Pharmacological validation of voluntary gait and mechanical sensitivity assays associated with inflammatory and neuropathic pain in mice

Highlights

• A neuropathic pain model causes prolonged changes in mouse voluntary gait patterns.

• An inflammatory pain model causes relatively transient gait changes.

• Gait changes induced by neuropathy/inflammation are not reversible with analgesics.

• A mechanical conflict-avoidance (MCA) test detects changes in these pain models.

• Neuropathy/inflammation-induced changes in MCA behavior are reversed by analgesics.

Abstract

The urgent need for more effective analgesic treatment options has prompted a re-evaluation of the behavioral tests used to assess pain in pre-clinical research, with an emphasis on inclusion of more voluntary, un-evoked behavioral assessments of pain. In order to validate voluntary gait analysis and a voluntary mechanical conflict-avoidance assay, we tested mouse models of neuropathy (spared nerve injury) and inflammation (complete Freund's adjuvant) alongside reflexive measures of mechanical and thermal hypersensitivity. To establish whether the observed changes in behavioral responses were pain-related, known analgesics (buprenorphine, gabapentin, carprofen) were also administered. Spared nerve injury persistently altered several gait indices, whereas complete Freund's adjuvant caused only transient changes. Furthermore, known analgesics could not reverse these gait changes, despite demonstrating their previously established efficacy in reflexive measures of mechanical and thermal hypersensitivity. In contrast, the mechanical conflict-avoidance assay demonstrated aversion in mice with neuropathy and inflammation-induced hypersensitivity, which could both be reversed by analgesics. We conclude that voluntary gait changes in rodent neuropathic and inflammatory pain models are not necessarily indicative of pain-related adaptations. On the other hand, mechanical conflict-avoidance represents a valid operant assay for quantifying pain-related behaviors in mice that can be reversed by known analgesics.

Introduction

Chronic pain in humans is a complex sensory and affective experience. A shift in pain-detection threshold and hypersensitivity to thermal/mechanical stimuli are key components of this experience. Historically, these aspects of stimulus-evoked assessment of pain-related behaviors - such as Hargreaves' and von Frey filament-based assessment of thermal and mechanical sensitivity (Hargreaves et al., 1988, Chaplan et al., 1994), tail-flick, paw flinching, licking/biting and guarding assessments (Cargill et al., 1985, Seltzer et al., 1990, Wheeler-Aceto et al., 1990) - have primarily been detected and quantified in preclinical animal models of painful pathologies. However, the ongoing failure to develop efficacious, new-generation analgesic drugs has led to a re-evaluation of the field's reliance on these evoked and/or reflexive assessments of pain-related behaviors in preclinical animal models (Mao, 2009, Berge, 2011, Percie du Sert and Rice, 2014, Clark, 2016).

In recent years in the area of pain research, newer assays of pain in rodent models, such as wheel running, burrowing, gait, ultrasonic vocalization, conditioned place preference etc. have been developed (Vrinten and Hamers, 2003, Li et al., 2004, Williams et al., 2008, King et al., 2009, Truin et al., 2009, Kurejova et al., 2010, Mogil et al., 2010, Andrews et al., 2012, Cobos et al., 2012, Huehnchen et al., 2013, Parvathy and Masocha, 2013, Ruan et al., 2013, Chiang et al., 2014, Muramatsu et al., 2014, Rutten et al., 2014, Sahbaie et al., 2014, Gould et al., 2016, Pitzer et al., 2016, Wodarski et al., 2016, Sheahan et al., 2017, Sugiyama et al., 2017) in an effort to improve the translational potential of preclinical findings (Barrot, 2012, Tappe-Theodor and Kuner, 2014, Clark, 2016). However, most of these newer and operant-based assays haven't shown universality across different pain types, such as inflammatory, neuropathic, and specific disease-related. In addition, while extensively tested, the added complexity of drug conditioning/addictive behavioral components, such as in conditioned place preference (Porreca and Navratilova, 2017) has led to questions on what facets of pain-related behavioral alterations are being measured in rodents. Therefore, it is vital to establish whether these newer measures of pain are capable of detecting quantifiable behavioral changes and/or adaptations that are unequivocally pain-related, as well as develop and validate newer methods to obtain such outcomes. One way to do this is to test an assay with well-established analgesic compounds, at appropriate doses, as positive controls.

Our study evaluated voluntary changes in gait and mechanical conflict-avoidance in a representative rodent model of neuropathic pain: spared nerve injury – SNI (Decosterd and Woolf, 2000), and inflammatory pain: complete Freund's Adjuvant – CFA (Larson et al., 1986). While we observed sustained changes in several voluntary gait indices in SNI mice, the effects of CFA administration on gait patterns were much less drastic and more transient. Furthermore, administration of the opioid buprenorphine (Christoph et al., 2005), gabapentin (Decosterd et al., 2004) or the NSAID carprofen (Adamson et al., 2010, Smeester et al., 2017) did not alter SNI or CFA-induced gait changes, respectively. This was despite these drugs being administered at doses shown to be effective in the more classical, evoked assays of hypersensitivity (von Frey testing for SNI, and von Frey and Hargreaves for CFA). In contrast, the voluntary mechanical conflict-avoidance (MCA) test did show significant SNI- and CFA-induced aversion behaviors, which were reversed by the same sets of well-established analgesics. Our findings suggest that gait data and specific changes in paw imaging-based gait indices must be analyzed carefully in rodent models of painful pathologies. Also, any alterations in gait should be validated with proven analgesic drugs at recommended doses. On the other hand, the MCA test constitutes a promising operant assay for quantifying pain-related behaviors in mice with pathologies that could be potentially painful.