Alexander H. Tuttle

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.

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 Freunds adjuvant, intraarticular kaolin-carrageenan, and laparotomy (post-operative pain) assays. The scales 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.

Spinal Cord Toll-Like Receptor 4 Mediates Inflammatory and Neuropathic Hypersensitivity in Male But Not Female Mice

The innate immune system is increasingly appreciated to play an important role in the mediation of chronic pain, and one molecule implicated in this process is the Toll-like receptor 4 (TLR4). Here, using pharmacological and genetic manipulations, we found that activating TLR4 in the spinal cord, with the agonist lipopolysaccharide (LPS), causes robust mechanical allodynia but only in male mice. Spinal LPS had no pain-producing effect in female mice. TLR4 also has a sex-specific role in inflammatory (complete Freunds adjuvant) and neuropathic (spared nerve injury) pain: pain behaviors were TLR4 dependent in males but TLR4 independent in females. The sex differences appear to be specific to the spinal cord, as LPS administered to the brain or the hindpaw produces equivalent allodynia in both sexes, and specific to pain, as intrathecal LPS produces equivalent hypothermia in both sexes. The involvement of TLR4 in pain behaviors in male mice is dependent on testosterone, as shown by gonadectomy and hormone replacement. We found no sex differences in spinal Tlr4 gene expression at baseline or after LPS, suggesting the existence of parallel spinal pain-processing circuitry in female mice not involving TLR4.

Remote Optogenetic Activation and Sensitization of Pain Pathways in Freely Moving Mice

We report a novel model in which remote activation of peripheral nociceptive pathways in transgenic mice is achieved optogenetically, without any external noxious stimulus or injury. Taking advantage of a binary genetic approach, we selectively targeted Nav1.8+ sensory neurons for conditional expression of channelrhodopsin-2 (ChR2) channels. Acute blue light illumination of the skin produced robust nocifensive behaviors, evoked by the remote stimulation of both peptidergic and nonpeptidergic nociceptive fibers as indicated by c-Fos labeling in laminae I and II of the dorsal horn of the spinal cord. A non-nociceptive component also contributes to the observed behaviors, as shown by c-Fos expression in lamina III of the dorsal horn and the expression of ChR2–EYFP in a subpopulation of large-diameter Nav1.8+ dorsal root ganglion neurons. Selective activation of Nav1.8+ afferents in vivo induced central sensitization and conditioned place aversion, thus providing a novel paradigm to investigate plasticity in the pain circuitry. Long-term potentiation was similarly evoked by light activation of the same afferents in isolated spinal cord preparations. These findings demonstrate, for the first time, the optical control of nociception and central sensitization in behaving mammals and enables selective activation of the same class of afferents in both in vivo and ex vivo preparations. Our results provide a proof-of-concept demonstration that optical dissection of the contribution of specific classes of afferents to central sensitization is possible. The high spatiotemporal precision offered by this non-invasive model will facilitate drug development and target validation for pain therapeutics.

Repeated Vulvovaginal Fungal Infections Cause Persistent Pain in a Mouse Model of Vulvodynia

The development of an animal model of vulvodynia indicates that recurrent candidiasis is a potential cause of the disorder. Progress in Parsing Vulvodynia, a Particularly Painful Malady The causes of some chronic pain syndromes have been mysterious. One of these is vulvodynia, in which the vulva becomes excruciatingly sensitive to touch. Now, Farmer and her colleagues reinforce one hypothesis as to its origins by showing that a vulvodynia-like state can be induced in mice by repeated yeast infections. The authors showed that two rounds of vulvar yeast infection had no effect on the response of mice to touch in the vulvar area, but after a third infection, 6 of 15 mice became hypersensitive. The vulvar tissue of hypersensitive mice was not inflamed or otherwise abnormal after microscopic inspection, but the affected mice did have particularly dense sympathetic and peptidergic vulvar innervations. A single, long infection could have the same effect and, as long as 70 days after the infection cleared up, the mice were hypersensitive to touch. Live yeast were not required, because injections of the yeast cell wall glucan zymosan produced the same chronic pain syndrome, as measured by hypersensitivity to touch. The re-creation of a vulvodynia-like state in mice by repeated infection with yeast raises key questions that need to be asked in further experiments. Does repeated exposure to infection induce hyperinnervation in some individuals? If so, what is the mechanistic basis of this effect? Is this innervation the cause of the chronic pain of vulvodynia? Some of these questions may be best asked in mice, but the ultimate goal is to understand this process in women well enough to effectively interfere with it. Provoked vestibulodynia, the most common form of vulvodynia (unexplained pain of the vulva), is a prevalent, idiopathic pain disorder associated with a history of recurrent candidiasis (yeast infections). It is characterized by vulvar allodynia (painful hypersensitivity to touch) and hyperinnervation. We tested whether repeated, localized exposure of the vulva to a common fungal pathogen can lead to the development of chronic pain. A subset of female mice subjected to recurrent Candida albicans infection developed mechanical allodynia localized to the vulva. The mice with allodynia also exhibited hyperinnervation with peptidergic nociceptor and sympathetic fibers (as indicated by increased protein gene product 9.5, calcitonin gene–related peptide, and vesicular monoamine transporter 2 immunoreactivity in the vaginal epithelium). Long-lasting behavioral allodynia in a subset of mice was also observed after a single, extended Candida infection, as well as after repeated vulvar (but not hind paw) inflammation induced with zymosan, a mixture of fungal antigens. The hypersensitivity and hyperinnervation were both present at least 3 weeks after the resolution of infection and inflammation. Our data show that infection can cause persistent pain long after its resolution and that recurrent yeast infection replicates important features of human provoked vulvodynia in the mouse.

Increasing placebo responses over time in U.s. clinical trials of neuropathic pain

Abstract Recent failures of clinical trials of novel analgesics designed to treat neuropathic pain have led to much speculation about the underlying reasons. One often discussed possibility is that the placebo response in these trials has increased in recent years, leading to lower separation between the drug and placebo arms. Whether this has indeed occurred has not yet been adequately addressed. Here, we extracted data from published randomized controlled trials (RCTs) of drugs for the treatment of chronic neuropathic pain over the years 1990 to 2013. We find that placebo responses have increased considerably over this period, but drug responses have remained stable, leading to diminished treatment advantage. This trend has been driven by studies conducted in the United States. Consideration of participant and study characteristics revealed that in the United States but not elsewhere, RCTs have increased in study size and length. These changes are associated with larger placebo response. Analysis of individual RCT time courses showed different kinetics for the treatment vs placebo responses, with the former evolving more quickly than the latter and plateauing, such that maximum treatment advantage was achieved within 4 weeks.

Social approach to pain in laboratory mice

Abstract It has been recently demonstrated that pain behavior in the mouse can be modulated by the presence of a conspecific, but what remains unclear is whether such pain behavior can serve the function of soliciting social approach. Using a novel social approach paradigm, we tested mice in various dyadic or triadic conditions, including “jailed” mice—some in pain via intraperitoneal injection of 0.9% acetic acid—and test mice free to approach or avoid the jailed mice. We observed a sex-specific effect whereby female, but not male, test mice approached a familiar same-sex conspecific in pain more frequently than an unaffected familiar or unfamiliar, but affected, conspecific. Despite a substantial literature emphasizing oxytocins role in affiliative and pair-bonding behavior, this effect was also observed in female mice lacking the oxytocin receptor, suggesting that pain-related social approach may not be mediated by oxytocin. Furthermore, we found that the frequency of contact by the test mouse was negatively correlated with the pain behavior of the jailed mouse, suggesting that proximity of a familiar unaffected conspecific may have analgesic properties.

Behavioral evidence for photophobia and stress-related ipsilateral head pain in transgenic Cacna1a mutant mice

&NA; We observed spontaneously emitted behaviors in 2 lines of Cacna1a mutant mice, suggesting that these mice experience stress‐induced lateralized head pain (ie, migraines). &NA; Migraine is a highly prevalent, disabling and complex episodic brain disorder whose pathogenesis is poorly understood, due in part to the lack of valid animal models. Here we report behavioral evidence of hallmark migraine features, photophobia and unilateral head pain, in transgenic knock‐in mice bearing human familial hemiplegic migraine, type 1 (FHM‐1) gain‐of‐function missense mutations (R192Q or S218L) in the Cacna1a gene encoding the CaV2.1 calcium channel &agr;1 subunit. Photophobia was demonstrated using a modified elevated plus maze in which the safe closed arms were brightly illuminated; mutant mice avoided the light despite showing no differences in the standard (anxiety) version of the test. Multiple behavioral measures suggestive of spontaneous head pain were found in 192Q mutants subjected to novelty and/or restraint stress. These behaviors were: (1) more frequent in mutant versus wildtype mice; (2) lateralized in mutant but not in wildtype mice; (3) more frequent in females versus males; and (4) dose‐dependently normalized by systemic administration of 2 different acute analgesics, rizatriptan and morphine. Furthermore, some of these behaviors were found to be more frequent and severe in 218L compared to 192Q mutants, consistent with the clinical presentation in humans. We suggest that Cacna1a transgenic mice can experience migraine‐related head pain and can thus serve as unique tools to study the pathogenesis of migraine and test novel antimigraine agents.

The Interaction Between Pain and Social Behavior in Humans and Rodents

Pain elicits behaviors in humans and nonhuman animals that serve as social cues. Pain behaviors serve a communicative function in humans, and this may be true as well in other animals. This review considers the current evidence for modulation of acute pain in different social contexts in humans and rodents, with a focus on dyadic social interactions. Increasing data supports the ability of social buffering, emotional contagion (a form of empathy), vicarious learning, and social stress to modulate pain sensitivity and pain behavior in mice and rats. As in humans, many of these social factors operate, and affect pain, in a sex-dependent manner. The development of a true social neuroscience of pain, with detailed explication of the underlying neurochemistry and genetics, now seems achievable.

The Nicotinic α6 Subunit Gene Determines Variability in Chronic Pain Sensitivity via Cross-inhibition of P2X2/3 Receptors

Finding that nicotinic receptors containing the α6 subunit, but not the α4, inhibit chronic pain points to a new set of potential therapeutic drugs. Which receptor underlies chronic pain? Pain, especially of the chronic variety, is not well controlled by current drugs, and recent clinical trials have been unsuccessful. By seeking genes with expression levels that correlate with a chronic pain–like test in mice, Wieskopf et al. show that we may have set our sights on the wrong target. Nicotinic receptors that contain the α6 subunit were highly expressed when chronic pain was low, and genetic experiments confirmed that this subunit is the cause. The α6 subunit was required for analgesia, whereas the α4 subunit—the target of recent drug development efforts—was not. A human genetic study showing that people with a certain allele in the α6 subunit gene are at increased risk of chronic pain lends confidence in the clinical relevance of these results. Chronic pain is a highly prevalent and poorly managed human health problem. We used microarray-based expression genomics in 25 inbred mouse strains to identify dorsal root ganglion (DRG)–expressed genetic contributors to mechanical allodynia, a prominent symptom of chronic pain. We identified expression levels of Chrna6, which encodes the α6 subunit of the nicotinic acetylcholine receptor (nAChR), as highly associated with allodynia. We confirmed the importance of α6* (α6-containing) nAChRs by analyzing both gain- and loss-of-function mutants. We find that mechanical allodynia associated with neuropathic and inflammatory injuries is significantly altered in α6* mutants, and that α6* but not α4* nicotinic receptors are absolutely required for peripheral and/or spinal nicotine analgesia. Furthermore, we show that Chrna6’s role in analgesia is at least partially due to direct interaction and cross-inhibition of α6* nAChRs with P2X2/3 receptors in DRG nociceptors. Finally, we establish the relevance of our results to humans by the observation of genetic association in patients suffering from chronic postsurgical and temporomandibular pain.