IUPHAR review: Navigating the role of preclinical models in pain research.

Chronic pain is a complex and challenging medical condition that affects millions of people worldwide. Understanding the underlying mechanisms of chronic pain is a key goal of preclinical pain research so that more effective treatment strategies can be developed. In this review, we explore nociception, pain, and the multifaceted factors that lead to chronic pain by focusing on preclinical models. We provide a detailed look into inflammatory and neuropathic pain models and discuss the most used animal models for studying the mechanisms behind these conditions. Additionally, we emphasize the vital role of these preclinical models in developing new pain-relief drugs, focusing on biologics and the therapeutic potential of NMDA and cannabinoid receptor antagonists. We also discuss the challenges of TRPV1 modulation for pain treatment, the clinical failures of neurokinin (NK)- 1 receptor antagonists, and the partial success story of Ziconotide to provide valuable lessons for preclinical pain models. Finally, we highlight the overall success and limitations of current treatments for chronic pain while providing critical insights into the development of more effective therapies to alleviate the burden of chronic pain.

Influence of Inflammatory Pain and Dopamine on Synaptic Transmission in the Mouse ACC.

Dopamine (DA) inhibits excitatory synaptic transmission in the anterior cingulate cortex (ACC), a brain region involved in the sensory and affective processing of pain. However, the DA modulation of inhibitory synaptic transmission in the ACC and its alteration of the excitatory/inhibitory (E/I) balance remains relatively understudied. Using patch-clamp recordings, we demonstrate that neither DA applied directly to the tissue slice nor complete Freund's adjuvant (CFA) injected into the hind paw significantly impacted excitatory currents (eEPSCs) in the ACC, when recorded without pharmacological isolation. However, individual neurons exhibited varied responses to DA, with some showing inhibition, potentiation, or no response. The degree of eEPSC inhibition by DA was higher in naïve slices compared to that in the CFA condition. The baseline inhibitory currents (eIPSCs) were greater in the CFA-treated slices, and DA specifically inhibited eIPSCs in the CFA-treated, but not naïve group. DA and CFA treatment did not alter the balance between excitatory and inhibitory currents. Spontaneous synaptic activity revealed that DA reduced the frequency of the excitatory currents in CFA-treated mice and decreased the amplitude of the inhibitory currents, specifically in CFA-treated mice. However, the overall synaptic drive remained similar between the naïve and CFA-treated mice. Additionally, GABAergic currents were pharmacologically isolated and found to be robustly inhibited by DA through postsynaptic D2 receptors and G-protein activity. Overall, the study suggests that CFA-induced inflammation and DA do not significantly affect the balance between excitatory and inhibitory currents in ACC neurons, but activity-dependent changes may be observed in the DA modulation of presynaptic glutamate release in the presence of inflammation.

Naked mole-rats lack cold sensitivity before and after nerve injury.

Neuropathic pain is a chronic disease state resulting from injury to the nervous system. This type of pain often responds poorly to standard treatments and occasionally may get worse instead of better over time. Patients who experience neuropathic pain report sensitivity to cold and mechanical stimuli. Since the nociceptive system of African naked mole-rats contains unique adaptations that result in insensitivity to some pain types, we investigated whether naked mole-rats may be resilient to sensitivity following nerve injury. Using the spared nerve injury model of neuropathic pain, we showed that sensitivity to mechanical stimuli developed similarly in mice and naked mole-rats. However, naked mole-rats lacked sensitivity to mild cold stimulation after nerve injury, while mice developed robust cold sensitivity. We pursued this response deficit by testing behavior to activators of transient receptor potential (TRP) receptors involved in detecting cold in naïve animals. Following mustard oil, a TRPA1 activator, naked mole-rats responded similarly to mice. Conversely, icilin, a TRPM8 agonist, did not evoke pain behavior in naked mole-rats when compared with mice. Finally, we used RNAscope to probe for TRPA1 and TRPM8 messenger RNA expression in dorsal root ganglia of both species. We found increased TRPA1 messenger RNA, but decreased TRPM8 punctae in naked mole-rats when compared with mice. Our findings likely reflect species differences due to evolutionary environmental responses that are not easily explained by differences in receptor expression between the species.

Social propinquity in rodents as measured by tube cooccupancy differs between inbred and outbred genotypes.

Existing assays of social interaction are suboptimal, and none measures propinquity, the tendency of rodents to maintain close physical proximity. These assays are ubiquitously performed using inbred mouse strains and mutations placed on inbred genetic backgrounds. We developed the automatable tube cooccupancy test (TCOT) based on propinquity, the tendency of freely mobile rodents to maintain close physical proximity, and assessed TCOT behavior on a variety of genotypes and social and environmental conditions. In outbred mice and rats, familiarity determined willingness to cooccupy the tube, with siblings and/or cagemates of both sexes exhibiting higher cooccupancy behavior than strangers. Subsequent testing using multiple genotypes revealed that inbred strain siblings do not cooccupy at higher rates than strangers, in marked contrast to both outbred and rederived wild mice. Mutant mouse strains with "autistic-like" phenotypes (Fmr1-/y and Eif4e Ser209Ala) displayed significantly decreased cooccupancy.

eIF2α phosphorylation controls thermal nociception.

A response to environmental stress is critical to alleviate cellular injury and maintain cellular homeostasis. Eukaryotic initiation factor 2 (eIF2) is a key integrator of cellular stress responses and an important regulator of mRNA translation. Diverse stress signals lead to the phosphorylation of the α subunit of eIF2 (Ser51), resulting in inhibition of global protein synthesis while promoting expression of proteins that mediate cell adaptation to stress. Here we report that eIF2α is instrumental in the control of noxious heat sensation. Mice with decreased eIF2α phosphorylation (eIF2α+/S51A) exhibit reduced responses to noxious heat. Pharmacological attenuation of eIF2α phosphorylation decreases thermal, but not mechanical, pain sensitivity, whereas increasing eIF2α phosphorylation has the opposite effect on thermal nociception. The impact of eIF2α phosphorylation (p-eIF2α) on thermal thresholds is dependent on the transient receptor potential vanilloid 1. Moreover, we show that induction of eIF2α phosphorylation in primary sensory neurons in a chronic inflammation pain model contributes to thermal hypersensitivity. Our results demonstrate that the cellular stress response pathway, mediated via p-eIF2α, represents a mechanism that could be used to alleviate pathological heat sensation.

Olfactory exposure to males, including men, causes stress and related analgesia in rodents.

We found that exposure of mice and rats to male but not female experimenters produces pain inhibition. Male-related stimuli induced a robust physiological stress response that results in stress-induced analgesia. This effect could be replicated with T-shirts worn by men, bedding material from gonadally intact and unfamiliar male mammals, and presentation of compounds secreted from the human axilla. Experimenter sex can thus affect apparent baseline responses in behavioral testing.

Inflammatory injury induces pain sensitization that is expressed beyond the site of injury in male (and not in female) mice.

Pain is a crucial protective mechanism for the body. It alerts us to potential tissue damage or injury and promotes the avoidance of harmful stimuli. Injury-induced inflammation and tissue damage lead to pain sensitization, which amplifies responses to subsequent noxious stimuli even after an initial primary injury has recovered. This phenomenon, commonly referred to as hyperalgesic priming, was investigated in male and female mice to determine whether it is specific to the site of previous injury. We used 10µl of 50 % Freund's complete adjuvant (CFA) administered to the left hind paw as a model of peripheral injury. Both male and female mice exhibited robust site-specific mechanical hypersensitivity after CFA, which resolved within one-week post-injection. After injury resolution, only male CFA-primed mice showed enhanced and prolonged mechanical sensitivity in response to a chemical challenge or a single 0.5 mA electric footshock. Among CFA-primed male mice, shock-induced mechanical hypersensitivity was expressed in both the left (previously injured) and the right (uninjured) hind paws, suggesting a pivotal role for altered centralized processes in the expression of pain sensitization. These findings indicate that pain history regulates sensory responses to subsequent mechanical and chemical pain stimuli in a sex-specific manner-foot-shock-induced hyperalgesic priming expression among male mice generalized beyond the initial injury site.

A single exposure to the predator odor 2,4,5-trimethylthiazoline causes long-lasting affective behavioral changes in female mice: Modulation by kappa opioid receptor signaling.

The volatile compound 2,4,5-trimethylthiazoline (TMT, a synthetic predator scent) triggers fear, anxiety, and defensive responses in rodents that can outlast the encounter. The receptor systems underlying the development and persistence of TMT-induced behavioral changes remain poorly characterized, especially in females. Kappa opioid receptors regulate threat generalization and fear conditioning and alter basal anxiety, but their role in unconditioned fear responses in females has not been examined. Here, we investigated the effects of the long-lasting kappa opioid receptor antagonist, nor-binalthorphinmine dihydrochloride (nor-BNI; 10 mg/kg), on TMT-induced freezing and conditioned place aversion in female mice. We also measured anxiety-like behavior in the elevated plus maze three days after TMT and freezing behavior when returned to the TMT-paired context ten days after the single exposure. We found that 35µl of 10 % TMT elicited a robust freezing response during a five-minute exposure in female mice. TMT evoked persistent fear as measured by conditioned place aversion, reduced entries into the open arm of the elevated plus maze, and increased general freezing behavior long after TMT exposure. In line with the known role of kappa-opioid receptors in threat generalization, we found that kappa-opioid receptor antagonism increased basal freezing but reduced freezing during TMT presentation. Together, these findings indicate that a single exposure to TMT causes long-lasting changes in fear-related behavioral responses in female mice and highlights the modulatory role of kappa-opioid receptor signaling on fear-related behavioral patterns in females.

Altered nociceptive behavior and emotional contagion of pain in mouse models of autism.

Individuals with autism spectrum disorder (ASD) have altered sensory processing but may ineffectively communicate their experiences. Here, we used a battery of nociceptive behavioral tests to assess sensory alterations in two commonly used mouse models of ASD, BTBR T+ Itpr3tf /J (BTBR), and fragile-X mental retardation-1 knockout (Fmr1-KO) mice. We also asked whether emotional contagion, a primitive form of empathy, was altered in BTBR and Fmr1 KO mice when experiencing pain with a social partner. BTBR mice demonstrated mixed nociceptive responses with hyporesponsivity to mechanical/thermal stimuli and intraplantar injections of formalin and capsaicin while displaying hypersensitivity on the acetic acid test. Fmr1-KO mice were hyposensitive to mechanical stimuli and intraplantar injections of capsaicin and formalin. BTBR and Fmr1-KO mice developed significantly less mechanical allodynia following intraplantar injections of complete Freund's adjuvant, while BTBR mice developed slightly more thermal hyperalgesia. Finally, as measured by the formalin and acetic acid writhing tests, BTBR and Fmr1-KO mice did not show emotional contagion of pain. In sum, our findings indicate that depending on the sensation, pain responses may be mixed, which reflects findings in ASD individuals.

Inflammatory Pain Alters Dopaminergic Modulation of Excitatory Synapses in the Anterior Cingulate Cortex of Mice.

Pain modulation of dopamine-producing nuclei is known to contribute to the affective component of chronic pain. However, pain modulation of pain-related cortical regions receiving dopaminergic inputs is understudied. The present study demonstrates that mice with chronic inflammatory injury of the hind paws develop persistent mechanical hypersensitivity and transient anxiety. Peripheral inflammation induced by injection of complete Freund's Adjuvant (CFA) induced potentiation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic receptor (AMPAR) currents with a presynaptic component in layer II/III of the ACC. After four days of inflammatory pain, the dopamine-mediated inhibition of AMPAR currents was significantly reduced in the ACC. Furthermore, dopamine enhanced presynaptic modulation of excitatory transmission, but only in mice with inflammatory pain. High-performance liquid chromatography (HPLC) analysis of dopamine tissue concentration revealed that dopamine neurotransmitter concentration in the ACC was reduced three days following CFA. Our results demonstrate that inflammatory pain induces activity-dependent changes in excitatory synaptic transmission and alters dopaminergic homeostasis in the ACC.

Alpha5GABAA receptor activity sets the threshold for long-term potentiation and constrains hippocampus-dependent memory.

Synaptic plasticity, which is the neuronal substrate for many forms of hippocampus-dependent learning, is attenuated by GABA type A receptor (GABA(A)R)-mediated inhibition. The prevailing notion is that a synaptic or phasic form of GABAergic inhibition regulates synaptic plasticity; however, little is known about the role of GABA(A)R subtypes that generate a tonic or persistent inhibitory conductance. We studied the regulation of synaptic plasticity by alpha5 subunit-containing GABA(A)Rs (alpha5GABA(A)Rs), which generate a tonic inhibitory conductance in CA1 pyramidal neurons using electrophysiological recordings of field and whole-cell potentials in hippocampal slices from both wild-type and null mutant mice for the alpha5 subunit of the GABA(A)R (Gabra5(-/-) mice). In addition, the strength of fear-associated memory was studied. The results showed that alpha5GABA(A)R activity raises the threshold for induction of long-term potentiation in a highly specific band of stimulation frequencies (10-20 Hz) through mechanisms that are predominantly independent of inhibitory synaptic transmission. The deletion or pharmacological inhibition of alpha5GABA(A)Rs caused no change in baseline membrane potential or input resistance but increased depolarization during 10 Hz stimulation. The encoding of hippocampus-dependent memory was regulated by alpha5GABA(A)Rs but only under specific conditions that generate moderate but not robust forms of fear-associated learning. Thus, under specific conditions, alpha5GABA(A)R activity predominates over synaptic inhibition in modifying the strength of both synaptic plasticity in vitro and certain forms of memory in vivo.

The Rat Grimace Scale: a partially automated method for quantifying pain in the laboratory rat via facial expressions.

We recently demonstrated the utility of quantifying spontaneous pain in mice via the blinded coding of facial expressions. As the majority of preclinical pain research is in fact performed in the laboratory rat, we attempted to modify the scale for use in this species. We present herein the Rat Grimace Scale, and show its reliability, accuracy, and ability to quantify the time course of spontaneous pain in the intraplantar complete Freund's adjuvant, intraarticular kaolin-carrageenan, and laparotomy (post-operative pain) assays. The scale's ability to demonstrate the dose-dependent analgesic efficacy of morphine is also shown. In addition, we have developed software, Rodent Face Finder®, which successfully automates the most labor-intensive step in the process. Given the known mechanistic dissociations between spontaneous and evoked pain, and the primacy of the former as a clinical problem, we believe that widespread adoption of spontaneous pain measures such as the Rat Grimace Scale might lead to more successful translation of basic science findings into clinical application.

Bridging the Translational Divide in Pain Research: Biological, Psychological and Social Considerations.

A gap exists between translating basic science research into effective pain therapies in humans. While preclinical pain research has primarily used animal models to understand biological processes, a lesser focus has been toward using animal models to fully consider other components of the pain experience, such as psychological and social influences. Herein, we provide an overview of translational studies within pain research by breaking them down into purely biological, psychological and social influences using a framework derived from the biopsychosocial model. We draw from a wide landscape of studies to illustrate that the pain experience is highly intricate, and every attempt must be made to address its multiple components and interactors to aid in fully understanding its complexity. We highlight our work where we have developed animal models to assess the cognitive and social effects on pain modulation while conducting parallel experiments in people that provide proof-of-importance for human pain modulation. In some instances, human pain research has sparked the development of novel animal models, with these animal models used to better understand the complexity of phenomena considered to be uniquely human such as placebo responses and empathy.

Characterizing Sex Differences in Depressive-Like Behavior and Glial Brain Cell Changes Following Peripheral Nerve Injury in Mice.

Chronic pain and depression are intimately linked; the combination of the two leads to higher health care costs, lower quality of life, and worse treatment outcomes with both conditions exhibiting higher prevalence among women. In the current study, we examined the development of depressive-like behavior in male and female mice using the spared nerve injury (SNI) model of neuropathic pain. Males displayed increased immobility on the forced-swim test - a measure of depressive-like behavior - 2 weeks following injury, while females developed depressive-like behavior at 3-week. Since the pathogenesis of chronic pain and depression may involve overlapping mechanisms including the activation of microglial cells, we explored glial cell changes in brain regions associated with pain processing and affect. Immunohistochemical analyses revealed that microglial cells were more numerous in female SNI mice in the contralateral ventral anterior cingulate cortex (ACC), a brain region important for pain processing and affect behavior, 2-week following surgery. Microglial cell activation was not different between any of the groups for the dorsal ACC or nucleus accumbens. Analysis of astrocyte density did not reveal any significant changes in glial fibrillary acidic protein (GFAP) staining in the ACC or nucleus accumbens. Overall, the current study characterized peripheral nerve injury induced depression-like behavior in male and female mice, which may be associated with different patterns of glial cell activation in regions important for pain processing and affect.

Prelimbic cortex glucocorticoid receptors regulate the stress-mediated inhibition of pain contagion in male mice.

Experiencing pain with a familiar individual can enhance one's own pain sensitivity, a process known as pain contagion. When experiencing pain with an unfamiliar individual, pain contagion is suppressed in males by activating the endocrine stress response. Here, we coupled a histological investigation with pharmacological and behavioral experiments to identify enhanced glucocorticoid receptor activity in the prelimbic subdivision of the medial prefrontal cortex as a candidate mechanism for suppressing pain contagion in stranger mice. Acute inhibition of glucocorticoid receptors in the prelimbic cortex was sufficient to elicit pain contagion in strangers, while their activation prevented pain contagion in cagemate dyads. Slice physiology recordings revealed enhanced excitatory transmission in stranger mice, an effect that was reversed by pre-treating mice with the corticosterone synthesis inhibitor metyrapone. Following removal from dyadic testing, stranger mice displayed enhanced affective-motivational pain behaviors when placed on an inescapable thermal stimulus, which were reversed by metyrapone. Together, our data suggest that the prelimbic cortex may play an integral role in modulating pain behavior within a social context and provide novel evidence towards the neural mechanism underlying the prevention of pain contagion.

Cage-lid hanging behavior as a translationally relevant measure of pain in mice.

ABSTRACT: The development of new analgesic drugs has been hampered by the inability to translate preclinical findings to humans. This failure is due in part to the weak connection between commonly used pain outcome measures in rodents and the clinical symptoms of chronic pain. Most rodent studies rely on the use of experimenter-evoked measures of pain and assess behavior under ethologically unnatural conditions, which limits the translational potential of preclinical research. Here, we addressed this problem by conducting an unbiased, prospective study of behavioral changes in mice within a natural homecage environment using conventional preclinical pain assays. Unexpectedly, we observed that cage-lid hanging, a species-specific elective behavior, was the only homecage behavior reliably impacted by pain assays. Noxious stimuli reduced hanging behavior in an intensity-dependent manner, and the reduction in hanging could be restored by analgesics. Finally, we developed an automated approach to assess hanging behavior. Collectively, our results indicate that the depression of hanging behavior is a novel, ethologically valid, and translationally relevant pain outcome measure in mice that could facilitate the study of pain and analgesic development.

Short-term memory impairment after isoflurane in mice is prevented by the α5 γ-aminobutyric acid type A receptor inverse agonist L-655,708.

BACKGROUND: Memory blockade is an essential component of the anesthetic state. However, postanesthesia memory deficits represent an undesirable and poorly understood adverse effect. Inhibitory α5 subunit-containing γ-aminobutyric acid subtype A receptors (α5GABAA) are known to play a critical role in memory processes and are highly sensitive to positive modulation by anesthetics. We postulated that inhibiting the activity of α5GABAA receptors during isoflurane anesthesia would prevent memory deficits in the early postanesthesia period. METHODS: Mice were pretreated with L-655,708, an α5GABAA receptor-selective inverse agonist, or vehicle. They were then exposed to isoflurane for 1 h (1.3%, or 1 minimum alveolar concentration, or air-oxygen control). Then, either 1 or 24 h later, mice were conditioned in fear-associated contextual and cued learning paradigms. In addition, the effect of L-655,708 on the immobilizing dose of isoflurane was studied. Motor coordination, sedation, anxiety, and the concentration of isoflurane in the brain at 5 min, 1 h, and 24 h after isoflurane were also examined. RESULTS: Motor and sensory function recovered within minutes after termination of isoflurane administration. In contrast, a robust deficit in contextual fear memory persisted for at least 24 h. The α5GABAA receptor inverse agonist, L-655,708, completely prevented memory deficits without changing the immobilizing dose of isoflurane. Trace concentrations of isoflurane were measured in the brain 24 h after treatment. CONCLUSIONS: Memory deficits occurred long after the sedative, analgesic, and anxiolytic effects of isoflurane subsided. L-655,708 prevented memory deficit, suggesting that an isoflurane interaction at α5GABAA receptors contributes to memory impairment during the early postanesthesia period.

D1 receptors in the anterior cingulate cortex modulate basal mechanical sensitivity threshold and glutamatergic synaptic transmission.

The release of dopamine (DA) into target brain areas is considered an essential event for the modulation of many physiological effects. While the anterior cingulate cortex (ACC) has been implicated in pain related behavioral processes, DA modulation of synaptic transmission within the ACC and pain related phenotypes remains unclear. Here we characterized a Crispr/Cas9 mediated somatic knockout of the D1 receptor (D1R) in all neuronal subtypes of the ACC and find reduced mechanical thresholds, without affecting locomotion and anxiety. Further, the D1R high-efficacy agonist SKF 81297 and low efficacy agonist (±)-SKF-38393 inhibit α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic receptor (AMPAR) currents in the ACC. Paradoxically, the D1R antagonists SCH-23390 and SCH 33961 when co-applied with D1R agonists produced a robust short-term synergistic depression of AMPAR currents in the ACC, demonstrating an overall inhibitory role for D1R ligands. Overall, our data indicate that absence of D1Rs in the ACC enhanced peripheral sensitivity to mechanical stimuli and D1R activation decreased glutamatergic synaptic transmission in ACC neurons.

The dichotomous role of epiregulin in pain.

It has recently been shown that epidermal growth factor receptor (EGFR) contributes to the pathogenesis of pain. We scanned genetic markers within genes coding for receptors of the EGFR family (EGFR, ERBB2, ERBB3, and ERBB4) and their ligands (AREG, BTC, EGF, EPGN, EREG, HBEGF, MUC4, NRG1, NRG2, NRG3, NRG4, and TGFA) for association with self-reported pain intensity in patients with chronic facial pain who participated in the Orofacial Pain: Prospective Evaluation and Risk Assessment (OPPERA) cohort. We found that only epiregulin (EREG) was associated with pain. The strongest effect was observed for a minor allele at rs6836436 in EREG, which was associated with lower chronic pain intensity. However, the same allele was associated with higher facial pain intensity among cases with recent onset of facial pain. Similar trends were observed in an independent cohort of UK Biobank (UKB) where the minor allele at rs6836436 was associated with a higher number of acute pain sites but a lower number of chronic pain sites. Expression quantitative trait loci analyses established rs6836436 as a loss-of-function variant of EREG. Finally, we investigated the functional role of EREG using mouse models of chronic and acute pain. Injecting mice with an EREG monoclonal antibody reversed established mechanosensitivity in the complete Freund's adjuvant and spared nerve injury models of chronic pain. However, the EREG monoclonal antibody prolonged allodynia when administered during the development of complete Freund's adjuvant-induced mechanosensitivity and enhanced pain behavior in the capsaicin model of acute pain.

Sex-specific effects of the histone variant H2A.Z on fear memory, stress-enhanced fear learning and hypersensitivity to pain.

Emerging evidence suggests that histone variants are novel epigenetic regulators of memory, whereby histone H2A.Z suppresses fear memory. However, it is not clear if altered fear memory can also modify risk for PTSD, and whether these effects differ in males and females. Using conditional-inducible H2A.Z knockout (cKO) mice, we showed that H2A.Z binding is higher in females and that H2A.Z cKO enhanced fear memory only in males. However, H2A.Z cKO improved memory on the non-aversive object-in-place task in both sexes, suggesting that H2A.Z suppresses non-stressful memory irrespective of sex. Given that risk for fear-related disorders, such as PTSD, is biased toward females, we examined whether H2A.Z cKO also has sex-specific effects on fear sensitization in the stress-enhanced fear learning (SEFL) model of PTSD, as well as associated changes in pain sensitivity. We found that H2A.Z cKO reduced stress-induced sensitization of fear learning and pain responses preferentially in female mice, indicating that the effects of H2A.Z depend on sex and the type of task, and are influenced by history of stress. These data suggest that H2A.Z may be a sex-specific epigenetic risk factor for PTSD susceptibility, with implications for developing sex-specific therapeutic interventions.