1. Introduction

Pain caused by bodily insults such as infection, surgery, or chemotherapy diminishes as the underlying condition resolves and injured tissue heals or chemotherapy treatment is completed. However, in a number of these patients, a transition occurs from acute to chronic pain.16,65 In patients receiving cancer chemotherapy, ∼25 to 30% experience chemotherapy-induced peripheral neuropathy (CIPN), with pain persisting for months or years after discontinuation of treatment,17,63 indicating that chemotherapy can produce long-lasting adverse alterations in pain pathways.

Stressful life events influence a variety of diseases through activation of the hypothalamic–pituitary–adrenal (HPA) and sympathoadrenal neuroendocrine stress axes.85 Repeated environmental stressors, especially when unpredictable, induce persistent hyperalgesic states, including hyperalgesic priming, a preclinical model of the transition from acute to chronic pain.4,26,31,35,41,45,79 We and others have demonstrated, in preclinical26,79 and clinical54,55,57,74 studies, the impact of stress and role of the neuroendocrine stress axes in CIPN pain. We have shown that rats exposed to unpredictable sound stress, using an experimental protocol that produces a chronic increase in plasma epinephrine and corticosterone,40,41 develop hyperalgesic priming,23,40 a neuroplastic change in nociceptors that markedly prolongs hyperalgesia produced by inflammatory mediators, prototypically prostaglandin E2 (PGE2),38 by a PKCɛ-dependent mechanism.40 We have also shown that the stress-induced neuroplasticity underlying hyperalgesic priming requires concerted action of glucocorticoids and catecholamines, acting at their cognate receptor, on nociceptors, to produce a switch in coupling of receptor G-protein signaling from Gs to Gi.23 Given the marked impact of stress on oxaliplatin-induced hyperalgesia79 and on nociceptor neuroplasticity,23,40,41,49 in this study, we evaluated the role of stress in hyperalgesic priming induced by oxaliplatin, including evaluation of the contribution of Gαi/o subunits, Gαi1, Gαi2, Gαi3, and Gαo, and a second messenger involved in hyperalgesic priming, PKCε.

2. Methods 2.1. Experimental animals

Experiments were performed on 230- to 430-g male Sprague–Dawley rats (Charles River Laboratories, Hollister, CA). Animals were housed in a controlled environment in the animal care facility at the University of California, San Francisco, under a 12-hour light/dark cycle, with food and water available ad libitum. Experimental protocols, approved by the UCSF Institutional Animal Care and Use Committee, adhered to the guidelines of the American Association of Laboratory Animal Care, the National Institutes of Health, and the Committee for Research and Ethical Issues of the International Association for the Study of Pain, for the use of animals in research. In the design of experiments, a concerted effort was made to minimize the number of animals used and their suffering. Because of the marked sexual dimorphism in the second messenger mechanisms of hyperalgesic priming,29,36,42 the additional extensive parallel experiments will be performed in female rats in a separate study.

2.2. Mechanical nociceptive threshold testing

Mechanical nociceptive threshold was quantified using an Ugo Basile Analgesy-meter (Stoelting, Wood Dale, IL), which applies a linearly increasing mechanical force to the dorsum of a rat's hind paw, as described previously.83 Rats were placed in cylindrical acrylic restrainers designed to provide ventilation and allow for hind leg extension through lateral ports, for the assessment of nociceptive threshold, with minimal stress. To acclimatize rats to the testing procedure, they were placed in restrainers for ∼40 minutes before starting each training session (3 consecutive days of training) and for ∼30 minutes before experimental manipulations. Nociceptive threshold was defined as the force, in grams, at which a rat withdrew its paw. Baseline nociceptive threshold was determined from the mean of 3 readings obtained before injections of test agents. Each experiment was performed on a different group of rats, and only 1 paw per rat was used in each experiment.

2.3. Drugs

The following compounds were used in the present experiments: oxaliplatin, cordycepin 5′-triphosphate sodium salt (protein translation inhibitor), SU6656 (Src family kinase inhibitor), U0126 (MAPK/ERK inhibitor), prostaglandin E2 (PGE2, direct-acting nociceptor-sensitizing agent commonly used to probe for the presence of hyperalgesic priming33,36,60), corticosterone, epinephrine bitartrate, and pertussis toxin (nonselective Gαi/o-protein inhibitor), purchased from Sigma-Aldrich (St. Louis, MO). The stock solution of PGE2 was prepared in 100% ethanol (1 μg/μL), and dilutions were made with physiological saline (0.9% NaCl) to produce the concentration used in experiments (100-ng PGE2/5 μL; 2% final ethanol concentration). All other drugs were dissolved in 100% DMSO (Sigma-Aldrich) and further diluted in saline containing 2% Tween 80 (Sigma-Aldrich). For all experiments, the final concentrations of DMSO and Tween 80 were 2%. All drugs were injected intradermally on the dorsum of 1 hind paw, in a volume of 5 μL, using a 30-gauge hypodermic needle adapted to a 50-μL Hamilton syringe (Reno, NV). To avoid mixing of distilled water and experimental drugs in the syringe, the injection of cordycepin, SU6656, U0126, and pertussis toxin was preceded by a hypotonic shock (2 μL of distilled water, separated in the syringe by a bubble of air) to facilitate entry of membrane-impermeable compounds into the nerve terminal. Dose selection was based on previous studies that established effectiveness at their targets when injected intradermally on the dorsum of the hind paw.3,8,22,23

2.4. Oxaliplatin chemotherapy-induced peripheral neuropathy hyperalgesic priming

Oxaliplatin was administered (2 mg/kg dissolved in 0.9% saline, 1 mL/kg) through a tail vein. To evaluate for the presence and time course of hyperalgesic priming, on days 21, 42, and 60 after oxaliplatin administration, hyperalgesia induced by PGE2 (100 ng/5 μL, i.d.) was evaluated; PGE2 hyperalgesia >4 hours indicates the presence of hyperalgesic priming.62

2.5. Surgical adrenalectomy

Under 3% isoflurane anesthesia, rats underwent bilateral excision of their adrenal gland (adrenalectomy), which removes both the medulla and cortex. After shaving their abdomen, anesthetized rats were placed on a thermal blanket, and their skin was swabbed with povidone-iodine solution in the area of the surgical field. To provide perioperative analgesia, rats were given meloxicam subcutaneously (5 mg/kg, s.c.), and bupivacaine was infiltrated into the skin preoperatively, in the area to be incised (5-8 mg/kg, i.d.). In each rat, bilateral flank wall incisions were made, and adrenal glands were visualized and excised; 5-0 silk suture was used to separately close the abdominal wall and skin incisions. Sham adrenalectomy, in which the adrenal glands were located and manipulated, but not excised, was performed in control rats.

2.6. Stress hormone replacement in adrenalectomized rats

To maintain basal levels of plasma corticosterone during the experimental period (∼25 days), adrenalectomized rats were supplied with corticosterone (25 µg/mL) and 0.45% saline in their drinking water (ad libitum) starting at the time of their adrenalectomy surgery. This stress hormone-replacement protocol simulates the phasic (circadian) corticosterone rhythm, normalizes basal adrenocorticotropic hormone (ACTH) and catecholamine levels,44,79 and prevents the weight loss observed in adrenalectomized rats that did not receive corticosterone replacement.1 To replace both hormones at levels observed in stressed rats, Alzet osmotic minipumps (Durect Corp, Cupertino, CA) delivered epinephrine at a rate of 5.4 μg/0.25 μL/hour, which produces plasma levels of 720 pg/mL in rats,82 and corticosterone was administered by a slow-release pellet, 100 mg of fused corticosterone, which produces a plasma corticosterone level of 32.6 mg⁄dL.81 Pumps and slow-release pellets, implanted subcutaneously in the interscapular region, delivered stress hormones over a period of 28 days. Oxaliplatin (2 mg/kg, i.v.) was injected 1 day after rats were submitted to adrenalectomy and hormone implants.

2.7. Receptor and second messenger antisense treatment

To investigate the role of the β2-adrenergic receptor, glucocorticoid receptor, PKCε, Gαi1, Gαi2, Gαi3, and Gαo in hyperalgesic priming induced by systemic administration of oxaliplatin, antisense (AS) and mismatch (MM), or sense (SE) (for Gαi/o proteins) oligodeoxynucleotides (ODNs) were administered intrathecally for 3 consecutive days. This procedure produces not only reversible inhibition of the expression of the relevant proteins in dorsal root ganglion (DRG) neurons but also modulation of nociceptive behavior.80 Immediately before their intrathecal administration, antisense and sense ODNs were lyophilized and reconstituted to a concentration of 6 μg/μL in 0.9% saline. To administer ODNs (120 μg/20 µL), rats were briefly anaesthetized with 2.5% isoflurane and a 30-gauge hypodermic needle inserted into the subarachnoid space, on the midline, between the L4 and L5 vertebrae. The intrathecal site of injection was confirmed by the elicitation of a tail flick, a reflex that is evoked by subarachnoid space access and bolus injection.53 The efficacy of this robust and reproducible method for intrathecal ODN administration53,76 is enhanced by a direct communication between the subarachnoid space and DRG in rats,39 facilitating access of ODNs to DRG cell bodies,46 as well as to central terminals of nociceptors. Antisense/mismatch/sense ODNs, synthesized by Life Technologies (Carlsbad, CA), were validated in previous studies. Oligodeoxynucleotide antisense sequences (5′-3′) were

β2-adrenergic receptor antisense: AAA GGC AGA AGG ATG TGC; mismatch sequence: ATA GCC TGA TGG AAG TCC.21 Glucocorticoid receptor antisense: TGG AGT CCA TTG GCA AAT; mismatch sequence: TGA AGT TCA GTG TCA ACT.21 PKCε antisense: GCC AGC TCG ATC TTG CGC CC; mismatch sequence: GCC AGC GCG ATC TTT CGC CC.61 Gαi1 antisense: AGA CCA CTG CTT TGT A; sense sequence: GGG GGA AGT AGG TCT TGG.6,32,70,75,78,86 Gαi2 antisense: CTT GTC GAT CAT CTT AGA; sense sequence: GGG GGA AGT AGG TCT TGG.32,70,75,78,86 Gαi3 antisense: AAG TTG CGG TCG ATC AT; sense sequence: GGG GGA AGT AGG TCT TGG.32,70,75,78,86 Gαo antisense: CGC CTT GCT CCG CTC; sense sequence: GGG GGA AGT AGG TCT TGG.32,70,75,78,86 2.8. Stress resilience induced by neonatal handling

To produce stress resilience in adult rats, we used a well-established model, neonatal handling.48,52 This protocol involves removing dams from their litters, daily from postnatal day 2 to 9, and gently handling, touching, and stroking pups for 15 minutes, after which litters were returned to the care of their dams, in their home cages. On postnatal day 21, pups were weaned, and females were culled. Male rats were housed 3 per cage to be used in experiments as adults.

2.9. Data analysis

In all experiments, the dependent variable was mean change in mechanical nociceptive paw-withdrawal threshold, expressed as percentage change from preintervention baseline. As specified in figure legends, the Student t test, or 1- or 2-way repeated-measures analysis of variance followed by Bonferroni post hoc test, was performed to compare the magnitude of the hyperalgesia induced by oxaliplatin in the different groups or to compare the effect produced by different treatments on the prolongation of PGE2-induced hyperalgesia (evaluated 4 hours after injection) with the duration of PGE2 hyperalgesia in control groups. Prism 9.0 (GraphPad Software, Inc, San Diego, CA) was used for the graphics and to perform statistical analyses. P < 0.05 was considered statistically significant. Data are presented as mean ± SEM.

3. Results 3.1. Oxaliplatin induces persistent hyperalgesic priming

To evaluate the role of stress in oxaliplatin CIPN, we first characterized 2 key features of CIPN pain, lowered mechanical nociceptive threshold (ie, hyperalgesia) and hyperalgesic priming (ie, prolongation of PGE2-induced hyperalgesia), in male rats.

When compared with the effect of vehicle (saline), oxaliplatin (a single injection of 2 mg/kg/i.v., day 0) produced a time-dependent decrease in mechanical nociceptive threshold (hyperalgesia) peaking 7 days after administration (∼30% decrease in mechanical nociceptive threshold) (Figs. 1A and B). This mechanical hyperalgesia was relatively stable for 28 days and was still present 42 days after administration of oxaliplatin.

Figure 1.:

Oxaliplatin induces long-lasting mechanical hyperalgesia and hyperalgesic priming in male rats. Male rats received a single intravenous injection of vehicle (0.9% saline) or oxaliplatin (2 mg/kg), and mechanical nociceptive threshold was evaluated before their administration (day 0) and on days 1, 7, 14, 21, 28, 42, and 60 after administration. Oxaliplatin-induced hyperalgesic priming was evaluated on days 21, 42, and 60. (A) The experimental protocol, with timing of treatments and nociceptive threshold measurements. (B) Oxaliplatin-induced mechanical hyperalgesia that lasted 42 days; *P<0.05, **P<0.01. (C) PGE2 (100 ng/5 μL, i.d.) was injected on the dorsum of 1 hind paw, and the mechanical nociceptive threshold was evaluated 30 minutes and 4 hours later to test for hyperalgesic priming. Hyperalgesic priming was evaluated before oxaliplatin and again 21, 42, and 60 days after oxaliplatin administration. PGE2-induced hyperalgesia at 30 minutes, and before administration of oxaliplatin, PGE2-induced hyperalgesia was no longer present at the fourth hour (30 minutes vs 4 hours, P = 0.0005, Student paired 2-tailed t test, t5 = 8.134). However, after treatment with oxaliplatin, the hyperalgesia induced by PGE2 was still present at the fourth hour 21, 42, and 60 days after oxaliplatin (for each time point, 30 minutes vs 4 hours, P = NS). Data are shown as mean ± SEM. PGE2, prostaglandin E2.

To test for the presence for hyperalgesic priming, PGE2 was injected intradermally, 21, 42, and 60 days after administration of oxaliplatin, and the mechanical nociceptive threshold was evaluated at the same site 30 minutes and 4 hours later. Hyperalgesia induced by PGE2 was prolonged, compared with preoxaliplatin treatment, at all 3 time points after administration (ie, 21, 42, and 60 days) of oxaliplatin (Fig. 1C), indicating the persistence of hyperalgesic priming unattenuated.7,28,36 Importantly, on day 60, at which time oxaliplatin-induced hyperalgesia was no longer present, hyperalgesic priming remained unattenuated.

3.2. Adrenalectomy prevents oxaliplatin-induced hyperalgesic priming

To evaluate the role of neuroendocrine stress axes in oxaliplatin CIPN chronic pain (hyperalgesic priming), we evaluated the impact of surgical adrenalectomy (Fig. 2A), which eliminates the final common pathway of the HPA (adrenal cortex) and sympathoadrenal (adrenal medulla) stress axes on hyperalgesic priming induced by oxaliplatin.

Figure 2.:

Role of neuroendocrine stress axes in oxaliplatin-induced hyperalgesia and priming. Male rats were submitted to sham bilateral adrenalectomy (adrenal intact animals), bilateral adrenalectomy only, or bilateral adrenalectomy plus stress hormone (epinephrine and corticosterone) replacements. Mechanical nociceptive threshold was evaluated before oxaliplatin administration (day 0) and again on day 21 after oxaliplatin administration. On day 21, after measurement of mechanical threshold, PGE2 (100 ng/paw) was injected intradermally to assess for hyperalgesic priming (ie, prolongation of PGE2-induced hyperalgesia). Mechanical nociceptive threshold was evaluated 30 minutes and 4 hours after PGE2. (A) The experimental protocol, with timing of treatments and nociceptive threshold measurements. (B) When the magnitude of oxaliplatin-induced hyperalgesia was evaluated on day 21, it was markedly attenuated in the oxaliplatin-treated adrenalectomized rats, compared with the adrenal intact group. However, in the adrenalectomized rats replaced with stress hormones, oxaliplatin-induced hyperalgesia was of similar magnitude to that observed in adrenal intact rats, supporting a contribution of stress hormones to oxaliplatin-induced hyperalgesia. Data are shown as mean ± SEM, treatment F(2,15) = 14.45; **P < 0.01: sham adrenalectomy group vs adrenalectomy group, ***P < 0.001: adrenalectomy group vs adrenalectomy plus stress hormone replacement (n = 6, 1-way repeated-measures ANOVA followed by Bonferroni post hoc test). (C) PGE2-induced hyperalgesia at 30 minutes, in all groups, that was no longer present at the fourth hour, in adrenalectomized rats. However, PGE2-induced hyperalgesia was still present 4 hours after its injection in the sham adrenalectomized group (adrenal intact animals) and in the adrenalectomized group that received replacement stress hormones, in support of a contribution of stress hormones to oxaliplatin-induced hyperalgesic priming. Data are shown as mean ± SEM, treatment F(2,15) = 14.45; **P < 0.01: sham adrenalectomy group vs adrenalectomized group, ***P < 0.001: adrenalectomy group vs adrenalectomy plus stress hormone replacement (n = 6, 1-way repeated-measures ANOVA followed by Bonferroni post hoc test). Data are shown as mean ± SEM, time F(1,30) = 5.922; treatment F(2,30) = 6.024; interaction F(2,30) = 4.143, **P < 0.01: sham adrenalectomy group vs adrenalectomy group, *P < 0.05: adrenalectomized group vs adrenalectomy plus stress hormones group (n = 6 rats/group), 2-way repeated-measures ANOVA followed by Bonferroni post hoc test. Adx, adrenalectomized; ANOVA, analysis of variance; PGE2, prostaglandin E2.

As we have previously shown,79oxaliplatin-induced hyperalgesia is markedly attenuated in adrenalectomized (Adx) rats evaluated 21 days after administration (Fig. 2B). We now show that in adrenalectomized rats receiving replacement of nonstress levels of corticosterone plus epinephrine, mechanical hyperalgesia was not statistically different from that seen in sham adrenalectomized rats (Fig. 2B). We also observed that oxaliplatin-induced hyperalgesic priming was also absent in adrenalectomized rats because there was no prolongation of PGE2 hyperalgesia measured 4 hours after its administration. In adrenalectomized rats receiving replacement corticosterone and epinephrine to produce levels seen in stressed rats,79oxaliplatin-induced hyperalgesic priming was reinstated (Fig. 2C). These data suggest that the hyperalgesia and hyperalgesic priming, the persistent form of CIPN pain, are strongly dependent on stress hormones at stress levels.

3.3. Contribution of nociceptor β2-adrenergic and glucocorticoid receptors to oxaliplatin-induced hyperalgesic priming

We have previously shown79 that male rats receiving intrathecal oligodeoxynucleotides antisense to glucocorticoid receptor (GR) and β2-adrenergic receptor (ADRB2) mRNA, to attenuate their level in nociceptors,21 both reversed oxaliplatin-induced hyperalgesia when measured on day 21. We now show that oxaliplatin-induced hyperalgesic priming was also markedly attenuated in rats receiving AS-ODNs (80 μg/20 μL administered intrathecally daily for 3 days starting 18 days after administration of oxaliplatin) (Fig. 3A), against ADRB2 mRNA (Fig. 3B) or against GR (Fig. 3C). Thus, ongoing GR and ADRB2 signaling is essential to produce oxaliplatin-induced hyperalgesic priming.

Figure 3.:

Role of the β2-adrenergic and glucocorticoid receptor in oxaliplatin-induced priming. Male rats received a single intravenous injection of oxaliplatin (2 mg/kg) on day 0, and starting on day 18, MM-ODNs or AS-ODNs (both 120 μg/20 μL, gray bar) to ADRB2 or GR mRNA were injected intrathecally for 3 consecutive days. On day 21 after administration of oxaliplatin, ∼17 hours after the last ODN injection, mechanical nociceptive threshold was measured, and PGE2 (100 ng/5 μL) was injected intradermally. Thirty minutes and 4 hours after injection of PGE2, the mechanical nociceptive threshold was again measured to assess for hyperalgesic priming. (A) The experimental protocol providing timing of treatments and nociceptive threshold measurements. (B) When the magnitude of oxaliplatin-induced hyperalgesia was evaluated on day 21, an inhibition in oxaliplatin-induced hyperalgesia (t 10 = 2.321, *P = 0.0427: ADRB2 AS-ODN–treated group vs ADRB2 MM-ODN–treated group, unpaired Student t test) and the prolongation of PGE2 hyperalgesia were observed in ADRB2 AS-ODN–treated group (time F(1,20) = 9.624; treatment F(1,20) = 2.274; interaction F(1,20) = 9.768, **P < 0.01: ADRB2 AS-ODN–treated group vs ADRB2 MM-ODN–treated group, 2-way repeated-measures ANOVA followed by Bonferroni post hoc test). Data are shown as mean ± SEM (n = 6 rats/group). These findings support the suggestion that oxaliplatin-induced hyperalgesia and hyperalgesic priming are β2-adrenergic receptor-dependent. (C) When the magnitude of oxaliplatin-induced hyperalgesia was evaluated on day 21, no differences between the GR AS-ODN–treated and MM-ODN–treated groups were observed (t 10 = 1.794, P = 0.1100: GR AS-ODN–treated group vs GR MM-ODN–treated group, unpaired Student t test). However, an inhibition of the prolongation of PGE2-induced hyperalgesia was observed in the GR AS-ODN–treated group, when compared with the GR MM-ODN–treated group (time F(1,20) = 6.420; treatment F(1,20) = 5.427; interaction F(1,20) = 10.10, ***P < 0.002: GR AS-ODN–treated group vs GR MM-ODN–treated group, 2-way repeated-measures ANOVA followed by Bonferroni post hoc test). Data are shown as mean ± SEM (n = 6 rats/group). These findings indicate that although oxaliplatin-induced hyperalgesia is glucocorticoid receptor-independent, in oxaliplatin-induced priming, this receptor plays a significant role. ADRB2, β2-adrenergic receptor; ANOVA, analysis of variance; AS, antisense; BL, antisense; GR, glucocorticoid receptor; ODN, oligodeoxynucleotide; ; MM, mismatch; PGE2, prostaglandin E2.

3.4. Stress resilience prevents oxaliplatin-induced hyperalgesia and hyperalgesic priming

To further evaluate the contribution of the HPA and sympathoadrenal stress axes to oxaliplatin CIPN, neonatal rats were exposed to a protocol that produces resilience to stress in the adult30,50,67 (Fig. 4A). We have previously shown79 that neonatal handling (daily, on postnatal days 2-9), which produces a stress-resilience phenotype in adult rats,5 prevented oxaliplatin-induced hyperalgesia in 8-week-old (adult) rats. We now show that oxaliplatin-induced hyperalgesic priming was also absent in neonatally handled adult rats because hyperalgesia was not present 4 hours after PGE2 administration (Fig. 4B). These data provide evidence that early life events associated with stress impact the chronification of oxaliplatin CIPN, as observed by a significant prevention in the induction of hyperalgesic priming.

Figure 4.:

Neonatal handling-induced stress resilience attenuates oxaliplatin-induced hyperalgesia and hyperalgesic priming in adult rats. Male rats were submitted to neonatal handling and as adults received oxaliplatin. (A) The experimental protocol providing timing of treatments and nociceptive threshold measurements. (B) Neonatal handling prevented oxaliplatin-induced hyperalgesia, when compared with control adult rats that received oxaliplatinneonatal handling, oxaliplatin vs oxaliplatin, ****P<0.0001, Student unpaired 2-tailed t test, t=6.883, and a marked inhibition in oxaliplatin-induced hyperalgesic priming. Data are shown as mean ± SEM neonatal handling, oxaliplatin vs oxaliplatin, at 4 h, **P<0.005, using 2-way ANOVA followed by Bonferroni post hoc test (n = 6 paws per group). ANOVA, analysis of variance; NH, neonatal handling (resilient); PGE2, prostaglandin E2.

3.5. Role of G-protein αi/o subunits in hyperalgesic priming

Given that the β2-adrenergic receptor can be coupled to multiple Gαi/o proteins13,51 that, in turn, signal through different second messengers, we initially evaluated the effect of pertussis toxin (PTX), which irreversibly inhibits all Gαi/o subunits, on oxaliplatin-induced mechanical hyperalgesia and hyperalgesic priming, administered 20 days after oxaliplatin. Although pertussis toxin had no effect on oxaliplatin-induced hyperalgesia (Figs. 5A and B), it completely reversed oxaliplatin-induced prolongation of PGE2 hyperalgesia (hyperalgesic priming) (Fig. 5C), suggesting the involvement of Gi proteins in the chronic form of oxaliplatin CIPN.

Figure 5.:

Role of GPCR Gi/o subunits in oxaliplatin-induced hyperalgesia and hyperalgesic priming. Male rats received a single intravenous injection of oxaliplatin (2 mg/kg), and the mechanical nociceptive threshold was evaluated before oxaliplatin administration (day 0) and again on day 20 after its administration. On day 20, after measurement of mechanical nociceptive threshold, vehicle (saline) or pertussis toxin, which inactivates diverse Gαi/o subunits, was injected intradermally (1 µg/5 µL). On day 21, ∼17 hours after pertussis toxin injection, mechanical nociceptive threshold was evaluated, and PGE2 (100 ng/paw) was then injected intradermally. In these experiments, mechanical nociceptive threshold was evaluated 30 minutes and 4 hours after injection of PGE2 to assess for hyperalgesic priming. (A) The experimental protocol providing timing of treatments and nociceptive threshold measurements. (B) When the magnitude of oxaliplatin-induced hyperalgesia was evaluated on day 21, no difference between the pertussis toxin–treated and vehicle-treated groups was observed (t 10 = 0.3922, P = 0.6817: pertussis toxin–treated group vs vehicle-treated group, unpaired Student t test). (C) However, inhibition of the prolongation of PGE2 was observed in the pertussis toxin–treated group, when compared with the vehicle-treated group (time F(1,20) = 14; treatment F(1,20) = 10.38; interaction F(1,20) = 6.415, **P < 0.01: for pertussis toxin–treated group vs vehicle-treated group, 2-way repeated-measures ANOVA followed by Bonferroni post hoc test). Data are shown as mean ± SEM (n = 6 rats/group). These findings indicate that although oxaliplatin-induced hyperalgesia is Gαi/o-GPCR subunit-independent, they contribute to oxaliplatin-induced hyperalgesic priming. ANOVA, analysis of variance; BL, baseline; GPCR, G protein-coupled receptor; Gα/o, G-protein subunit α/o; PGE2, prostaglandin E2.

To evaluate the role of individual Gαi/o subunits in oxaliplatin-induced mechanical hyperalgesic priming, we used intrathecal administration of oligodeoxynucleotides antisense to Gαi1, Gαi2, Gαi3, or Gαo mRNA to selectively attenuate each Gαi/o subunit. Rats were treated intrathecally with AS-ODN or SE-ODN to Gαi1, Gαi2, Gαi3, or Gαo mRNA, daily for 3 days, beginning 18 days after oxaliplatin administration (Fig. 6A). On, day 21, ∼17 hours after the third injection of ODNs, PGE2 was injected intradermally, and the mechanical nociceptive threshold was evaluated, at the injection site, 30 minutes and 4 hours later. Hyperalgesic priming (ie, PGE2 hyperalgesia at the fourth hour) was absent in rats treated with Gαi1 (Fig. 6B) and Gαo (Fig. 6C) mRNA but was present in rats treated with ODN antisense to Gαi3 and Gαi2 mRNA (Figs. 6D and E). These data indicate that oxaliplatin-induced hyperalgesic priming is Gαi1- and Gαo-dependent and Gαi3- or Gαi2-independent.

Figure 6.:

Role of GPCR Gi/o subunits in oxaliplatin-induced priming. Male rats received a single intravenous injection of oxaliplatin (2 mg/kg), and on day 18 after its administration, SE-ODNs (120 μg/20 μL, black bar) or AS-ODNs (120 μg/20 μL, gray bar) against Gαi1, Gαi2, Gαi3, or Gαo, mRNA was injected intrathecally for 3 consecutive days. On day 21, ∼17 hours after the last ODN injection, mechanical nociceptive threshold was measured, and then PGE2 (100 ng/5 μL) was injected intradermally. Thirty minutes and 4 hours after PGE2, the mechanical nociceptive threshold was again measured to assess for hyperalgesic priming. (A) The experimental protocol providing timing of treatments and nociceptive threshold measurements. (B) An inhibition of the prolongation of PGE2 effect was observed in the Gαi1 AS-ODN–treated group, when compared with the Gαi1 SE-ODN group (time F(1,20) = 26.36; treatment F(1,20) = 16.25; interaction F(1,20) = 8.369, **P < 0.01: Giα 1 AS-ODN–treated group vs Gαi1 SE-ODN–treated group, 2-way repeated-measures ANOVA followed by Bonferroni post hoc test). Data are shown as mean ± SEM (n = 6 rats/group). These findings indicate that oxaliplatin-induced hyperalgesic priming is Gαi1-dependent. (C) An inhibition of the prolongation of PGE2 hyperalgesia was observed in the Goα AS-ODN–treated group, when compared with the Goα SE-ODN–treated group (time F(1,20) = 46.27; treatment F(1,20) = 58.32; interaction F(1,20) = 42.99, **P < 0.0001: Goα AS-ODN–treated group vs Gαo SE-ODN–treated group, 2-way repeated-measures ANOVA followed by Bonferroni post hoc test). Data are shown as mean ± SEM (n= 6 rats/group). These findings indicate that oxaliplatin-induced hyperalgesic