment, F_(1,10)= 12.19, p = 0.0058; Bonferroni's multiple post hoc comparisons test: *p = 0.0205 and ****p < 0.0001 (MOR MM-ODN vs AS-ODN). N = 6 rats per group. C, D, Two additional groups of male rats received paclitaxel (1 mg/kg, i.p., on Days 0, 2, 4, and 6). One group was then treated intrathecally with AS-ODN against MOR mRNA and the other with MM-ODN (both 120 μg in 20 μl/day) once daily starting 3 d after the last dose of paclitaxel, for seven doses, over 10 d. Mechanical nociceptive threshold was evaluated from Day 0 before to 28 after the first dose of paclitaxel. Compared with the MOR MM-ODN-treated group, in the MOR AS-ODN-treated group, paclitaxel CIPN hyperalgesia was markedly attenuated, and this attenuation was undiminished during the 28 d testing period. C, Data is presented as percentage change from baseline, mean ± SEM. Two-way repeated-measures ANOVA, time × MOR AS-ODN interaction, F_(6,60)= 75.99, p < 0.0001; MOR AS-ODN treatment, F_(1,10)= 117.5, p < 0.0001; Bonferroni's multiple post hoc comparisons test: ****p < 0.0001 (MOR MM-ODN vs AS-ODN). D, Data is presented in mechanical threshold (grams), mean ± SEM. Two-way repeated-measures ANOVA, time × MOR AS-ODN interaction, F₍₇₇₆₀₎= 80.04, p < 0.0001; MOR AS-ODN treatment, F_(1,10)= 6.26, p = 0.0056; Bonferroni's multiple post hoc comparisons test: **p = 0.0014, ^##p = 0.0036, and ^aap = 0.0025 [MOR MM-ODN vs AS-ODN]). N = 6 rats per group.

We also evaluated whether MOR AS-ODN can reverse paclitaxel CIPN. Male rats received paclitaxel (1 mg/kg, i.p.), on Days 0, 2, 4, and 6, and mechanical nociceptive threshold was measured from Days 0 to 28. MOR AS-ODN or MM-ODN (120 μg/20 μl, i.t.) was administered intrathecally for 4 consecutive days and then three more doses were administered, one every other day for a total of seven doses. Treatment with ODN started 3 d after administration of the fourth dose of paclitaxel. In MOR AS-ODN-treated rats, paclitaxel-induced hyperalgesia was inhibited (Fig. 2C,D), when compared with its control MOR MM-ODN-treated group. The reversal of paclitaxel CIPN by MOR AS-ODN persisted at time points when the level of MOR in DRG would be expected to have recovered. These findings support the suggestion that similar to oxaliplatin, maintenance of paclitaxel CIPN is MOR-dependent.

Systemic morphine inhibits oxaliplatin-induced hyperalgesia (CIPN)

Opioid analgesics are used clinically to treat neuropathic pain, including for CIPN (Shah et al., 2018; Jordan et al., 2020). We tested whether an analgesic dose of morphine attenuates oxaliplatin CIPN. Rats were treated with oxaliplatin (2 mg/kg, i.v.), and 14 d later, when CIPN hyperalgesia was fully established, an analgesic dose of morphine (3 mg/kg, s.c.; Araldi et al., 2019) was administered. Mechanical nociceptive threshold was measured before and 14 d after oxaliplatin and then 15, 30, 45, 60 min, and 24 h after systemic morphine. In rats with oxaliplatin CIPN, this dose of morphine produced a robust increase in mechanical nociceptive threshold, measured 15, 30, 45, and 60 min after administration (F_(4,45)= 118.4; p < 0.0001; two-way repeated-measures ANOVA); by 24 h, hyperalgesia returned to the premorphine level (Fig. 3A). We have previously shown that an analgesic dose of morphine, in naive-control rats, produced a similar increase in mechanical nociceptive threshold (Araldi et al., 2019). On the other hand, morphine administered intradermally, at a dose that inhibits PGE₂-induced hyperalgesia in naive-control rats (10 μg/5 μl, i.d.; unpublished data), did not affect oxaliplatin-induced hyperalgesia (Fig. 3B; F_(1,10)= 2.341; p = 0.1570; two-way repeated-measures ANOVA), supporting the suggestion that inhibition of oxaliplatin hyperalgesia with systemic morphine is due to its action in the CNS and/or at the central terminal of the nociceptor, in the central nervous system (CNS), but not at the peripheral terminal. While in rats with oxaliplatin CIPN, anti-hyperalgesia induced by systemic morphine is mediated mainly by its action in the CNS (Aicher et al., 2000; Abbadie et al., 2001), it is also dependent on MOR present on the peripheral terminal of nociceptor (Ferrari et al., 2019).

Figure 3.

Systemic but not intradermal morphine attenuates oxaliplatin-induced hyperalgesia. A, Male rats were treated with oxaliplatin (2 mg/kg, i.v.). Fourteen days later, one group received subcutaneous (s.c.) saline (vehicle) and the other group an analgesic dose of morphine (3 mg/kg, s.c.). Mechanical nociceptive threshold was measured before and 0.5 h, 1 h, and 14 d after oxaliplatin and then 15, 30, 45, 60, and 1440 min after systemic morphine or its vehicle. The rats treated with systemic morphine demonstrate marked attenuation of oxaliplatin-induced hyperalgesia, 15, 30, 45, and 60 min after its administration (data is presented as percentage change from baseline, mean ± SEM. Two-way repeated-measures ANOVA, time × morphine interaction, F_(4,25)= 659.2, p < 0.0001; morphine treatment, F_(1,25)= 10941, p < 0.0001; Bonferroni's multiple post hoc comparisons test: ****p < 0.0001 (vehicle vs morphine)], returning to premorphine mechanical threshold values at 1,440 min (24 h) later. Mechanical nociceptive threshold (in grams, mean ± SEM) for the saline and morphine groups, respectively: before oxaliplatin (140.0 ± 3.83 and 141.67 ± 3.81 g); 24 h after oxaliplatin (99.67 ± 2.39 and 98.33 ± 2.80 g); 14 d after oxaliplatin (95.33 ± 1.43 and 93.0 ± 1.24 g). After subcutaneous administration of saline or morphine, respectively (15 min: 90.0 ± 2.0 and 233.33 ± 6.67 g; 30 min: 89.33 ± 2.46 and 231.67 ± 5.43 g; 45 min: 92.0 ± 2.97 and 235.0 ± 3.42 g; 60 min: 91.33 ± 2.72 and 231.67 ± 4.01 g; and 1,440 min: 93.33 ± 2.51 and 92.67 ± 2.57 g). N = 6 rats per group. B, To differentiate central (CNS) from peripheral (PNS) effects of morphine on oxaliplatin-induced CIPN hyperalgesia, one group of rats received intravenous oxaliplatin (2 mg/kg, i.v.), followed 14 d later by intradermal saline (vehicle, 5 μl) and another group intradermal morphine (10 μg/5 μl). Mechanical nociceptive threshold was measured before and 14 d after oxaliplatin and then 30 min after intradermal morphine or saline. Morphine, administered intradermally, did not affect oxaliplatin-induced hyperalgesia (data is presented as percentage change from baseline, mean ± SEM. Two-way repeated-measures ANOVA, time x morphine interaction, F_(1,10)= 0.00156, p = 0.9693; morphine treatment, F_(1,10)= 2.341, p = 0.1570). These data support the suggestion that inhibition of oxaliplatin-induced hyperalgesia by systemic/subcutaneous morphine is due to its action at sites in the CNS. Mechanical nociceptive threshold (in grams, mean ± SEM) for the saline and morphine groups, respectively: before oxaliplatin (141.93 ± 4.29 and 140.0 ± 4.52 g); 14 d after oxaliplatin (99.83 ± 5.23 and 98.07 ± 3.27 g); and 30 min after vehicle (saline) or morphine (96.23 ± 4.81 and 100.4 ± 2.91 g). N = 6 rats per group.

MOR antisense prevents hyperalgesic priming induced by oxaliplatin

In addition to hyperalgesia, oxaliplatin induces hyperalgesic priming (priming; Staurengo-Ferrari et al., 2023), a neuroplastic change in nociceptors that contributes to the transition from acute to chronic pain (Reichling and Levine, 2009; Kandasamy and Price, 2015). Priming is characterized by the marked prolongation of hyperalgesia produced by inflammatory mediators, prototypically prostaglandin E₂ (PGE₂; Aley et al., 2000). To determine if nociceptor MOR contributes to priming associated with CIPN, rats received intrathecal MOR AS-ODN or as a control MM-ODN (120 μg/20 μl, i.t.), daily for 4 consecutive days, and then three more doses, one every other day (total of seven doses). Approximately 17 h after the third dose of ODN, oxaliplatin was administered (2 mg/kg, i.v.), and mechanical nociceptive threshold measured from Day −3 (before the first intrathecal administration of ODNs) to Day 10. On Day 10 (4 d after the last dose of MOR AS-ODN or MM-ODN), at which time oxaliplatin-induced hyperalgesia is markedly attenuated in MOR AS-ODN-treated rats, PGE₂ was injected intradermally (100 ng/5 μl) and mechanical nociceptive threshold measured, at its injection site, 30 min and 4 h later. Enhancement of CIPN hyperalgesia, measured 30 min after PGE₂, was present in both MOR MM- and AS-ODN-treated rats. However, hyperalgesic priming, measured as prolongation of PGE₂-induced hyperalgesia (at the fourth hour) was markedly attenuated in rats treated with MOR AS-ODN, compared with the MM-ODN-treated control group (Fig. 4A,B; F_(1,10)= 15.35; p < 0.0001; two-way repeated-measures ANOVA). These findings support the suggestion that hyperalgesic priming, as well as CIPN hyperalgesia, induced by oxaliplatin, are nociceptor MOR dependent.

Figure 4.

Hyperalgesic priming induced by oxaliplatin is MOR dependent. Groups of male rats received AS-ODN against MOR mRNA or MM-ODN (120 μg/20 μl, i.t.), daily for 4 consecutive days, and then three more doses, one every other day (total of 7 doses). Approximately 17 h after the third dose of AS- or MM-ODN, oxaliplatin was administered (2 mg/kg, i.v.) to induce CIPN, and mechanical nociceptive threshold measured from Day −3 (before the first intrathecal administration of ODNs) to 10. On Day 10 (4 d after the last intrathecal ODN administration), at which time oxaliplatin-induced hyperalgesia was markedly attenuated in MOR AS-ODN-treated rats (**p = 0.0019), PGE₂ was injected (100 ng/5 μl, i.d.), and mechanical nociceptive threshold measured, at its injection site, 30 min and 4 h after PGE₂ injection. Hyperalgesia 30 min after PGE₂ was present in both the MOR MM- and AS-ODN-treated groups of rats. However, hyperalgesic priming, measured as prolongation of PGE₂-induced hyperalgesia (at the fourth hour), was markedly attenuated in rats treated with MOR AS-ODN. A, Data presented in mechanical threshold (g): Two-way repeated-measures ANOVA, time × MOR AS-ODN, F_(3,30)= 30.16, p < 0.0001; MOR AS-ODN treatment, F_(1,10)= 28.40, p < 0.0001; Bonferroni's multiple post hoc comparisons test: **p = 0.0019, ****p < 0.0001 (MOR MM-ODN vs AS-ODN). B, The same data in A here presented as percentage change from baseline: Two-way repeated-measures ANOVA, time × MOR AS-ODN, F_(1,10)= 43.26, p < 0.0001; MOR AS-ODN treatment, F_(1,10)= 15.35, p < 0.0001; Bonferroni's multiple post hoc comparisons test: ****p < 0.0001 (MOR MM-ODN vs AS-ODN). These findings support the suggestion that oxaliplatin-induced hyperalgesic priming, as well as oxaliplatin hyperalgesia (CIPN), are MOR dependent. N = 6 rats per group.

Oxaliplatin also produces OIH

We have classified hyperalgesic priming as having at least two mechanistically distinct forms: Type I is maintained by protein translation (i.e., it is permanently reversed by exposure to cordycepin, administered in the vicinity of the nociceptor terminal, peripheral, or central; Ferrari et al., 2013, 2019), while maintenance of Type II is Src and mitogen-activated protein kinase (MAPK) dependent (Araldi et al., 2017, 2018c); coadministration of their inhibitors reverses Type II priming. Since repeated administration of the selective MOR agonist DAMGO induces hyperalgesic priming and hyperalgesia (OIH), which share mechanisms in common (Araldi et al., 2018a), and oxaliplatin also induces priming (Staurengo-Ferrari et al., 2023), we evaluated if oxaliplatin also produces OIH. Mechanical nociceptive threshold was measured before and 14 d after oxaliplatin (2 mg/kg, i.v.). On Day 14, rats received an intradermal injection of vehicle (saline), the protein translation inhibitor (cordycepin), the combination of a Src and a MAPK inhibitor (SU6656 + U0126, respectively), or a selective MOR antagonist (CTOP), followed 10 min later by intradermal morphine (1 μg/5 μl, i.d.) or its vehicle saline (5 μl, i.d.; Fig. 5). Of note, the intradermal injection of this dose of morphine does not affect nociceptive threshold in naive-control rats (Table 1). In Figure 5 the black bars represent the mechanical nociceptive threshold before the intravenous administration of oxaliplatin and the red bars the decrease in mechanical nociceptive threshold 14 d after administration of oxaliplatin. In contrast, oxaliplatin-treated rats that received intradermal morphine (1 μg, i.d.) displayed a dramatic further decrease in nociceptive threshold (−49.3%, rose compared with black bars), in addition to the pre-existing oxaliplatin hyperalgesia (−35.5%, rose compared with red bars). When oxaliplatin-treated rats received either intradermal cordycepin (blue bar) or the combination of Src and MAPK inhibitors (green bar) followed 10 min later by intradermal morphine, cordycepin did not affect morphine OIH while the combination of a Src and MAPK inhibitor completely abolished it (F_(3,20)= 19.01; p < 0.0001; two-way repeated-measures ANOVA). The combination of the Src and MAPK inhibitor also abolished oxaliplatin-induced hyperalgesia (green bar), which was also markedly attenuated by cordycepin (blue bar). To evaluate if OIH in rats with oxaliplatin CIPN is MOR dependent, oxaliplatin-treated rats received a selective MOR antagonist, CTOP, intradermally, followed 10 min later by morphine, administered at the same site. While CTOP completely prevents hyperalgesia induced by morphine (OIH) in rats with CIPN (beige bar), it does not affect oxaliplatin hyperalgesia (beige bar; F_(2,15)= 13.06; p = 0.0005; two-way repeated-measures ANOVA).

Figure 5.

The coadministration of an inhibitor of Src and MAPK or a selective MOR antagonist, markedly attenuate morphine-induced hyperalgesia (OIH) in rats with oxaliplatin CIPN. Intravenous administration of oxaliplatin (2 mg/kg, i.v.) to male rats produces a 24% decrease in mechanical nociceptive threshold when measured 14 d after administration of oxaliplatin; comparing black (the average paw-withdrawal threshold before oxaliplatin: 133.11 ± 1.48 g) and red bars (the average paw-withdrawal threshold 14 d after oxaliplatin: 100.97 ± 1.28 g; ****p < 0.0001, t₍₄₁₎= 27.2, paired Student's t test; this statistic analysis is not shown in the figure). Fourteen days after oxaliplatin administration, vehicle (saline, 5 μl), a protein translational inhibitor (cordycepin (1 μg/5 μl), a combination of a Src and a MAPK inhibitor (SU6656 + U0126; 1 μg/2 μl each), or a selective MOR antagonist (CTOP, 1 μg/5 μl) was administered intradermally (i.d.), followed 10 min later by morphine (1 μg/5 μl, i.d.) or its vehicle saline (5 μl, i.d.), administered at the site of nociceptive threshold testing. Mechanical nociceptive threshold was then measured 30 min after intradermal morphine. Rats that received intradermal morphine, 14 d after oxaliplatin, demonstrate a robust further decrease in mechanical nociceptive threshold (rose bar). Cordycepin (blue bar), which reverses Type I priming, did not attenuate this morphine-induced hyperalgesia in rats with oxaliplatin CIPN. However, the combination of a Src and a MAPK inhibitor (green bar), which reverses Type II priming, or CTOP (beige bar) prevents morphine-induced hyperalgesia in oxaliplatin-treated rats [data is presented in mechanical threshold (g): Two-way repeated-measures ANOVA, time × treatments, F_(1,20)= 19.18, p = 0.0003; inhibitors/antagonist treatment, F_(3,20)= 19.01, p < 0.0001; Bonferroni's multiple post hoc comparisons test: ****p < 0.0001 (inhibitors/antagonist followed by intradermal morphine vs 14 d after oxaliplatin)]. Additional groups of oxaliplatin-treated rats received cordycepin (blue bar), the combination of a Src and a MAPK inhibitor (green bar), or CTOP (beige bar), each administered intradermally (same doses and volumes listed above), and mechanical nociceptive threshold was measured 30 min after administration of the inhibitors. All rats received oxaliplatin (2 mg/kg, i.v.) 14 d prior. This set of experiments was performed to evaluate the effect of these drugs on oxaliplatin-induced hyperalgesia. While the combination of a Src and a MAPK inhibitor and cordycepin markedly inhibits oxaliplatin-induced hyperalgesia, CTOP does not [data is presented in mechanical threshold (g): Two-way repeated-measures ANOVA, time × treatments, F_(1,15)= 155.4, p < 0.0001; inhibitors/antagonist treatment, F_(2,15)= 13.06, p = 0.0005; Bonferroni's multiple post hoc comparisons test: ****p < 0.0001 (inhibitors/antagonist vs 14 d after oxaliplatin)]. N = 6 rats per group.

Interdependence of MOR and TLR4 in oxaliplatin OIH

Since cross talk between MOR and TLR4 is been well described (Hutchinson et al., 2011; Meng et al., 2013; Araldi et al., 2019; Zhang et al., 2020) and ODN antisense for TLR4 prevents and reverses CIPN (Araldi et al., 2024), we evaluated for interdependence of MOR and TLR4 in OIH produced by oxaliplatin. All rats received oxaliplatin (2 mg/kg, i.v.); the black bars represent the mechanical nociceptive threshold before intravenous administration of oxaliplatin and the red bars the decrease in mechanical nociceptive threshold 14 d after oxaliplatin. Fourteen days after oxaliplatin, rats received vehicle (saline; rose bar) or the TLR4 antagonist (LPS-RS Ultrapure; blue bar), intradermally, followed 10 min later by intradermal morphine. When LPS-RS Ultrapure was injected intradermally followed 10 min later by morphine (blue bar), at the same site, in oxaliplatin CIPN rats, it completely prevented OIH (Fig. 6; F_(2,15)= 20.38; p < 0.0001; two-way repeated-measures ANOVA). However, intradermal administration of the TLR4 antagonist, by itself, did not affect oxaliplatin CIPN hyperalgesia (blue bar), when compared with hyperalgesia 14 d after oxaliplatin (red bar; Fig. 6).

Figure 6.

The selective TLR4 antagonist LPS-RS Ultrapure inhibits morphine-induced hyperalgesia (OIH) in rats with oxaliplatin CIPN. Male rats treated with oxaliplatin (2 mg/kg, i.v.) demonstrate a 25% decrease in mechanical nociceptive threshold, when evaluated 14 d later, comparing black (the average paw-withdrawal threshold before oxaliplatin: 136.81 ± 1.81 g) and red bars (the average paw-withdrawal threshold 14 d after oxaliplatin: 102.55 ± 1.40 g; ****p < 0.0001, t₍₁₇₎= 17.99, paired Student's t test; this statistic analysis is not shown in the figure). Fourteen days after administration of oxaliplatin, vehicle (saline, 5 μl, i.d.) or a TLR4 antagonist (LPS-RS Ultrapure; 1 μg/5 μl, i.d.) was administered, followed 10 min later by morphine (1 μg/5 μl, i.d.), all administered at the site of nociceptive threshold testing. Mechanical nociceptive threshold was evaluated again 30 min after intradermal morphine. The group of rats that received intradermal morphine, 14 d after oxaliplatin, demonstrate a robust decrease in nociceptive threshold (rose bar), opioid-induced hyperalgesia (OIH). In oxaliplatin-treated rats, intradermal LPS-RS Ultrapure (blue bar) prevents morphine-induced hyperalgesia, while LPS-RS Ultrapure followed 10 min later by saline (5 μl, i.d.) did not affect oxaliplatin-induced hyperalgesia [data is presented in mechanical threshold (g): Two-way repeated-measures ANOVA, time × treatments, F_(1,15)= 74.14, p < 0.0001; TLR4 antagonist treatment, F_(2,15)= 20.38, p < 0.0001; Bonferroni's multiple post hoc comparisons test: ****p < 0.0001 (TLR4 antagonist followed by intradermal morphine vs 14 d after oxaliplatin followed by intradermal morphine)]. N = 6 rats per group.

Discussion

In the present experiments, we have demonstrated that intrathecal administration of ODN antisense to MOR mRNA—an intervention shown to decrease MOR in rat DRGs (Ferrari et al., 2019)—can both prevent and reverse pain associated with CIPN induced by two chemotherapy drugs, oxaliplatin and paclitaxel, which produce their antitumor effects by very different mechanisms, and also attenuate hyperalgesic priming induced by oxaliplatin. We also found that in rats with oxaliplatin CIPN, systemic morphine (3 mg/kg, s.c.) temporarily attenuates CIPN pain, while a dose of intradermal morphine (10 μg), which attenuates PGE₂-induced hyperalgesia (unpublished data), does not affect CIPN hyperalgesia. Additionally, a dose of intradermal morphine (1 μg), which does not change nociceptive threshold in naive rats, now induces mechanical hyperalgesia (OIH). This chemotherapy-induced OIH was markedly attenuated by (1) intradermal administration of inhibitors of second messengers that reverse Type II hyperalgesic priming (i.e., the combination of a Src and a MAPK inhibitor), (2) a TLR4 antagonist, as well as (3) a selective MOR antagonist.

CIPN pain is a frequent complication of many classes of antitumor drugs that have different mechanisms of action, used to treat diverse forms of cancer. Resistance of CIPN pain to currently available neuropathic pain treatments indicates the necessity to consider novel treatment modalities. Currently, the three main classes of medications used to treat CIPN pain are the following: (1) the antiepileptics, most commonly the gabapentinoids (Maihofner et al., 2021); (2) serotonin–noradrenaline reuptake inhibitor (SNRI) antidepressants such as duloxetine and amitriptyline (Maihofner et al., 2021); and (3) opioid analgesics, most commonly MOR agonists (Nagashima et al., 2014; Maihofner et al., 2021). While it is generally accepted that opioids are less effective for treating neuropathic pain (Smith, 2012), including pain associated with CIPN (Nagashima et al., 2014; Maihofner et al., 2021), compared with pain associated with trauma and inflammation, conditions in which MOR agonists are routinely used to treat moderate-to-severe pain, mechanisms underlying this loss of efficacy of opioid analgesics in the treatment of CIPN pain, remain to be elucidated.

It has recently been demonstrated that Toll-like receptor 4 (TLR4) plays an important role in CIPN pain induced by the clinically important chemotherapy agents studied in the present experiments, oxaliplatin and paclitaxel (Illias et al., 2022; Araldi et al., 2024). Since many clinically used opioid analgesics are agonists at TLR4 (Hutchinson et al., 2010a, b, 2011; Wang et al., 2012; Jacobsen et al., 2014), as well as MOR, and TLR4 and MOR, both present on nociceptors (Barajon et al., 2009; Corder et al., 2017), have well-described cross talk (Hutchinson et al., 2011; Meng et al., 2013; Araldi et al., 2019; Zhang et al., 2020), we examined the hypothesis that MOR, as well as TLR4, play a role in CIPN pain induced by representatives of two classes of neurotoxic chemotherapy drugs, platinum-based (oxaliplatin) and a taxanes (paclitaxel), whose effect on cancer cells and neurons are thought to be mediated by very different mechanisms (Quasthoff and Hartung, 2002; Argyriou et al., 2008; Boyette-Davis et al., 2015). CIPN produced by both classes of neurotoxic chemotherapy drugs was prevented by MOR AS-ODN, even at time points when the inhibitory effect of antisense on MOR levels is no longer present. This is similar to our results using TLR4 and RAGE antisense to prevent and reverse CIPN induced by oxaliplatin, paclitaxel, and bortezomib (Araldi et al., 2024). We also demonstrated that hyperalgesia in established oxaliplatin and paclitaxel CIPN could be reversed by MOR AS-ODN. These findings support MOR dependence of both the induction and maintenance of CIPN. However, the mechanism by which transient attenuation of nociceptor MOR is able to produce a prolonged reversal of CIPN remains to be established. Of note in this regard, we recently demonstrated that transiently attenuating TLR4 in DRG, using TLR4 antisense, also led to a persistent prevention and reversal of CIPN (Araldi et al., 2024). Since MOR and TLR4 are both found in neuronal lipid rafts (Bruno et al., 2018; Nehr-Majoros et al., 2024), the use of antisense ODN to inhibit MOR and TLR4 protein synthesis might have a long-term impact on the functional organization of the lipid raft. This could explain why, even when protein levels for MOR and TLR4 return to pre-antisense levels, CIPN is still inhibited.

Current therapeutic guidelines relegate strong opioids as a third-line treatment for neuropathic pain, in part due to safety concerns (Finnerup et al., 2015), including the potential to facilitate the progression to chronic pain (Bigal et al., 2008; Hooten et al., 2015; Armstrong et al., 2020), development of hyperalgesia (OIH), and pain exacerbation with their repeated administration (Mori et al., 2014). When we evaluated the analgesic effect of systemic morphine for the treatment of pain associated with oxaliplatin CIPN, as expected, morphine produced an increase in mechanical nociceptive threshold (i.e., was antihyperalgesic/analgesic). Of note, the analgesia induced by systemic morphine is, in large part, mediated by its action in the CNS (Aicher et al., 2000; Abbadie et al., 2001), which includes contribution at the central terminal of the nociceptor (Ferrari et al., 2019), located in the spinal dorsal horn. Importantly, unlike its role in OIH, opioid-induced antihyperalgesia is not nociceptor TLR4 dependent (Araldi et al., 2019).

In addition to neuropathic pain, we have previously shown that the same neurotoxic chemotherapy agents (i.e., oxaliplatin and paclitaxel) also induce hyperalgesic priming (Staurengo-Ferrari et al., 2023), which is a form of long-lasting neuroplasticity in nociceptor function, characterized by marked prolongation of the mechanical hyperalgesia induced by diverse inflammatory mediators, prototypically characterized using PGE₂ (Aley et al., 2000; Reichling and Levine, 2009; Ferrari et al., 2013; Araldi et al., 2015, 2018c, 2019; Staurengo-Ferrari et al., 2023). When oxaliplatin-treated rats subsequently received MOR AS-ODN, the prolongation of hyperalgesia, measured at the fourth hour after PGE₂, but not the acute hyperalgesia, measured 30 min after injection of PGE₂, was markedly attenuated, supporting the suggestion that the maintenance of hyperalgesic priming induced by chemotherapy agents is MOR dependent.

Since priming develops in rats with CIPN, we evaluated if OIH is also present in rats with CIPN, a phenomenon that could contribute to a net decrease in the pain relief by opioid analgesics. To evaluate for OIH in rats treated with oxaliplatin, 14 d prior, morphine was administered intradermally, a dose (1 μg) that does not affect nociceptive threshold in naive control rats. We found that this dose of morphine produces hyperalgesia (OIH) in rats with oxaliplatin-induced CIPN, establishing a side effect of opioid analgesics that can develop following cancer CIPN. Since hyperalgesic priming and OIH have shared mechanisms (Araldi et al., 2018a), we tested the hypothesis that in oxaliplatin-treated rats, inhibitors of hyperalgesic priming would also attenuate the hyperalgesia induced by intradermal morphine (OIH). We have previously identified two types of priming. Type I priming is maintained by protein translation in the peripheral and central terminals of nociceptors (Ferrari et al., 2013), while Type II priming is maintained by the combined action of Src and MAPK, also at both nociceptor terminals (Araldi et al., 2017, 2018c). In the present study, we found that the combination of a Src and MAPK inhibitor markedly attenuated OIH in rats with oxaliplatin CIPN. Importantly, this combination of second-messenger inhibitors abolished both morphine- and oxaliplatin-induced hyperalgesia, as well as oxaliplatin-induced hyperalgesic priming (Staurengo-Ferrari et al., 2023). On the other hand, cordycepin, which reverses Type I priming, attenuated oxaliplatin-induced priming (Staurengo-Ferrari et al., 2023) and did not affect OIH produced by intradermal morphine in oxaliplatin-treated rats. And, the MOR selective antagonist, CTOP, inhibited OIH in oxaliplatin-treated rats, without affecting oxaliplatin hyperalgesia, corroborating our previous finding that OIH is dependent on MOR, present in nociceptor peripheral terminals (Ferrari et al., 2019). We also demonstrated that while a highly selective TLR4 antagonist (LPS-RS Ultrapure), administered intradermally at the site of nociceptive threshold testing, did not affect hyperalgesia, in rats with oxaliplatin CIPN, it eliminated OIH produced by intradermal morphine. These findings are in line with our previous study demonstrating that inhibition of nociceptor TLR4 attenuates OIH (Araldi et al., 2019).

Collectively, our findings support the suggestion that MOR, present in primary afferent nociceptors, plays a pivotal role in CIPN pain and chemotherapy-induced hyperalgesic priming. In addition to neuropathic pain (CIPN) and hyperalgesic priming, chemotherapy-treated rats develop OIH that is ameliorated by interventions that reverse Type II hyperalgesic priming. We propose that priming and OIH occur in the setting of CIPN and contribute to the attenuated response to opioid analgesics in neuropathic pain.