measuring-changes-tactile-sensitivity-hind-paw-mice-using-an

Measuring Changes in Tactile Sensitivity in the Hind Paw of Mice Using an Electronic von Frey Apparatus

Measuring inflammation-induced changes in thresholds of hind paw withdrawal from mechanical pressure is a useful technique to assess changes in pain ...

isolation-infiltrating-leukocytes-from-mouse-skin-using-enzymatic

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

Macalester College

University of Minnesota

Mast cells mediate allergies, hypersensitivities, host defense, and venom neutralization. An area of recent interest is the contribution of mast cells to inflammatory pain. Here we found that specific, local activation of mast cells produced plantar hyperalgesia in mice. Basic secretagogue compound 48/80 induced plantar mast cell degranulation accompanied by thermal hyperalgesia, tissue edema, and neutrophil influx in the hindpaws of ND4 Swiss mice. Blocking mast cell degranulation, neutrophil extravasation, and histamine signaling abrogated these responses. Compound 48/80 also produced edema, pain, and neutrophil influx in WT C57BL/6 but not in genetically mast cell-deficient C57BL/6-Kit(W-sh)(/)(W-sh) mice. These responses were restored following plantar reconstitution with bone marrow-derived cultured mast cells.

Mast cell degranulation mediates compound 48/80-induced hyperalgesia in mice.

The interplay among pain, allergy and dysregulated inflammation promises to yield significant conceptual advances in immunology and chronic pain. Hapten-mediated contact hypersensitivity reactions are used to model skin allergies in rodents but have not been utilized to study associated changes in pain perception in the affected skin. Here we characterized changes in mechanical hyperalgesia in oxazolone-sensitized female mice challenged with single and repeated labiar skin exposure to oxazolone. Female mice were sensitized with topical oxazolone on their flanks and challenged 1-3 times on the labia. We then measured mechanical sensitivity of the vulvar region with an electronic pressure meter and evaluated expression of inflammatory genes, leukocyte influx and levels of innervation in the labiar tissue. Oxazolone-sensitized mice developed vulvar mechanical hyperalgesia after a single labiar oxazolone challenge. Hyperalgesia lasted up to 24 hours along with local influx of neutrophils, upregulation of inflammatory cytokine gene expression, and increased density of cutaneous labiar nerve fibers. Three daily oxazolone challenges produced vulvar mechanical hyperalgesic responses and increases in nerve density that were detectable up to 5 days post-challenge even after overt inflammation resolved. This persistent vulvar hyperalgesia is resonant with vulvodynia, an understudied chronic pain condition that is remarkably prevalent in 18-60 year-old women. An elevated risk for vulvodynia has been associated with a history of environmental allergies. Our pre-clinical model can be readily adapted to regimens of chronic exposures and long-term assessment of vulvar pain with and without concurrent inflammation to improve our understanding of mechanisms underlying subsets of vulvodynia and to develop new therapeutics for this condition.

Contact hypersensitivity to oxazolone provokes vulvar mechanical hyperalgesia in mice.

Neuro-inflammatory circuits in the tissue regulate the complex pathophysiology of pain. Protective nociceptive pain serves as an early warning system against noxious environmental stimuli. Tissue-resident mast cells orchestrate the increased thermal sensitivity following injection of basic secretagogue compound 48/80 in the hind paw tissues of ND4 mice. Here we investigated the effects of pre-treatment with TNF-α neutralizing antibody on compound 48/80-provoked thermal hyperalgesia.

TNF-alpha neutralizing antibody blocks thermal sensitivity induced by compound 48/80-provoked mast cell degranulation.

Mast cells are important first responders in protective pain responses that provoke withdrawal from intense, noxious environmental stimuli, in part because of their sentinel location in tissue-environment interfaces. In chronic pain disorders, the proximity of mast cells to nerves potentiates critical molecular cross-talk between these two cell types that results in their synergistic contribution to the initiation and propagation of long-term changes in pain responses via intricate signal networks of neurotransmitters, cytokines and adhesion molecules. Both in rodent models of inflammatory pain and chronic pain disorders, as well as in increasing evidence from the clinic, it is abundantly clear that understanding the mast cell-mediated mechanisms underlying protective and maladaptive pain cascades will lead to improved understanding of mast cell biology as well as the development of novel, targeted therapies for the treatment and management of debilitating pain conditions.

Mast cells: versatile gatekeepers of pain.

Cellular and molecular mediators of immune responses are increasingly implicated in acute and chronic pain pathophysiologies. Here we demonstrate that passive cutaneous IgE/Ag anaphylaxis provokes increased thermal sensitivity in the hind paw tissue of mice. The murine anti-DNP IgE antibodies SPE-7 and ɛ26 are known to induce differential cytokine production in bone marrow cultured mast cells in vitro without antigen challenge. We found a novel, antigen-dependent heterogeneity in the thermal pain responses elicited in the hind paws between SPE-7 and ɛ26 sensitized DNP-challenged mice. Mice experienced pronounced hind paw thermal sensitivity lasting 6h after DNP challenge when sensitized with SPE-7 but not ɛ26 IgE. The two IgE clones induced equivalent hind paw edema, neutrophil influx, cytokine production, and reduction in tissue histamine content in vivo, and bound to the same or overlapping epitopes on the DNP antigen in vitro. Therefore IgE antibodies against the same antigen can induce comparable inflammation, yet contribute to markedly different anaphylaxis-associated pain within an allergic response, suggesting that non-canonical IgE binding partners such as sensory neurons may play a role in allergy-related pain responses.

Clonal differences in IgE antibodies affect cutaneous anaphylaxis-associated thermal sensitivity in mice.

The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have higher transposon density (25%-50%) than euchromatic reference regions (3%-11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% versus 11%-27%). F element genes have larger coding spans, more coding exons, larger introns, and lower codon bias. Comparison of the Effective Number of Codons with the Codon Adaptation Index shows that, in contrast to the other species, codon bias in D. grimshawi F element genes can be attributed primarily to selection instead of mutational biases, suggesting that density and types of transposons affect the degree of local heterochromatin formation. F element genes have lower estimated DNA melting temperatures than D element genes, potentially facilitating transcription through heterochromatin. Most F element genes (~90%) have remained on that element, but the F element has smaller syntenic blocks than genome averages (3.4-3.6 versus 8.4-8.8 genes per block), indicating higher rates of inversion despite lower rates of recombination. Overall, the F element has maintained characteristics that are distinct from other autosomes in the Drosophila lineage, illuminating the constraints imposed by a heterochromatic milieu.

Drosophila Muller F Elements Maintain a Distinct Set of Genomic Properties Over 40 Million Years of Evolution.

Programmed death-1 (PD-1) promotes T cell tolerance. Despite therapeutically targeting this pathway for chronic infections and tumors, little is known about how different T cell subsets are affected during blockade. We examined PD-1/PD ligand 1 (PD-L1) regulation of self-antigen-specific CD4 and CD8 T cells in autoimmune-susceptible models. PD-L1 blockade increased insulin-specific effector CD4 T cells in type 1 diabetes. However, anergic islet-specific CD4 T cells were resistant to PD-L1 blockade. Additionally, PD-L1 was critical for induction, but not maintenance, of CD8 T cell intestinal tolerance. PD-L1 blockade enhanced functionality of effector T cells, whereas established tolerant or anergic T cells were not dependent on PD-1/PD-L1 signaling to remain unresponsive. This highlights the existence of Ag-experienced T cell subsets that do not rely on PD-1/PD-L1 regulation. These findings illustrate how positive treatment outcomes and autoimmunity development during PD-1/PD-L1 inhibition are linked to the differentiation state of a T cell.

ArticleTotal : 2	Year
Publication title Cited by 105	2013
Publication title Cited by 33	2016

Article	Year
Mast cell degranulation mediates compound 48/80-induced hyperalgesia in mice. Biochemical and biophysical research communications\| PubMed ID: 22828511	2012
Contact hypersensitivity to oxazolone provokes vulvar mechanical hyperalgesia in mice. PloS one\| PubMed ID: 24205293	2013
TNF-alpha neutralizing antibody blocks thermal sensitivity induced by compound 48/80-provoked mast cell degranulation. F1000Research\| PubMed ID: 24555087	2013
Mast cells: versatile gatekeepers of pain. Molecular immunology\| PubMed ID: 24666768	2015
Clonal differences in IgE antibodies affect cutaneous anaphylaxis-associated thermal sensitivity in mice. Immunology letters\| PubMed ID: 25149207	2014
Drosophila Muller F Elements Maintain a Distinct Set of Genomic Properties Over 40 Million Years of Evolution. G3 (Bethesda, Md.)\| PubMed ID: 25740935	2015
Cutting edge: identification of autoreactive CD4+ and CD8+ T cell subsets resistant to PD-1 pathway blockade. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 25769925	2015

Tijana Martinov

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