Journal: BMC Immunology

Article Title: TRPV1 + neurons alter Staphylococcus aureus skin infection outcomes by affecting macrophage polarization and neutrophil recruitment

doi: 10.1186/s12865-023-00584-x

Figure Lengend Snippet: S. aureus activates cutaneous TRPV1 + neurons to release CGRP. a DRG was isolated from WT mice and stained with DAPI (blue), TRPV1 (green), and CGRP (red); scale bars, 200 μm. b Primary DRG neurons isolated from 6-week-old WT mice were stained with DAPI (blue), TRPV1 (green), and CGRP (red); white arrows show colocalization of TRPV1 with CGRP; scale bars, 50 μm. c CGRP release from dorsal skin tissue of TRPV1 −/− and WT mice before and 8 h after infection ( n = 4/group); One-way ANOVA with Tukey’s post hoc test. d Pretreatment of mice with RTX or PBS and CGRP release from dorsal skin tissue before and 8 h after infection ( n = 4/group); One-way ANOVA with Tukey’s post hoc test. e DRG neurons were stimulated with different concentrations of S. aureus supernatant or capsaicin for 30 min, and the release of CGRP was analyzed ( n = 4/group). One-way ANOVA with Tukey’s post hoc test. Data were pooled from two or three independent experiments

Article Snippet: Then, after treatment with CGRP (100 nM, GenScript, #RP11095) or vehicle for 2 h, M1 polarization was induced with IFN-γ (10 ng/ml, Novoprotein, #CM41), M2 polarization was induced with IL-4 (40 ng/ml, Novoprotein, #CK74), and subsequent experiments were performed 24 h later.

Techniques: Isolation, Staining, Infection

Journal: BMC Immunology

Article Title: TRPV1 + neurons alter Staphylococcus aureus skin infection outcomes by affecting macrophage polarization and neutrophil recruitment

doi: 10.1186/s12865-023-00584-x

Figure Lengend Snippet: CGRP regulates the polarization of BMDMs and the release of inflammatory factors. a - c BMDMs cultured in vitro were induced to M1 polarization with IFN-γ and M2 polarization with IL-4 The polarized macrophages were treated with CGRP or PBS and stained with DAPI (blue), CD80 (green), and CD206 (red); the ratio of CD80 + and CD206 + was analyzed under different intervention conditions. No difference in the confluency of BMDMs was observed among different groups. Scale bars, 20 μm. One-way ANOVA with Tukey’s post hoc test. d - f Expression levels of TNFα, IL-1β, and IL-10 in BMDMs after polarization in vitro under different experimental conditions; one-way ANOVA with Tukey posttests. Data were pooled from two or three independent experiments

Article Snippet: Then, after treatment with CGRP (100 nM, GenScript, #RP11095) or vehicle for 2 h, M1 polarization was induced with IFN-γ (10 ng/ml, Novoprotein, #CM41), M2 polarization was induced with IL-4 (40 ng/ml, Novoprotein, #CK74), and subsequent experiments were performed 24 h later.

Techniques: Cell Culture, In Vitro, Staining, Expressing

Journal: BMC Immunology

Article Title: TRPV1 + neurons alter Staphylococcus aureus skin infection outcomes by affecting macrophage polarization and neutrophil recruitment

doi: 10.1186/s12865-023-00584-x

Figure Lengend Snippet: Graphical abstract. In Staphylococcus aureus skin infection, local TRPV1 + neurons release CGRP to inhibit neutrophil recruitment and regulate macrophage polarization, resulting in the aggravation of local infection

Article Snippet: Then, after treatment with CGRP (100 nM, GenScript, #RP11095) or vehicle for 2 h, M1 polarization was induced with IFN-γ (10 ng/ml, Novoprotein, #CM41), M2 polarization was induced with IL-4 (40 ng/ml, Novoprotein, #CK74), and subsequent experiments were performed 24 h later.

Techniques: Infection

Journal: Annals of Neurology

Article Title: Anti‐migraine Calcitonin Gene–Related Peptide Receptor Antagonists Worsen Cerebral Ischemic Outcome in Mice

doi: 10.1002/ana.25831

Figure Lengend Snippet: Olcegepant worsens the cerebral blood flow deficit and outcome of 12‐, 15‐, or 20‐minute focal cerebral ischemia. (A) Experimental timeline. (B) 2,3,5‐Triphenyltetrazolium chloride–stained coronal brain sections showing the infarct ( blue arrows ) after 12‐minute middle cerebral artery occlusion (MCAO) after pretreatment with a single dose of olcegepant (1mg/kg), and normal brain 20‐minute MCAO with vehicle injection. (C) Stroke risk (%) in vehicle (Veh) and olcegepant (Olce) groups after 12‐, 15‐, and 20‐minute MCAO. Data represent the percentage of infarct absence (transient ischemic attack [TIA], gray portion ) or presence (stroke, black portion ). Actual numbers of stroke/total are also provided in parentheses under the bars. Data were analyzed using multiple logistic regression (independent variables: treatment, ischemia duration; dependent variable: infarct presence) for the entire cohort, as well as using Fisher exact test in the 12‐minute MCAO group. (D) Representative cerebral blood flow tracing during MCAO and reperfusion measured by laser Doppler flowmetry. Gray shades represent typical time segments used to quantify the CBF during ischemia and after reperfusion. Lower panel shows the residual CBF (% of baseline) during different stages of MCAO in vehicle (n = 18) and olcegepant (1mg/kg, n = 19) arms ( p = 0.029, 2‐way repeated measures analysis of variance). Data are from pooled 12‐, 15‐, and 20‐minute MCAO experiments. Numbers within the gray bars represent the relative difference between treatment arms calculated as (CBF Olce /CBF Veh ) − 1. AD = anoxic depolarization; CCAO = common carotid artery occlusion; CCAR = common carotid artery reperfusion; MCAR = middle cerebral artery reperfusion. Seven mice in the vehicle arm and 1 mouse in the olcegepant arm were excluded from analyses based on predetermined criteria (see Materials and Methods). There was no mortality.

Article Snippet: Animals were treated with the small molecule CGRP receptor antagonists olcegepant (BIBN4096, 0.1 or 1mg/kg, 97.5% purity, intravenous in acute and intraperitoneal in chronic treatment; Tocris Bioscience, Bristol, UK) or rimegepant (BMS‐927711, 10 or 100mg/kg, intraperitoneal; 99.08% purity; MedChemExpress, Monmouth Junction, NJ), either with a single dose 10 minutes before ischemia onset (olcegepant and rimegepant) or with 10 once‐daily doses over 2 weeks followed by a single dose 10 minutes before ischemia onset (olcegepant only).

Techniques: Staining, Injection

Journal: Annals of Neurology

Article Title: Anti‐migraine Calcitonin Gene–Related Peptide Receptor Antagonists Worsen Cerebral Ischemic Outcome in Mice

doi: 10.1002/ana.25831

Figure Lengend Snippet: Olcegepant worsens the outcomes after 60‐minute focal cerebral ischemia. (A) Experimental timeline. (B) 2,3,5‐Triphenyltetrazolium chloride–stained coronal brain sections showing a typical infarct (unstained tissue) after 60‐minute middle cerebral artery occlusion (MCAO). (C) Infarct volume (mm 3 ) and neurologic deficit score ( p = 0.001 and p = 0.011, respectively, 2‐way analysis of variance [ANOVA]). Data from individual animals are shown ( triangles = male; circles = female) along with their group median ( red lines ). Mean ages were 5.3 ± 1.4 and 5.7 ± 1.5 months in the vehicle (Veh) and olcegepant (Olce; 1mg/kg) groups, respectively (all wild type; mean ± standard deviation). (D) Residual cerebral blood flow (CBF; % of baseline) during MCAO ( p = 0.223, 2‐way repeated measures ANOVA). Numbers within the gray bars represent the relative difference between treatment arms calculated as (CBF Olce /CBF Veh ) − 1. Two mice in the vehicle arm and 1 mouse in the olcegepant arm were excluded from analyses based on predetermined criteria (see Materials and Methods). There was no mortality.

Article Snippet: Animals were treated with the small molecule CGRP receptor antagonists olcegepant (BIBN4096, 0.1 or 1mg/kg, 97.5% purity, intravenous in acute and intraperitoneal in chronic treatment; Tocris Bioscience, Bristol, UK) or rimegepant (BMS‐927711, 10 or 100mg/kg, intraperitoneal; 99.08% purity; MedChemExpress, Monmouth Junction, NJ), either with a single dose 10 minutes before ischemia onset (olcegepant and rimegepant) or with 10 once‐daily doses over 2 weeks followed by a single dose 10 minutes before ischemia onset (olcegepant only).

Techniques: Staining, Standard Deviation

Journal: Annals of Neurology

Article Title: Anti‐migraine Calcitonin Gene–Related Peptide Receptor Antagonists Worsen Cerebral Ischemic Outcome in Mice

doi: 10.1002/ana.25831

Figure Lengend Snippet: Prolonged treatment with olcegepant worsens the outcomes after 60‐minute focal cerebral ischemia. (A) Experimental timeline. (B) Infarct volume (mm 3 ) and neurologic deficit scores ( p < 0.001 and p = 0.007, respectively, 2‐way analysis of variance). Data from individual animals are shown along with their group mean for infarct and median for neurologic score ( red lines ). All mice were 2 months old, male, and wild type. Two mice in the vehicle (Veh) and 3 mice in the olcegepant (Olce) 0.1mg/kg and olcegepant 1mg/kg arms died prior to outcome assessments. There were no exclusions. [Color figure can be viewed at www.annalsofneurology.org ]

Article Snippet: Animals were treated with the small molecule CGRP receptor antagonists olcegepant (BIBN4096, 0.1 or 1mg/kg, 97.5% purity, intravenous in acute and intraperitoneal in chronic treatment; Tocris Bioscience, Bristol, UK) or rimegepant (BMS‐927711, 10 or 100mg/kg, intraperitoneal; 99.08% purity; MedChemExpress, Monmouth Junction, NJ), either with a single dose 10 minutes before ischemia onset (olcegepant and rimegepant) or with 10 once‐daily doses over 2 weeks followed by a single dose 10 minutes before ischemia onset (olcegepant only).

Techniques:

Journal: Annals of Neurology

Article Title: Anti‐migraine Calcitonin Gene–Related Peptide Receptor Antagonists Worsen Cerebral Ischemic Outcome in Mice

doi: 10.1002/ana.25831

Figure Lengend Snippet: Stroke outcome in familial hemiplegic migraine type 1 (FHM1) mice after a single dose of olcegepant. (A) Experimental timeline. (B) Infarct volume (mm 3 ) and neurologic deficit scores in experiment testing olcegepant 1mg/kg ( p = 0.017 and p = 0.374, respectively, 2‐way analysis of variance [ANOVA]). Data from individual animals are shown ( triangles = male; circles = female) along with their group median ( red lines ). Mean ages were 6.4 ± 4.5 and 7.0 ± 6.5 months in vehicle (Veh) and olcegepant (Olce) groups, respectively (all FHM1 mutants; mean ± standard deviation [SD]). (C) Infarct volume (mm 3 ) and neurologic deficit scores in experiment testing olcegepant 0.1mg/kg ( p = 0.356 and p = 0.440, respectively, 2‐way ANOVA). Data from individual animals are shown ( triangles = male; circles = female) along with their group median ( red lines ). (D) Residual cerebral blood flow (CBF; % of baseline) during middle cerebral artery occlusion (MCAO; p = 0.019, 2‐way repeated measures ANOVA). Mean ages were 13.2 ± 3.9 and 12.3 ± 2.3 months in the vehicle and olcegepant (1mg/kg) groups, respectively (all FHM1 mutants; mean ± SD). (E) Residual CBF (% of baseline) during MCAO ( p = 0.052, 2‐way repeated measures ANOVA). Numbers within the gray bars represent the relative difference between treatment arms calculated as (CBF Olce /CBF Veh ) − 1. Seven mice in the vehicle, 3 mice in the olcegepant 0.1mg/kg, and 6 in the olcegepant 1mg/kg arms were excluded from analyses based on predetermined criteria (see Materials and Methods). Two mice in the vehicle, 1 in olcegepant 0.1mg/kg, and 2 in the olcegepant 1mg/kg arms died prior to outcome assessments. AD = anoxic depolarization; CCAO = common carotid artery occlusion; CCAR = common carotid artery reperfusion; MCAR = middle cerebral artery reperfusion. [Color figure can be viewed at www.annalsofneurology.org ]

Article Snippet: Animals were treated with the small molecule CGRP receptor antagonists olcegepant (BIBN4096, 0.1 or 1mg/kg, 97.5% purity, intravenous in acute and intraperitoneal in chronic treatment; Tocris Bioscience, Bristol, UK) or rimegepant (BMS‐927711, 10 or 100mg/kg, intraperitoneal; 99.08% purity; MedChemExpress, Monmouth Junction, NJ), either with a single dose 10 minutes before ischemia onset (olcegepant and rimegepant) or with 10 once‐daily doses over 2 weeks followed by a single dose 10 minutes before ischemia onset (olcegepant only).

Techniques: Standard Deviation

Journal: Annals of Neurology

Article Title: Anti‐migraine Calcitonin Gene–Related Peptide Receptor Antagonists Worsen Cerebral Ischemic Outcome in Mice

doi: 10.1002/ana.25831

Figure Lengend Snippet: Baseline hemodynamic parameters in young male wild‐type mice after a single dose of olcegepant. Baseline blood pressure (BP; p = 0.177), heart rate (HR; p = 0.917), and cerebral blood flow (CBF; p = 0.441) were measured over a period of 30 minutes after vehicle or olcegepant (1mg/kg) treatment (both groups: 2.6 ± 0.2 months of age, all male, wild‐type mice). Two‐way repeated measures analysis of variance, n = 5 per group. BPM = beats per minute.

Article Snippet: Animals were treated with the small molecule CGRP receptor antagonists olcegepant (BIBN4096, 0.1 or 1mg/kg, 97.5% purity, intravenous in acute and intraperitoneal in chronic treatment; Tocris Bioscience, Bristol, UK) or rimegepant (BMS‐927711, 10 or 100mg/kg, intraperitoneal; 99.08% purity; MedChemExpress, Monmouth Junction, NJ), either with a single dose 10 minutes before ischemia onset (olcegepant and rimegepant) or with 10 once‐daily doses over 2 weeks followed by a single dose 10 minutes before ischemia onset (olcegepant only).

Techniques:

Journal: Annals of Neurology

Article Title: Anti‐migraine Calcitonin Gene–Related Peptide Receptor Antagonists Worsen Cerebral Ischemic Outcome in Mice

doi: 10.1002/ana.25831

Figure Lengend Snippet: Pharmacological characterization of olcegepant and rimegepant in mouse aortas, human coronary arteries, and human middle meningeal arteries. (A) Concentration–response curves to rat α–calcitonin gene–related peptide (CGRP) in mouse aortas in the absence or presence of olcegepant (1μM, 3μM, and 10μM; 3–6 females and 3–5 males) with the corresponding Schild plot (pA 2 = 7.22, 2 females and 3 males). (B) Concentration–response curves to rat α‐CGRP in mouse aortas in the absence or presence of rimegepant (3μM, 10μM, 30μM; 2–5 females and 2–4 males) with the corresponding Schild plot (pA 2 = 6.24, 3 females and 4 males). (C) Concentration–response curves to human α‐CGRP in human coronary arteries in the absence or presence of rimegepant (1nM, 10nM, 100nM, 1μM; 2–3 females and 3–4 males) with the corresponding Schild plot and pK b values (3 females and 4 males). (D) Concentration–response curves to human α‐CGRP in human middle meningeal arteries in the absence or presence of rimegepant (1nM, 10nM, 100nM; 2 females and 1–2 males) with the corresponding Schild plot (pA 2 = 10.02; 2 females and 2 males). All data are represented as mean ± standard error of the mean. DR‐1 = Dose Ratio–1; KCL = kaliumchloride; M = Molar

Article Snippet: Animals were treated with the small molecule CGRP receptor antagonists olcegepant (BIBN4096, 0.1 or 1mg/kg, 97.5% purity, intravenous in acute and intraperitoneal in chronic treatment; Tocris Bioscience, Bristol, UK) or rimegepant (BMS‐927711, 10 or 100mg/kg, intraperitoneal; 99.08% purity; MedChemExpress, Monmouth Junction, NJ), either with a single dose 10 minutes before ischemia onset (olcegepant and rimegepant) or with 10 once‐daily doses over 2 weeks followed by a single dose 10 minutes before ischemia onset (olcegepant only).

Techniques: Concentration Assay