Journal: Molecular Pain

Article Title: Systemic administration of a β2-adrenergic receptor agonist reduces mechanical allodynia and suppresses the immune response to surgery in a rat model of persistent post-incisional hypersensitivity

doi: 10.1177/1744806921997206

Figure Lengend Snippet: Effects of β2 adrenergic receptor (AR) agonist on enhanced spinal microglia activation in a rat model of persistent postoperative pain. Sections of spinal cord were collected from rats 8 days following plantar incision and following treatment with DβH-saporin to deplete spinal noradrenergic terminals or control IgG-saporin. Rats were chronically administered clenbuterol (0.5 mg/kg, 2×/day, i.p.) or saline vehicle 6 days prior to and for 8 days after plantar incision. Depletion of spinal noradrenergic fibers was verified immunohistochemically with an antibody against dopamine β hydroxylase (DβH, (a)–(c)). Representative confocal images of IBA1-IR (blue, (d)–(f)) and phospho-p38 MAPK-IR (purple, (g)–(i)) in the ipsilateral spinal cord of incision rats. Localization of p38 MAPK in microglia was confirmed by colocalization with an antibody against the cell surface antigen CD11b (green, inset in (h)). Quantification of IBA1-IR in ipsilateral and contralateral spinal cord of rats with incision (j). Data represent mean ± SEM, n = 3 rats per group. Two way ANOVA indicated effect of group: p < 0.001 but not side p = 0.184 or interaction: p = 0.59 with SNK pairwise comparisons *p < 0.001 versus Incision+ DβH-saporin+ vehicle. Quantification of phospho-p38 MAPK microglial in the ipsilateral and contralateral spinal cord of rats with incision (k). Data represent mean ± SEM, n = 3 rats per group. Two way ANOVA indicate effect of group: p = 0.002 but not side p = 0.398 or interaction: p = 0.67 with SNK pairwise comparisons * p < 0.005 versus Incision+ DβH-saporin+ vehicle.

Article Snippet: We used a previously characterized antibody against β2 adrenergic receptors (AAR-016, β2-AR, 1:1000, rabbit anti-mouse, Alomone Labs; Jerusalem, Israel).

Techniques: Activation Assay

Journal: Molecular Pain

Article Title: Systemic administration of a β2-adrenergic receptor agonist reduces mechanical allodynia and suppresses the immune response to surgery in a rat model of persistent post-incisional hypersensitivity

doi: 10.1177/1744806921997206

Figure Lengend Snippet: Beta 2-adrenergic receptor immunoreactivity in the spinal cord of rats under naïve conditions and two days following plantar incision. Transverse section of L4 spinal cord of rat reacted with antibody against β2 adrenergic receptor ((a), β2-AR, green). There is a high density of immunoreactivity in cellular profiles throughout dorsal and ventral horn. There is also dense immunoreactivity in axon terminals within the lateral portion of the superficial laminae (arrow) and ependymal cells in the vicinity of the central canal (Arrowhead). Note lack of staining for β2-AR in motor neurons within the ventral horn (asterisk). Higher magnification confocal images show β2-AR-IR ((c), green) is present in a subpopulation of neurons ((d), NeuN, purple) in the dorsal spinal cord. Most β2-AR-IR cellular profiles colocalized with NeuN with the exception of a few non-neuronal profiles with morphology typical of microglia (arrows, (c)–(f)). β2-AR-IR non-neuronal cellular profiles in the spinal cord colocalized with the microglial marker IBA1 (red, (e) and (f)). Arrows in F indicate IBA1 negative neuronal cellular profiles. Representative images of β2 mRNA and DAPI in the dorsal spinal cord (g) with high power image showing colocalization with a subset of nuclei (h).

Article Snippet: We used a previously characterized antibody against β2 adrenergic receptors (AAR-016, β2-AR, 1:1000, rabbit anti-mouse, Alomone Labs; Jerusalem, Israel).

Techniques: Staining, Marker

Journal: Molecular Pain

Article Title: Systemic administration of a β2-adrenergic receptor agonist reduces mechanical allodynia and suppresses the immune response to surgery in a rat model of persistent post-incisional hypersensitivity

doi: 10.1177/1744806921997206

Figure Lengend Snippet: β2-adrenergic receptor immunoreactivity (β2AR-IR) in hindpaw of rats under naïve conditions and following plantar incision. Skin sections were obtained from the hind paw of naïve rats and incision rats two days following surgery. Sixteen-μm-thick sections were stained with antibodies against β2AR-IR (green, (a)–(c)), IBA1 (red, (d)–(f)) to label all monocytes/and macrophage, CD68 (blue, (g)–(i)) for activated M1 macrophage) and DAPI ((j) and (k)) to label all nuclei. β2-AR IR was present in keratinocytes of both naïve and incision rats. Two days following plantar incision there were increased β2-AR IR cellular profiles in predominantly the dermal layers of the skin. Higher magnification confocal images ((c), (f), (i), and (l)) indicate colocalization of β2-AR in IBA1 + cells and a subset of which express CD68-IR. Note in naïve skin IBA1-IR was primarily present at the epidermal/dermal interface and had reduced dermal cellularity (DAPI + cells) compared to skin adjacent to the wound in incision rats.

Article Snippet: We used a previously characterized antibody against β2 adrenergic receptors (AAR-016, β2-AR, 1:1000, rabbit anti-mouse, Alomone Labs; Jerusalem, Israel).

Techniques: Staining

Journal: Cell reports

Article Title: β2-adrenergic receptor signaling regulates metabolic pathways critical to myeloid-derived suppressor cell function within the TME

doi: 10.1016/j.celrep.2021.109883

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: anti–mouse β2-AR (Catalog orb15065) , Biorbyt , Catalog orb15065.

Techniques: Recombinant, RNA Sequencing, Knock-Out, Software

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Chronic psychosocial stress compromises the immune response and endochondral ossification during bone fracture healing via β-AR signaling

doi: 10.1073/pnas.1819218116

Figure Lengend Snippet: Immune response in the fracture callus 3 d after femur osteotomy following 19 d of CSC housing. (A) Quantification of Ly6G+, (B) CD8+, and (C) F4/80+ cells in the fracture callus of SHC and CSC mice on d3 after fracture. (D) Representative image of fracture callus stained for Ly6G (scale bars: 50 µm), (E) CD8 (scale bars: 50 µm), and (F) F4/80 (scale bars: 50 µm) of SHC and CSC mice. (G) Relative gene expression of TH analyzed by qPCR from fracture hematoma homogenates. Values were normalized to B2M. (H) Representative image of fracture callus stained for TH in SHC and CSC mice on d3 after femur osteotomy. (Scale bars: 50 µm.) (I) Representative images of fracture calli from SHC (Left) and CSC (Right) mice on d3 after fracture, double-stained for TH and Ly6G: TH (Upper), Ly6G (Middle), merge (Lower). (Scale bars: 100 µm; inlay: 50 µm.) Data are presented as mean + SD. SHC, n = 4–7; CSC, n = 4–7. *0.05 > P > 0.01.

Article Snippet: Immunofluorescence staining for TH and β2-AR, as well as double stainings for TH and Ly6G and β2-AR and ENPP1 was performed using the following antibodies: rabbit anti-mouse β2-AR antibody (sc-569, Santa Cruz), rabbit anti-mouse TH antibody (AB152, Millipore), rat anti-mouse Ly6G (127632, BioLegend), goat anti-mouse ENPP1/PC1 (SAB 2500355, Sigma), goat-anti rabbit IgG-biotin (sc-3840, Santa Cruz), donkey anti-rabbit IgG AF594 ( {"type":"entrez-nucleotide","attrs":{"text":"A21207","term_id":"583479","term_text":"A21207"}} A21207 , Invitrogen), goat anti-rat IgG-biotin (A10517, Life Technologies), donkey anti-goat IgG-biotin (A10518, Life Technologies), and FITC-streptavidin (40201, BioLegend).

Techniques: Staining, Gene Expression

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Chronic psychosocial stress compromises the immune response and endochondral ossification during bone fracture healing via β-AR signaling

doi: 10.1073/pnas.1819218116

Figure Lengend Snippet: Callus composition and vascularization on d10 after femur osteotomy following 19 d of CSC housing. (A) Bone area/total area (BA/TA) ratio, (B) cartilage area/total area (CA/TA) ratio, (C) fibrous tissue area/total area (FA/TA) ratio, and (D) whole callus area of SHC and CSC mice on d10 after fracture. (E) Representative staining of fracture calli stained with Safranin-O. (Scale bars: 500 µm.) (F) Relative gene expression of Runx2, VEGF, and Sox2 analyzed by qPCR following LCM of the cartilage-to-bone transition zone of the fracture callus. Values were normalized to B2M. (G) Representative image of fracture callus stained for Runx2 (scale bars: 50 µm), (H) VEGF (scale bars: 50 µm), and (I) Sox2 (scale bars: 100 µm) in SHC and CSC mice on d10 after fracture. (J) Relative vascular area of transition zone. (K) Representative image of Safranin-O staining of the transition zone in SHC and CSC mice on d10 after fracture. (Scale bars: 50 µm.) (L) Representative images of fracture calli from SHC and CSC mice on d10 after fracture, double stained for β2-AR and ENPP1: β2-AR (Left), ENPP1 (Middle), merge (Left). (Scale bars: 100 µm and inlay, 50 µm.) Data are presented as mean + SD. SHC, n = 5–8; CSC, n = 5–8. *0.05 > P > 0.01; **0.01 > P > 0.001.

Article Snippet: Immunofluorescence staining for TH and β2-AR, as well as double stainings for TH and Ly6G and β2-AR and ENPP1 was performed using the following antibodies: rabbit anti-mouse β2-AR antibody (sc-569, Santa Cruz), rabbit anti-mouse TH antibody (AB152, Millipore), rat anti-mouse Ly6G (127632, BioLegend), goat anti-mouse ENPP1/PC1 (SAB 2500355, Sigma), goat-anti rabbit IgG-biotin (sc-3840, Santa Cruz), donkey anti-rabbit IgG AF594 ( {"type":"entrez-nucleotide","attrs":{"text":"A21207","term_id":"583479","term_text":"A21207"}} A21207 , Invitrogen), goat anti-rat IgG-biotin (A10517, Life Technologies), donkey anti-goat IgG-biotin (A10518, Life Technologies), and FITC-streptavidin (40201, BioLegend).

Techniques: Staining, Gene Expression

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Chronic psychosocial stress compromises the immune response and endochondral ossification during bone fracture healing via β-AR signaling

doi: 10.1073/pnas.1819218116

Figure Lengend Snippet: Callus composition and vascularization on d10 after femur osteotomy following 19 d of CSC housing. (A) Bone area/total area (BA/TA) ratio, (B) cartilage area/total area (CA/TA) ratio, (C) fibrous tissue area/total area (FA/TA) ratio, and (D) whole callus area of SHC and CSC mice on d10 after fracture. (E) Representative staining of fracture calli stained with Safranin-O. (Scale bars: 500 µm.) (F) Relative gene expression of Runx2, VEGF, and Sox2 analyzed by qPCR following LCM of the cartilage-to-bone transition zone of the fracture callus. Values were normalized to B2M. (G) Representative image of fracture callus stained for Runx2 (scale bars: 50 µm), (H) VEGF (scale bars: 50 µm), and (I) Sox2 (scale bars: 100 µm) in SHC and CSC mice on d10 after fracture. (J) Relative vascular area of transition zone. (K) Representative image of Safranin-O staining of the transition zone in SHC and CSC mice on d10 after fracture. (Scale bars: 50 µm.) (L) Representative images of fracture calli from SHC and CSC mice on d10 after fracture, double stained for β2-AR and ENPP1: β2-AR (Left), ENPP1 (Middle), merge (Left). (Scale bars: 100 µm and inlay, 50 µm.) Data are presented as mean + SD. SHC, n = 5–8; CSC, n = 5–8. *0.05 > P > 0.01; **0.01 > P > 0.001.

Article Snippet: Immunofluorescence staining for TH and β2-AR, as well as double stainings for TH and Ly6G and β2-AR and ENPP1 was performed using the following antibodies: rabbit anti-mouse β2-AR antibody (sc-569, Santa Cruz), rabbit anti-mouse TH antibody (AB152, Millipore), rat anti-mouse Ly6G (127632, BioLegend), goat anti-mouse ENPP1/PC1 (SAB 2500355, Sigma), goat-anti rabbit IgG-biotin (sc-3840, Santa Cruz), donkey anti-rabbit IgG AF594 ( {"type":"entrez-nucleotide","attrs":{"text":"A21207","term_id":"583479","term_text":"A21207"}} A21207 , Invitrogen), goat anti-rat IgG-biotin (A10517, Life Technologies), donkey anti-goat IgG-biotin (A10518, Life Technologies), and FITC-streptavidin (40201, BioLegend).

Techniques: Staining, Expressing