Attenuation of Unevoked Mechanical and Cold Pain Hypersensitivities Associated With Experimental Neuropathy in Mice by Angiotensin II Type-2 Receptor Antagonism

Abstract

Recent findings from a phase II clinical trial showed analgesic effects of an angiotensin II type-2 receptor (AT2R) antagonist in postherpetic neuralgia patients. This study aimed to investigate whether AT2R antagonism could provide effective analgesia in voluntary measures of unevoked/ongoing pain-like behaviors in mice with experimental neuropathy. Mice were subjected to spared nerve injury to induce neuropathy and tested in 2 operant behavioral tests to measure ongoing mechanical and cold pain hypersensitivities. Systemic administration of an AT2R antagonist provided effective analgesia in these behavioral measures of mechanical and cold pain in spared nerve injury mice, suggesting its effectiveness in neuropathic pain.

The lack of a precise mechanistic understanding of neuropathic pain has undoubtedly hampered the development of effective analgesics. The fact that such pain is managed inadequately by existing drugs^1,2 further strengthens the need for the development of new treatment options. One such potential compound, an angiotensin II type-2 receptor (AT2R) antagonist (EMA401), has shown effective pain relief in postherpetic neuralgia patients in a phase II clinical trial.³ Several recent reports have also shown analgesic efficacy of AT2R antagonists in reflexive measures of mechanical pain hypersensitivity in preclinical models of neuropathic and cancer pain.^4–8 However, growing concerns over a mismatch between pain assessments that rely on evoked or reflexive end points (such as reflexive withdrawal from a noxious stimulus) and clinical outcomes has resulted in a reevaluation of the field’s reliance on such measures.⁹ Given that hypersensitivity to mechanical and cold stimuli is a common manifestation of pain in multiple neuropathic conditions,² we aimed to test the analgesic effectiveness of AT2R antagonism in more clinically relevant, ongoing pain-like behaviors in preclinical models of neuropathic pain: a mechanical conflict-avoidance (MCA) paradigm^10,11 and a temperature place preference assay.¹² Using the spared nerve injury (SNI) model of experimental neuropathy in mice, we corroborate the effectiveness of AT2R antagonism in attenuating voluntary unevoked/ongoing pain-like behaviors, a finding that could prove beneficial in furthering the development of AT2R antagonists for multiple neuropathic pain conditions.

METHODS

Mice

All experiments involving mice and the procedures followed therein were approved by the Institutional Animal Studies Committee of Washington University in St Louis in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Every effort was made to minimize the number of mice used and their suffering. Mice were maintained on a 12:12 light/dark cycle with food and water ad libitum. Equal numbers of male and female C57BL/6J mice (Stock No: 000664; Jackson Lab, Bar Harbor, ME) of 8–14 weeks of age were used throughout. Preliminary experiments showed no sex-related differences in pain sensitivity or drug efficacy. As a result, male and female data were pooled. Experimenters were blinded to mouse sham/surgery conditions and saline/drug injection types during the conduct of experiments, data recordings, and analyses.

Mouse SNI Model of Neuropathy

Mice underwent surgery as part of SNI-induced neuropathy as described previously.^13,14 Briefly, mice were anesthetized, and the biceps femoris muscle was separated to access the branches of the sciatic nerve. The common peroneal and tibial nerve branches were ligated with silk suture, and approximately 1 mm of nerve was removed distally. After wound closure and administration of postoperative analgesia, mice were monitored for signs of distress and allowed to recover on a heating pad before return to the home cage. Sham-operated controls received the incision, muscle separation, and postoperative analgesia alone. Experimentation began on these mice 8 days later. After sham/surgery, the animals were randomly distributed to individual experimental and treatment groups.

MCA Assay

Voluntary MCA was assessed following a protocol detailed recently.^10,11 Mice (1 at a time) were acclimated to the MCA unit 3 times for 5 minutes each before the beginning of baseline recordings. Rodents with neuropathic pain conditions, such as SNI and chronic constriction injury, exhibit increased escape latencies, which could be reversed with analgesics such as morphine and gabapentin, suggesting this as a measure of voluntary pain behavior.^10,11

Warm–Cool Place Avoidance Assay

A warm–cool place avoidance test was performed as a voluntary cold hypersensitivity pain behavior due to neuropathy in mice,¹² following published procedures, with minor modifications. Mice were acclimated for 15 minutes twice to a connected pair of temperature-controlled metal plates maintained at 30°C, with a white Plexiglas perimeter box surrounding both plates (Cold/Hot Plate Analgesia Meter; Columbus Instruments, Columbus, OH). For testing, both plates were set to 30°C, and then a mouse was placed at the junction of both plates, from where it had access to move freely for 10 minutes and was video recorded from above. After running this test on all mice in an experimental cohort, the process was repeated with the temperature of the test plate set to 20°C. The total time spent on the test temperature side/plate only (30°C or 20°C) was quantified by offline analysis of individual video files.

Statistical Analysis

Based on the magnitude of behavioral changes reported in prior studies utilizing similar behavioral assays, power analysis was performed using the online BioMath software (New York, NY; http://www.biomath.info/). Data are analyzed with GraphPad Prism 7.0 (GraphPad Software, Inc, La Jolla, CA) using 2-way analysis of variance with Tukey’s multiple comparisons post hoc test. Spike height and surgery/drug treatment groups served as the 2 factors, with escape latency as the measured outcome. P < .05 in each set of data comparisons was considered statistically significant. No data points were excluded from statistical analysis.

RESULTS

Antagonists of AT2R have recently been shown to provide effective analgesia in mouse neuropathic pain models, using reflexive measures of hindpaw withdrawal threshold to mechanical stimuli.^5–7 We tested whether an analgesic action of AT2R antagonism could be observed in operant behavioral assessment of unevoked/voluntary/ongoing pain in mice, using the recently developed and validated MCA assay for voluntary measures of mechanical pain hypersensitivity (Figure A).^10,11 An effective sample size for these experiments was determined as ≥6 per experimental group. At a higher spike height (5 mm) in the conflict chamber, SNI mice showed a significant increase in the latency to escape from the lit chamber 8 days postsurgery, compared to sham-operated controls (SNI + saline: 34.97 s, 95% confidence interval [CI], 9.42–60.53; Sham + PD123319: 11.24 s, 95% CI, 7.64–14.84; Figure B). This increased escape latency in SNI mice was ≈100% inhibited by systemic administration of the AT2R antagonist PD123319 (10 mg/kg; intraperitoneal [i.p.]; Tocris-Biotechne, Minneapolis, MN) (SNI + PD123319: 10.52 s, 95% CI, 6.87–14.16; Figure B). Surgery/drug treatment yielded an “F” ratio of F_(2,51) = 5.733, P = .0057. The spike height yielded an “F” ratio of F_(2,51) = 8.583, P = .0006. The interaction between factors was significant (F_(4,51) = 4.797, P = .0023).

Figure.

Attenuation of voluntary mechanical and cold pain hypersensitivities in mice with experimental neuropathy by an AT2R antagonist. A, Experimental scheme depicting unevoked/nonreflexive mechanical hypersensitivity assessment using an MCA system in mice that are subjected to sham or SNI surgery. B, Systemic administration of an AT2R antagonist, PD123319 (10 mg/kg; i.p.), attenuates SNI-induced increases in escape latency from lit chamber to mechanical conflict chamber. Raw data are presented as points with respective shapes, and mean ± SEM is presented as superimposed bars; **P < .01 versus respective baseline values or sham + PD123319 groups; ^##P < .01 versus SNI + saline group. C, Experimental scheme depicting unevoked/nonreflexive cold hypersensitivity assessment using warm/cool plate place avoidance system in mice that are subjected to sham/SNI surgery. D, Systemic administration of PD123319 (10 mg/kg; i.p.) attenuates SNI-induced avoidance duration to cool-temperature chamber. Data are presented as mean ± SEM; ns, **P < .01 and ***P < .001, versus sham + PD123319 group; and ^###P < .001, versus SNI + saline group. AT2R indicates angiotensin II type-2 receptor; MCA, mechanical conflict-avoidance; ns, not significant; SEM, standard error of the mean; SNI, spared nerve injury.

Previous reports have shown maximal attenuation of reflexive mechanical pain hypersensitivity in mice with experimental neuropathy at this concentration, which did not result in any hemodynamic or other central side effects.⁶ Similarly, in separate cohorts of mice without prior exposure/baseline to the MCA assay, administration of PD123319 (10 mg/kg; i.p.) attenuated the SNI-induced increase in lit chamber escape latency (SNI + saline: 35.85 s, 95% CI, 12.61–59.09; Sham + PD123319: 6.90 s, 95% CI, 4.32–9.49; SNI + PD123319: 8.39 s, 95% CI, 7.45–9.33; Supplemental Digital Content 1, Figure 1, http://links.lww.com/AA/C605). Surgery/drug treatment yielded an “F” ratio of F_(2,51) = 3.423, P = .0403. The spike height yielded an “F” ratio of F_(2,51) = 7.96, P = .001. The interaction between factors was significant (F₍₄,₅₁₎ = 2.778, P = .0365).

Experimental models of neuropathy in mice have been shown to induce reflexive cold hypersensitivity on hind-paws.¹⁵ We next performed a thermal place preference/avoidance assay to better interrogate voluntary ongoing cold hypersensitivity associated with SNI (Figure C). Eight days postsurgery, SNI mice exhibited significantly increased aversion to the test chamber when set to 20°C versus sham/surgery controls, shown as percent time spent in the test chamber (Figure D; P < .0001 on day 8 postsurgery). This increased aversion persisted at postoperative day 11, which was significantly attenuated 2 hours post-PD123319 administration (10 mg/kg; i.p.), and was subsequently restored 24 hours later, at 12 days postsurgery (Figure D; P = .0008 at 2 hours after the administration drug versus vehicle). A similar reduction in SNI-induced cold aversion was observed in separate cohorts of mice at postoperative day 16 with systemic administration of buprenorphine (0.025 mg/kg; i.p.) (Supplemental Digital Content 2, Figure 2, http://links.lww.com/AA/C606), suggesting that this cold avoidance is indeed a valid measure of voluntary/ongoing cold pain behavior.

DISCUSSION

Antagonists of AT2R represent a novel class of analgesics that have demonstrated efficacy in preclinical models of neuropathic and inflammatory pain.^4–7 However, the over-reliance of preclinical pain research on assessments that use reflexive end points has come under recent scrutiny and is suggested to contribute to translational failures in analgesic drug developments.⁹ Here, we show that reversal of pain-related behaviors by AT2R antagonism is conserved in an experimental neuropathy model in mice subjected to 2 distinct unevoked behavioral assays. Specifically, the AT2R antagonist PD123319 attenuated increases in MCA and cold avoidance in much the same way as previous reports have shown for other, well-established analgesics, such as morphine and pregabalin.^10,11 These findings are consistent with the observation that mechanical and cold hypersensitivities are common characteristics reported in multiple neuropathic pain conditions.²

It is important to note that the AT2R antagonist used in this study (PD123319) was slightly modified to improve oral bioavailability to generate EMA401,¹⁶ the compound used in the phase II clinical trial for treatment of postherpetic neuralgia.³ A number of mechanisms underlying the analgesic efficacy of AT2R antagonism have been proposed.^4,5,17,18 That said, this study and prior reports using the same dose and route of PD123319 administration show attenuation of mechanical and cold hypersensitivities in experimental neuropathy in mice.^5,7,17 This finding confirms that the analgesic action of AT2R antagonism is conserved across the different subtypes of EMA compounds used. Furthermore, while MCA and cold preference/avoidance showed SNI-induced changes that were reversible with analgesics, not all unevoked assays have been reported to show similar results. For example, alterations in gait patterns observed after SNI remained unaffected by administration of buprenorphine or PD123319.^7,11 This finding indicates that the choice of test (as well as the appropriateness of the disease model) is an important consideration with unevoked assessments of ongoing pain and that study designs incorporating several types of assays—both evoked and unevoked in nature—would perhaps prove more informative. In summary, our findings using voluntary measures of pain sensitivity solidify the observation that AT2R antagonism is a promising analgesic target for the treatment of neuropathic pain.