Protease-Induced Excitation of Dorsal Root Ganglion Neurons in Response to Acute Perturbation of the Gut Microbiota Is Associated With Visceral and Somatic Hypersensitivity.

BACKGROUND & AIMS: Abdominal pain is a major symptom of diseases that are associated with microbial dysbiosis, including irritable bowel syndrome and inflammatory bowel disease. Germ-free mice are more prone to abdominal pain than conventionally housed mice, and reconstitution of the microbiota in germ-free mice reduces abdominal pain sensitivity. However, the mechanisms underlying microbial modulation of pain remain elusive. We hypothesized that disruption of the intestinal microbiota modulates the excitability of peripheral nociceptive neurons. METHODS: In vivo and in vitro assays of visceral sensation were performed on mice treated with the nonabsorbable antibiotic vancomycin (50 µg/mL in drinking water) for 7 days and water-treated control mice. Bacterial dysbiosis was verified by 16s rRNA analysis of stool microbial composition. RESULTS: Treatment of mice with vancomycin led to an increased sensitivity to colonic distension in vivo and in vitro and hyperexcitability of dorsal root ganglion (DRG) neurons in vitro, compared with controls. Interestingly, hyperexcitability of DRG neurons was not restricted to those that innervated the gut, suggesting a widespread effect of gut dysbiosis on peripheral pain circuits. Consistent with this, mice treated with vancomycin were more sensitive than control mice to thermal stimuli applied to hind paws. Incubation of DRG neurons from naive mice in serum from vancomycin-treated mice increased DRG neuron excitability, suggesting that microbial dysbiosis alters circulating mediators that influence nociception. The cysteine protease inhibitor E64 (30 nmol/L) and the protease-activated receptor 2 (PAR-2) antagonist GB-83 (10 µmol/L) each blocked the increase in DRG neuron excitability in response to serum from vancomycin-treated mice, as did the knockout of PAR-2 in NaV1.8-expressing neurons. Stool supernatant, but not colonic supernatant, from mice treated with vancomycin increased DRG neuron excitability via cysteine protease activation of PAR-2. CONCLUSIONS: Together, these data suggest that gut microbial dysbiosis alters pain sensitivity and identify cysteine proteases as a potential mediator of this effect.

Advanced Dynamic Weight Bearing as an Observer-independent Measure of Hyperacute Hypersensitivity in Mice.

Background: Standard methods assessing pain in rodents are often observer dependent, potentially resulting in biased outcomes. Advanced dynamic weight bearing (ADWB) offers an observer-independent approach that can provide objective, reliable data in preclinical pain research. Aims: The aim of this study was to characterize the use of ADWB in assessing murine responses to allyl isothiocyanate (AITC)-induced hyperacute hypersensitivity and identify best practices for use of the device. Methods: Male C57BL/6J mice received intraplantar injections of saline or 0.1% AITC solution and were assessed using the ADWB system; simultaneous observer-dependent durations of paw licking and biting were measured. ADWB data were analyzed using the proprietary software from Bioseb and correlated to observer-dependent results, with parameters assessed to optimize data collected. Results: ADWB detected pain-directed changes in weight and surface area distribution in AITC-treated mice, with paw weight and surface area placement correlating to paw licking and biting. Optimization of adjustable threshold parameters allowed for reduced coefficients of variability and increased duration of validated data. Conclusions: The ADWB assay provides an efficient and unbiased measure of chemical-induced hyperacute hypersensitivity in mice. ADWB detection parameters influence amount of validated data and variability, a consideration for data analysis in future studies.

Contexte: Les méthodes standard d'évaluation de la douleur chez les rongeurs dépendent souvent de l'observateur, ce qui peut fausser les résultats. La mise en charge dynamique avancée offre une approche indépendante de l'observateur qui peut fournir des données objectives et fiables dans la recherche préclinique sur la douleur.Objectifs: L'objectif de cette étude était de caractériser l'utilisation de la mise en charge dynamique avancée dans l'évaluation des réponses murines à l'hypersensibilité hyperaiguë induite par l'isothiocyanate d'allyle et de répertorier les meilleures pratiques d'utilisation de l'appareil.Méthodes: Des souris C57BL/6J mâles ont reçu des injections intraplantaires de solution saline ou de solution d'isothiocyanate d'allyle à 0,1 % et ont été évaluées à l'aide du système de mise en charge dynamique avancée; les durées simultanées de léchage et de morsure des pattes, dépendantes de l'observateur, ont été mesurées. Les données obtenues par la mise en charge dynamique avancée ont été analysées à l'aide du logiciel propriétaire de Bioseb et corrélées aux résultats dépendants de l'observateur, avec des paramètres évalués pour optimiser les données collectées.Résultats: L'essai réalisé à l'aide de la mise en charge dynamique avancée a détecté des changements de poids et de distribution de surface liés à la douleur chez les souris traitées à l'isothiocyanate d'allyle, le poids des pattes et le placement de la surface étant corrélés au léchage et à la morsure des pattes. L'optimisation des paramètres de seuil ajustables a permis de réduire les coefficients de variabilité et d'augmenter la durée des données validées.Conclusion: L'essai réalisé à l'aide de la mise en charge dynamique avancée fournit une mesure efficace et impartiale de l'hypersensibilité hyperaiguë induite par les produits chimiques chez la souris. Les paramètres de détection du système de mise en charge dynamique avancée influencent la quantité de données validées et la variabilité, ce qui doit être pris en compte pour l'analyse des données dans les études futures.

Spinal Cord Injury in the Mouse Using the Infinite Horizon Spinal Cord Impactor.

Death of central nervous system neurons is a critical feature of spinal cord injury (SCI). The Infinite Horizon spinal cord impactor can be used to create both contusion and compression SCI, with a high degree of reproducibility. The device can also be positioned at different locations along the spinal cord to evaluate how the location of injury may alter neuronal death and functional recovery. A mouse with a successful SCI can experience a period of loss of bladder function, weight loss, and paralysis of the hind limbs, with more severe injuries resulting in further deficits. This chapter describes the surgical protocol along with pre- and postoperative care, as well as mitigation strategies for any setbacks that might occur.

Skin-resident dendritic cells mediate postoperative pain via CCR4 on sensory neurons.

Inflammatory pain, such as hypersensitivity resulting from surgical tissue injury, occurs as a result of interactions between the immune and nervous systems with the orchestrated recruitment and activation of tissue-resident and circulating immune cells to the site of injury. Our previous studies identified a central role for Ly6Clow myeloid cells in the pathogenesis of postoperative pain. We now show that the chemokines CCL17 and CCL22, with their cognate receptor CCR4, are key mediators of this response. Both chemokines are up-regulated early after tissue injury by skin-resident dendritic and Langerhans cells to act on peripheral sensory neurons that express CCR4. CCL22, and to a lesser extent CCL17, elicit acute mechanical and thermal hypersensitivity when administered subcutaneously; this response abrogated by pharmacological blockade or genetic silencing of CCR4. Electrophysiological assessment of dissociated sensory neurons from naïve and postoperative mice showed that CCL22 was able to directly activate neurons and enhance their excitability after injury. These responses were blocked using C 021 and small interfering RNA (siRNA)-targeting CCR4. Finally, our data show that acute postoperative pain is significantly reduced in mice lacking CCR4, wild-type animals treated with CCR4 antagonist/siRNA, as well as transgenic mice depleted of dendritic cells. Together, these results suggest an essential role for the peripheral CCL17/22:CCR4 axis in the genesis of inflammatory pain via direct communication between skin-resident dendritic cells and sensory neurons, opening therapeutic avenues for its control.

Spinal cord injury in mice affects central and peripheral pathology in a severity-dependent manner.

ABSTRACT: Chronic pain is a common medical complication experienced by those living with spinal cord injury (SCI) and leads to worsened quality of life. The pathophysiology of SCI pain is poorly understood, hampering the development of safe and efficacious therapeutics. We therefore sought to develop a clinically relevant model of SCI with a strong pain phenotype and characterize the central and peripheral pathology after injury. A contusion (50 kdyn) injury, with and without sustained compression (60 seconds) of the spinal cord, was performed on female C57BL/6J mice. Mice with compression of the spinal cord exhibited significantly greater heat and mechanical hypersensitivity starting at 7 days postinjury, concomitant with reduced locomotor function, compared with those without compression. Immunohistochemical analysis of spinal cord tissue revealed significantly less myelin sparing and increased macrophage activation in mice with compression compared with those without. As measured by flow cytometry, immune cell infiltration and activation were significantly greater in the spinal cord (phagocytic myeloid cells and microglia) and dorsal root ganglia (Ly6C+ monocytes) after compression injury. We also decided to investigate the gastrointestinal microbiome, as it has been shown to be altered in patients with SCI and has recently been shown to play a role in immune system maturation and pain. We found increased dysbiosis of the gastrointestinal microbiome in an injury severity-dependent manner. The use of this contusion-compression model of SCI may help advance the preclinical assessment of acute and chronic SCI pain and lead to a better understanding of mechanisms contributing to this pain.

The gut-brain axis and beyond: Microbiome control of spinal cord injury pain in humans and rodents.

Spinal cord injury (SCI) is a devastating injury to the central nervous system in which 60 to 80% of patients experience chronic pain. Unfortunately, this pain is notoriously difficult to treat, with few effective options currently available. Patients are also commonly faced with various compounding injuries and medical challenges, often requiring frequent hospitalization and antibiotic treatment. Change in the gut microbiome from the "normal" state to one of imbalance, referred to as gut dysbiosis, has been found in both patients and rodent models following SCI. Similarities exist in the bacterial changes observed after SCI and other diseases with chronic pain as an outcome. These changes cause a shift in the regulation of inflammation, causing immune cell activation and secretion of inflammatory mediators that likely contribute to the generation/maintenance of SCI pain. Therefore, correcting gut dysbiosis may be used as a tool towards providing patients with effective pain management and improved quality of life.

Chronic mechanical hypersensitivity in experimental autoimmune encephalomyelitis is regulated by disease severity and neuroinflammation.

Chronic pain severely affects quality of life in more than half of people living with multiple sclerosis (MS). A commonly-used model of MS, experimental autoimmune encephalomyelitis (EAE), typically presents with hindlimb paralysis, neuroinflammation and neurodegeneration. However, this paralysis may hinder the use of pain behavior tests, with no apparent hypersensitivity observed post-peak disease. We sought to adapt the classic actively-induced EAE model to optimize its pain phenotype. EAE was induced with MOG35-55/CFA and 100-600 ng pertussis toxin (PTX), and mice were assessed for mechanical, cold and thermal sensitivity over a 28-day period. Spinal cord tissue was collected at 14 and 28 days post-injection to assess demyelination and neuroinflammation. Only mice treated with 100 ng PTX exhibited mechanical hypersensitivity. Hallmarks of disease pathology, including demyelination, immune cell recruitment, cytokine expression, glial activation, and neuronal damage were higher in EAE mice induced with moderate (200 ng) doses of pertussis toxin, compared to those treated with low (100 ng) levels. Immunostaining demonstrated activated astrocytes and myeloid/microglial cells in both EAE groups. These results indicate that a lower severity of EAE disease may allow for the study of pain behaviors while still presenting with disease pathology. By using this modified model, researchers may better study the mechanisms underlying pain.

γδ T Cells Modulate Myeloid Cell Recruitment but Not Pain During Peripheral Inflammation.

Circulating immune cells, which are recruited to the site of injury/disease, secrete various inflammatory mediators that are critical to nociception and pain. The role of tissue-resident immune cells, however, remains poorly characterized. One of the first cells to be activated in peripheral tissues following injury are γδT cells, which serve important roles in infection, disease, and wound healing. Using a mouse line lacking these cells, we sought to identify their contribution to inflammatory pain. Three distinct models of peripheral inflammatory pain were used: intraplantar injection of formalin (spontaneous inflammatory pain), incisional wound (acute inflammatory pain), and intraplantar injection of complete Freund's adjuvant (chronic inflammatory pain). Our results show that absence of γδT cells does not alter baseline sensitivity, nor does it result in changes to mechanical or thermal hypersensitivity after tissue injury. Myeloid cell recruitment did show differential changes between models of acute and chronic inflammatory pain. These results were consistent in both male and female mice, suggesting that there are no sex differences in these outcomes. This comprehensive characterization suggests that γδT cells do not contribute to basal sensitivity or the development and maintenance of inflammatory pain.

Skin-Resident γδ T Cells Exhibit Site-Specific Morphology and Activation States.

Skin-resident γδ T cells play an important role in maintaining the immune barrier at the epithelial surface. Their roles in wound healing, regulation of immune response to injury, and reepithelialization have been characterized extensively in the mouse, though their function in human skin remains largely unknown. Human skin-resident γδ T cells sparsely populate the skin and are often small and rounded in appearance. Those in the mouse ear and back, which line the dermal barrier, are highly arborized cells with many processes extending from the cell body. To date, these cells have been studied primarily in the mouse ear and back; however, it is important to further identify and characterize γδ T cells in other body sites to better understand their function and study their contribution to injury and disease. We developed a novel method to visualize these cells in the skin (whole-mount and cryosections) that when combined with flow cytometry allowed us to assess differences in skin-resident γδ T cell numbers, morphology, and activation state in the ear, back, and footpad (chosen for their importance in immunological and pain research). In comparing cell length, number of dendritic processes, and expression of the activation marker CD69, we found that γδ T cell morphology and activation states vary significantly among the three tissue environments. Specifically, γδ T cells in the footpad are smaller, have fewer processes, and show the highest levels of activation compared to back- and ear-resident cells. Our observations suggest that our understanding of skin-resident γδ T cell functionality, drawn from the experiments performed in the ear and back tissue, may not be applicable to all skin environments. The footpad-resident cells also more closely resemble γδ T cells in human skin, suggesting that cells in this tissue environment may serve as a better translational model when studying γδ T cell function/activity.