Journal: Journal of Neuroinflammation

Article Title: Morphine immunomodulation prolongs inflammatory and postoperative pain while the novel analgesic ZH853 accelerates recovery and protects against latent sensitization

doi: 10.1186/s12974-019-1480-x

Figure Lengend Snippet: Immunohistochemistry following CFA and drug. Tissue was taken 25 days after CFA in the CFA-then-drug paradigm and 21 days after CFA in the drug-then-CFA paradigm. a CFA then drug. Staining at this time point did not show higher microglial activation in any group, but pp38 was decreased by ZH853, and IL-1β and CGRP were increased by morphine relative to vehicle and ZH853. Astrocyte activation and P2X7Rs were increased in morphine-treated animals compared to ZH853-treated animals. b Drug then CFA. Microglial activation was greater in animals pretreated with morphine- versus vehicle- and ZH853-treated animals. pp38 was increased by morphine and decreased by ZH853, producing a significant difference between drugs. IL-1β was significantly decreased by ZH853 relative to morphine and vehicle. CGRPR staining was increased by morphine relative to vehicle- or ZH853-treated animals. Astrocyte activation was decreased by ZH853 relative to vehicle and morphine, and P2X7R was increased by morphine relative to vehicle and ZH853. n indicated in bar graphs. Error bars indicate SEM. One-way ANOVAs were performed for each marker. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: After two washes in PBS and blocking with 5% normal horse serum/0.3% Triton X-100, sections were incubated in the following primary antibodies: calcitonin gene-related peptide (CGRP) (rabbit, T-4032, Peninsula Labs, San Carlos, CA), glial fibrillary acidic protein monoclonal (GFAP) (mouse, Astro6 MA5-12023, ThermoFisher, Carlsbad, CA), Anti-CD11b/c (OX42) (rabbit, CBL1512-100UG, Millipore Sigma, St. Louis, MO), purinergic receptor 7 (P2X7R) (rabbit, #APR-008, Alomone Labs, Jerusalem, Israel), phosphorylated-p38 MAP kinase (pp38) (rabbit, #4511, Cell Signaling Technology, Danvers, MA), or interleukin-1beta (IL-1β) (rabbit, ab9787, Abcam, Cambridge, MA) and were incubated for 24 h at 4 °C on a slow rocker.

Techniques: Immunohistochemistry, Staining, Activation Assay, Marker

Journal: Journal of Neuroinflammation

Article Title: Morphine immunomodulation prolongs inflammatory and postoperative pain while the novel analgesic ZH853 accelerates recovery and protects against latent sensitization

doi: 10.1186/s12974-019-1480-x

Figure Lengend Snippet: Representative images of IHC from the Drug-then-CFA paradigm. Each analysis was done on images acquired with the same exposure, gain, and aperture across treatment groups, but exposure and gain were adjusted for each antibody. All images were taken on a × 10 objective and microglia were imaged at × 20 for the inset of OX42. OX42 labels microglia, pp38 labels phosphorylated p38 Map Kinase, IL-1β labels interleukin-1beta, CGRP labels α-calcitonin gene-related peptide, Astro6 labels glial fibrillary acidic protein (GFAP), and P2X7R labels the P2X purinoceptor 7

Article Snippet: After two washes in PBS and blocking with 5% normal horse serum/0.3% Triton X-100, sections were incubated in the following primary antibodies: calcitonin gene-related peptide (CGRP) (rabbit, T-4032, Peninsula Labs, San Carlos, CA), glial fibrillary acidic protein monoclonal (GFAP) (mouse, Astro6 MA5-12023, ThermoFisher, Carlsbad, CA), Anti-CD11b/c (OX42) (rabbit, CBL1512-100UG, Millipore Sigma, St. Louis, MO), purinergic receptor 7 (P2X7R) (rabbit, #APR-008, Alomone Labs, Jerusalem, Israel), phosphorylated-p38 MAP kinase (pp38) (rabbit, #4511, Cell Signaling Technology, Danvers, MA), or interleukin-1beta (IL-1β) (rabbit, ab9787, Abcam, Cambridge, MA) and were incubated for 24 h at 4 °C on a slow rocker.

Techniques:

Journal: Journal of Neuroinflammation

Article Title: Morphine immunomodulation prolongs inflammatory and postoperative pain while the novel analgesic ZH853 accelerates recovery and protects against latent sensitization

doi: 10.1186/s12974-019-1480-x

Figure Lengend Snippet: Regression analyses of IHC markers versus von Frey scores. Individual scores for both von Frey at the time point just prior to perfusion and integrated density (or cell count for pp38) from both paradigms of CFA and drug treatment were plotted for all drug groups. pp38 and P2X7R are consistently correlated with pain. P values are listed on each graph

Article Snippet: After two washes in PBS and blocking with 5% normal horse serum/0.3% Triton X-100, sections were incubated in the following primary antibodies: calcitonin gene-related peptide (CGRP) (rabbit, T-4032, Peninsula Labs, San Carlos, CA), glial fibrillary acidic protein monoclonal (GFAP) (mouse, Astro6 MA5-12023, ThermoFisher, Carlsbad, CA), Anti-CD11b/c (OX42) (rabbit, CBL1512-100UG, Millipore Sigma, St. Louis, MO), purinergic receptor 7 (P2X7R) (rabbit, #APR-008, Alomone Labs, Jerusalem, Israel), phosphorylated-p38 MAP kinase (pp38) (rabbit, #4511, Cell Signaling Technology, Danvers, MA), or interleukin-1beta (IL-1β) (rabbit, ab9787, Abcam, Cambridge, MA) and were incubated for 24 h at 4 °C on a slow rocker.

Techniques: Cell Counting

Journal: Journal of Neuroinflammation

Article Title: Morphine immunomodulation prolongs inflammatory and postoperative pain while the novel analgesic ZH853 accelerates recovery and protects against latent sensitization

doi: 10.1186/s12974-019-1480-x

Figure Lengend Snippet: Schematic of postulated mechanisms of microglial activation by morphine but not by ZH853. Proposed mechanisms by which morphine activates glia or enhances trauma-induced activation include (1) induction of neuronal CGRP that activates microglial CGRP receptors , (2) binding at toll-like receptors (TLRs) alone or in concert with damage- or pathogen-associated molecular patterns (DAMPs, PAMPS) , and (3) activation of purinergic P2X receptors including by upregulation through MOR (but see ). Each of these mechanisms causes phosphorylation of the mitogen-activated protein kinase (MAPK) p38 to increase transcription and translation of cytokines, including interleukin-1β (IL-1β , example shown), P2X7R activation also induces NOD-like receptor family, pyrin domain containing 3 (NLRP3) to become a complex (inflammasome) that contains and releases active caspase-1 which cleaves pro-IL-1β into the active cytokine IL-1β . IL-1β activates IL-1 receptors on astrocytes and neurons, further increasing inflammation. Ultimately, this proinflammatory pathway causes dorsal horn plasticity that leads to central sensitization and increased pain

Article Snippet: After two washes in PBS and blocking with 5% normal horse serum/0.3% Triton X-100, sections were incubated in the following primary antibodies: calcitonin gene-related peptide (CGRP) (rabbit, T-4032, Peninsula Labs, San Carlos, CA), glial fibrillary acidic protein monoclonal (GFAP) (mouse, Astro6 MA5-12023, ThermoFisher, Carlsbad, CA), Anti-CD11b/c (OX42) (rabbit, CBL1512-100UG, Millipore Sigma, St. Louis, MO), purinergic receptor 7 (P2X7R) (rabbit, #APR-008, Alomone Labs, Jerusalem, Israel), phosphorylated-p38 MAP kinase (pp38) (rabbit, #4511, Cell Signaling Technology, Danvers, MA), or interleukin-1beta (IL-1β) (rabbit, ab9787, Abcam, Cambridge, MA) and were incubated for 24 h at 4 °C on a slow rocker.

Techniques: Activation Assay, Binding Assay