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

NMDA Receptor Activation Underlies the Loss of Spinal Dorsal Horn Neurons and the Transition to Persistent Pain after Peripheral Nerve Injury

Peripheral nerve lesions provoke apoptosis in the dorsal horn of the spinal cord. The cause of cell death, the involvement of neurons, and the relevance for the processing of somatosensory information are controversial. Here, we demonstrate in a mouse model of sciatic nerve injury that glutamate-induced neurodegeneration and loss of γ-aminobutyric acid (GABA)ergic interneurons in the superficial dorsal horn promote the transition from acute to chronic neuropathic pain. Conditional deletion of Grin1, the essential subunit of N-methyl-d-aspartate-type glutamate receptors (NMDARs), protects dorsal horn neurons from excitotoxicity and preserves GABAergic inhibition. Mice deficient in functional NMDARs exhibit normal nociceptive responses and acute pain after nerve injury, but this initial increase in pain sensitivity is reversible. Eliminating NMDARs fully prevents persistent pain-like behavior. Reduced pain in mice lacking proapoptotic Bax confirmed the significance of neurodegeneration. We conclude that NMDAR-mediated neuron death contributes to the development of chronic neuropathic pain

Relationship between galactomannan structure and physicochemical properties of films produced thereof

In this work five sources of galactomannans, Adenanthera pavonina, Cyamopsis tetragonolobus, Caesalpinia pulcherrima, Ceratonia siliqua and Sophora japonica, presenting mannose/galactose ratios of 1.3, 1.7, 2.9, 3.4 and 5.6, respectively, were used to produce galactomannan-based films. These films were characterized in terms of: water vapour, oxygen and carbon dioxide permeabilities (WVP, O 2 P and CO 2 P); moisture content, water solubility, contact angle, elongation-at-break (EB), tensile strength (TS) and glass transition temperature (T g ). Results showed that films properties vary according to the galactomannan source (different galactose distribution) and their mannose/galactose ratio. Water affinity of mannan and galactose chains and the intermolecular interactions of mannose backbone should also be considered being factors that affect films properties. This work has shown that knowing mannose/galactose ratio of galactomannans is possible to foresee galactomannan-based edible films properties.The authors thank the fellowship (SFRH/BPD/ 72753/2010) from the Fundação para a Ciência e Tecnologia (FCT, Portugal). The authors also thank the FCT Strategic Project of UID/BIO/ 04469/2013 unit, the project RECI/BBB-EBI/0179/2012 (FCOMP-010124-FEDER-027462) and the project BBioInd - Biotechnology and Bioengineering for improved Industrial and Agro-Food processes, REF. NORTE-07-0124-FEDER-000028 Co-funded by the Programa OperacionalRegionaldoNorte(ON.2–ONovoNorte),QREN,FEDER. Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico – FUNCAP, CE Brazil (CI1-0080-00055.01.00/13)

Differential Gene Expression in the EphA4 Knockout Spinal Cord and Analysis of the Inflammatory Response Following Spinal Cord Injury

Mice lacking the axon guidance molecule EphA4 have been shown to exhibit extensive axonal regeneration and functional recovery following spinal cord injury. To assess mechanisms by which EphA4 may modify the response to neural injury a microarray was performed on spinal cord tissue from mice with spinal cord injury and sham injured controls. RNA was purified from spinal cords of adult EphA4 knockout and wild-type mice four days following lumbar spinal cord hemisection or laminectomy only and was hybridised to Affymetrix All-Exon Array 1.0 GeneChips™. While subsequent analyses indicated that several pathways were altered in EphA4 knockout mice, of particular interest was the attenuated expression of a number of inflammatory genes, including Arginase 1, expression of which was lower in injured EphA4 knockout compared to wild-type mice. Immunohistological analyses of different cellular components of the immune response were then performed in injured EphA4 knockout and wildtype spinal cords. While numbers of infiltrating CD3+ T cells were low in the hemisection model, a robust CD11b+ macrophage/microglial response was observed post-injury. There was no difference in the overall number or spread of macrophages/activated microglia in injured EphA4 knockout compared to wild-type spinal cords at 2, 4 or 14 days post-injury, however a lower proportion of Arginase-1 immunoreactive macrophages/activated microglia was observed in EphA4 knockout spinal cords at 4 days post-injury. Subtle alterations in the neuroinflammatory response in injured EphA4 knockout spinal cords may contribute to the regeneration and recovery observed in these mice following injury

Silencing Nociceptor Neurons Reduces Allergic Airway Inflammation

Lung nociceptors initiate cough and bronchoconstriction. To elucidate if these fibers also contribute to allergic airway inflammation, we stimulated lung nociceptors with capsaicin and observed increased neuropeptide release and immune cell infiltration. In contrast, ablating Nav1.8(+) sensory neurons or silencing them with QX-314, a charged sodium channel inhibitor that enters via large-pore ion channels to specifically block nociceptors, substantially reduced ovalbumin- or house-dust-mite-induced airway inflammation and bronchial hyperresponsiveness. We also discovered that IL-5, a cytokine produced by activated immune cells, acts directly on nociceptors to induce the release of vasoactive intestinal peptide (VIP). VIP then stimulates CD4(+) and resident innate lymphoid type 2 cells, creating an inflammatory signaling loop that promotes allergic inflammation. Our results indicate that nociceptors amplify pathological adaptive immune responses and that silencing these neurons with QX-314 interrupts this neuro-immune interplay, revealing a potential new therapeutic strategy for asthma

Toxin-Based Models to Investigate Demyelination and Remyelination.

Clinical myelin diseases, and our best experimental approximations, are complex entities in which demyelination and remyelination proceed unpredictably and concurrently. These features can make it difficult to identify mechanistic details. Toxin-based models offer lesions with predictable spatiotemporal patterns and relatively discrete phases of damage and repair: a simpler system to study the relevant biology and how this can be manipulated. Here, we discuss the most widely used toxin-based models, with a focus on lysolecithin, ethidium bromide, and cuprizone. This includes an overview of their respective mechanisms, strengths, and limitations and step-by-step protocols for their use

Spatio-Temporal Progression of Grey and White Matter Damage Following Contusion Injury in Rat Spinal Cord

Cellular mechanisms of secondary damage progression following spinal cord injury remain unclear. We have studied the extent of tissue damage from 15 min to 10 weeks after injury using morphological and biochemical estimates of lesion volume and surviving grey and white matter. This has been achieved by semi-quantitative immunocytochemical methods for a range of cellular markers, quantitative counts of white matter axonal profiles in semi-thin sections and semi-quantitative Western blot analysis, together with behavioural tests (BBB scores, ledged beam, random rung horizontal ladder and DigiGait™ analysis). We have developed a new computer-controlled electronic impactor based on a linear motor that allows specification of the precise nature, extent and timing of the impact. Initial (15 min) lesion volumes showed very low variance (1.92±0.23 mm3, mean±SD, n = 5). Although substantial tissue clearance continued for weeks after injury, loss of grey matter was rapid and complete by 24 hours, whereas loss of white matter extended up to one week. No change was found between one and 10 weeks after injury for almost all morphological and biochemical estimates of lesion size or behavioural methods. These results suggest that previously reported apparent ongoing injury progression is likely to be due, to a large extent, to clearance of tissue damaged by the primary impact rather than continuing cell death. The low variance of the impactor and the comprehensive assessment methods described in this paper provide an improved basis on which the effects of potential treatment regimes for spinal cord injury can be assessed

Genome-wide association studies with experimental validation identify a protective role for B lymphocytes against chronic post-surgical pain

Background: Chronic post-surgical pain (CPSP) significantly impacts patients' recovery and quality of life. Although environmental risk factors are well-established, genetic risk remains less understood. Methods: A meta-analysis of genome-wide association studies followed by partitioned heritability was performed on 1350 individuals across five surgery types: hysterectomy, mastectomy, abdominal, hernia, and knee. In subsequent animal studies, withdrawal thresholds to evoked mechanical stimulation were measured in Rag1 null mutant and wild-type mice after plantar incision and laparotomy. Cell sorting by flow cytometry tracked recruitment of immune cell types. Results: We discovered 77 genome-wide significant single-nucleotide polymorphism (SNP) hits, distributed among 24 loci and 244 genes. Meta-analysis of all cohorts estimated a SNP-based narrow-sense heritability for CPSP at ∼39%, indicating a substantial genetic contribution. Partitioned heritability analysis across a wide variety of tissues revealed enrichment of heritability in immune system-related genes, particularly those associated with B and T cells. Rag1 null mutant mice lacking both T and B cells exhibited exacerbated and prolonged allodynia up to 42 days after surgery, which was rescued by B-cell transfer. Recruitment patterns of B cells but not T cells differed significantly during the first 7 days after injury in the footpad, lymph nodes, and dorsal root ganglia. Conclusions: These findings suggest a key protective role for the adaptive immune system in the development of chronic post-surgical pain