Journal: Arthritis & Rheumatology (Hoboken, N.j.)

Article Title: Peripheral Calcitonin Gene-Related Peptide Receptor Activation and Mechanical Sensitization of the Joint in Rat Models of Osteoarthritis Pain

doi: 10.1002/art.38656

Figure Lengend Snippet: Primer and probe sequences used for the TaqMan real-time quantitative polymerase chain reaction analyses

Article Snippet: MIA (Sigma), rat α-CGRP (Sigma), rat CGRP 8–37 (Tocris Bioscience), and 2% Fluoro-Gold (Fluorochrome) were dissolved in sterile saline.

Techniques: Real-time Polymerase Chain Reaction, Sequencing

Journal: Arthritis & Rheumatology (Hoboken, N.j.)

Article Title: Peripheral Calcitonin Gene-Related Peptide Receptor Activation and Mechanical Sensitization of the Joint in Rat Models of Osteoarthritis Pain

doi: 10.1002/art.38656

Figure Lengend Snippet: Calcitonin gene-related peptide (CGRP)–induced mechanical sensitization of an increased proportion of knee afferents following monosodium iodoacetate (MIA) injection. A and B, CGRP significantly increased the mechanical evoked firing rates (action potentials per second [APs/s]) of joint afferents in saline-treated (A) and MIA-treated (B) rats ( P < 0.001 by 2-way analysis of variance with Bonferroni post hoc test). Values are the mean ± SEM maximum sensitizing effects of CGRP over each 1-hour recording period (n = 9 MIA-treated rats; n = 5 saline-treated rats). C, CGRP administration reduced mechanical thresholds (minimum stimulus evoking ≥2 action potentials) in saline-treated rats to a level not significantly different from that in MIA-treated rats ( P > 0.05 for comparisons of 0.5 μg or 10 μg of CGRP versus controls, by Mann-Whitney t -test). Mechanical thresholds in MIA-treated rats were unaltered by CGRP administration. Each circle represents an individual rat (n = 7–9 MIA treated and n = 5 saline treated); horizontal lines show the median. D, The percentage of fibers sensitized by CGRP was greater in MIA-treated rats than in saline-treated rats ( P < 0.001 by Fisher's exact test). ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001 by Mann-Whitney t -test. vFH = von Frey hair; NS = not significant.

Article Snippet: MIA (Sigma), rat α-CGRP (Sigma), rat CGRP 8–37 (Tocris Bioscience), and 2% Fluoro-Gold (Fluorochrome) were dissolved in sterile saline.

Techniques: Injection, Saline, MANN-WHITNEY

Journal: Arthritis & Rheumatology (Hoboken, N.j.)

Article Title: Peripheral Calcitonin Gene-Related Peptide Receptor Activation and Mechanical Sensitization of the Joint in Rat Models of Osteoarthritis Pain

doi: 10.1002/art.38656

Figure Lengend Snippet: Inhibition of knee afferent mechanical evoked firing by the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP 8–37 in monosodium iodoacetate (MIA)–treated rats. Representative examples of knee afferent mechanical evoked (8-, 10-, or 15-gm von Frey monofilaments) response data recorded before and after administration of 10 μg of CGRP 8–37 in a saline-treated rat (A) and an MIA-treated rat (B) are shown. The inhibitory effects of CGRP 8–37 were more pronounced following MIA treatment. Results are represented as rate histograms (1-second bins) of action potential firing of knee afferents (single units) in response to stimulation at the indicated von Frey monofilament level. Insets show an overlay of a representative fiber recording with the corresponding conduction velocity (CV; in meters/second), demonstrating single-fiber recordings; both are Aδ fibers.

Article Snippet: MIA (Sigma), rat α-CGRP (Sigma), rat CGRP 8–37 (Tocris Bioscience), and 2% Fluoro-Gold (Fluorochrome) were dissolved in sterile saline.

Techniques: Inhibition, Saline

Journal: Arthritis & Rheumatology (Hoboken, N.j.)

Article Title: Peripheral Calcitonin Gene-Related Peptide Receptor Activation and Mechanical Sensitization of the Joint in Rat Models of Osteoarthritis Pain

doi: 10.1002/art.38656

Figure Lengend Snippet: Ablation of monosodium iodoacetate (MIA)–induced and medial meniscus transection (MMT)–induced mechanical sensitization of joint afferents by the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP 8–37. CGRP 8–37 had no effect on mechanically evoked responses (action potentials per second [APs/s]) of joint afferents in saline-treated (A) and sham-operated (D) rats, whereas it significantly inhibited mechanically evoked responses in MIA-treated (B) and MMT-operated (E) rats ( P < 0.0001 for each comparison versus its corresponding control, by 2-way analysis of variance with Bonferroni post hoc test). Values are the mean ± SEM responses over each 1-hour recording period (n = 8 saline-treated, 6 sham-operated, 9 MIA-treated, and 8 MMT-operated rats). CGRP 8–37 had no effect on mechanical thresholds (minimum stimulus evoking ≥2 action potentials) in saline-treated (C) or sham-operated (F) rats. However, in MIA-treated (C) and MMT-operated (F) rats, thresholds in the presence of 10 μg of CGRP 8–37 were increased, reversing the osteoarthritis-induced reduction of mechanical thresholds. Each circle represents an individual rat (n = 6–9 per group); horizontal lines show the median. ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001, by Mann-Whitney t -test. vFH = von Frey hair.

Article Snippet: MIA (Sigma), rat α-CGRP (Sigma), rat CGRP 8–37 (Tocris Bioscience), and 2% Fluoro-Gold (Fluorochrome) were dissolved in sterile saline.

Techniques: Saline, Comparison, Control, MANN-WHITNEY

Journal: Arthritis & Rheumatology (Hoboken, N.j.)

Article Title: Peripheral Calcitonin Gene-Related Peptide Receptor Activation and Mechanical Sensitization of the Joint in Rat Models of Osteoarthritis Pain

doi: 10.1002/art.38656

Figure Lengend Snippet: Expression of mRNA for calcitonin gene-related peptide (CGRP) receptor components in knee joint–innervating L3–L4 dorsal root ganglia (DRGs) and modulation of the levels following treatment with monosodium iodoacetate (MIA). A, Expression of mRNA for CGRP was significantly reduced 28 days after MIA treatment. B, Calcitonin-like receptor (CLR) expression was increased 28 days after MIA treatment. C, Levels of receptor activity–modifying protein 1 (RAMP-1) were unaltered, both on day 14 and on day 28 after MIA treatment. D, Levels of mRNA for CGRP receptor component protein (CRCP) were reduced 14 days after MIA treatment. There was a small but significant increase in CRCP expression 28 days after MIA treatment. Results were normalized to β-actin expression (which was unchanged between groups and treatments). Data are shown as box plots. Each box represents the 25th to 75th percentiles (n = 6–8 rats per group). Lines inside the boxes represent the median. Lines outside the boxes represent the 10th and 90th percentiles. ∗ = P < 0.05; ∗∗ = P < 0.01 by Mann-Whitney t -test.

Article Snippet: MIA (Sigma), rat α-CGRP (Sigma), rat CGRP 8–37 (Tocris Bioscience), and 2% Fluoro-Gold (Fluorochrome) were dissolved in sterile saline.

Techniques: Expressing, Activity Assay, MANN-WHITNEY

Journal: Arthritis & Rheumatology (Hoboken, N.j.)

Article Title: Peripheral Calcitonin Gene-Related Peptide Receptor Activation and Mechanical Sensitization of the Joint in Rat Models of Osteoarthritis Pain

doi: 10.1002/art.38656

Figure Lengend Snippet: Coexpression of the calcitonin gene-related peptide (CGRP) receptor components calcitonin-like receptor (CLR) and receptor component protein (CRCP [RCP]) by knee afferent neurons and increased CLR expression following treatment with monosodium iodoacetate (MIA). A1–A4, CLR is expressed on knee afferents (arrows) and non–knee afferent neuronal cell bodies (∗) in ipsilateral L4 dorsal root ganglia (DRGs). Slides incubated with anti–neuronal nuclei (anti-NeuN) antibodies or with Fluoro-Gold (FG) are also shown. Bar = 34 μm. B1–B4, CRCP is coexpressed with CLR on knee afferent neurons (arrows) and non–knee afferent neurons (∗). Bar = 40 μm. C, The numbers of medium (30–40 μm in diameter) and large (40–50 μm in diameter) CLR-positive knee afferents were significantly increased in MIA-treated rats. CLR-positive knee afferent cell counts were analyzed according to diameter ranges similar to those described in a previously published report . Values are the mean ± SEM of 7 rats per group. ∗ = P < 0.05 by Mann-Whitney t -test.

Article Snippet: MIA (Sigma), rat α-CGRP (Sigma), rat CGRP 8–37 (Tocris Bioscience), and 2% Fluoro-Gold (Fluorochrome) were dissolved in sterile saline.

Techniques: Expressing, Incubation, MANN-WHITNEY

Journal: British Journal of Pharmacology

Article Title: Endocannabinoids modulate human blood–brain barrier permeability in vitro

doi: 10.1111/bph.13106

Figure Lengend Snippet: The effects of increasing concentrations of AEA on BBB permeability measured by TEER (A) with corresponding AUC (B) ( n = 9 inserts from three separate experiments). The effects of capsazepine (Cpz) (C) ( n = 7 inserts from three separate experiments) or AM630 (D) ( n = 7–8 inserts from three separate experiments) or CGRP (E) ( n = 5–6 inserts from three separate experiments) on the effect of AEA (10 μM). The effects of dexamethasone ( n = 6) and the CB 2 agonist HU308 on BBB permeability over time (F) and expressed as AUC (G). Data are given as mean ± SEM. * * * P < 0.001, * * P < 0.01, * P < 0.05; AEA compared with vehicle-treated inserts; †† P < 0.01; AEA and antagonist compared with AEA alone; # P < 0.05, ## P < 0.01; HU308 compared with vehicle; one-way anova with Dunnett's (B) or Bonferroni's test (C,D,E).

Article Snippet: AM251, AM630, GW6471, GW9662 (all 100 nM), capsazepine, O-1918 (both 1 μM) (all dissolved in dimethyl sulfoxide) and CGRP 8–37 (2 μM, dissolved in distilled water) were all purchased from Tocris and URB597 (1 μM, dissolved in dimethyl sulfoxide) was purchased from Sigma (Dorset, UK).

Techniques: Permeability

Journal: British Journal of Pharmacology

Article Title: Endocannabinoids modulate human blood–brain barrier permeability in vitro

doi: 10.1111/bph.13106

Figure Lengend Snippet: Expression profiling of potential target sites of action in HA and HBMEC cells. Shown are the ethidium bromide-stained gels of the products obtained by RT-PCR using primers specific for PPARα, PPARγ, CB 1 receptors, CB 2 receptors, TRPV1 channels, CGRP receptors and the control gene HPRT. cDNAs generated in the presence (+) or absence (−) of reverse transcriptase on total RNA from HA or HBMEC cells were used as template for the PCRs. The 100 bp DNA ladder was used in all gels except for PPARα and PPARγ where a 10 bp ladder was used. Sizes are in base pairs.

Article Snippet: AM251, AM630, GW6471, GW9662 (all 100 nM), capsazepine, O-1918 (both 1 μM) (all dissolved in dimethyl sulfoxide) and CGRP 8–37 (2 μM, dissolved in distilled water) were all purchased from Tocris and URB597 (1 μM, dissolved in dimethyl sulfoxide) was purchased from Sigma (Dorset, UK).

Techniques: Expressing, Staining, Reverse Transcription Polymerase Chain Reaction, Control, Generated, Reverse Transcription

Journal: British Journal of Pharmacology

Article Title: Endocannabinoids modulate human blood–brain barrier permeability in vitro

doi: 10.1111/bph.13106

Figure Lengend Snippet:

Article Snippet: AM251, AM630, GW6471, GW9662 (all 100 nM), capsazepine, O-1918 (both 1 μM) (all dissolved in dimethyl sulfoxide) and CGRP 8–37 (2 μM, dissolved in distilled water) were all purchased from Tocris and URB597 (1 μM, dissolved in dimethyl sulfoxide) was purchased from Sigma (Dorset, UK).

Techniques:

Journal: Arthritis & Rheumatology (Hoboken, N.j.)

Article Title: Calcitonin Gene‐Related Peptide–Expressing Sensory Neurons and Spinal Microglial Reactivity Contribute to Pain States in Collagen‐Induced Arthritis

doi: 10.1002/art.39082

Figure Lengend Snippet: Development of CIA is associated with an increase in calcitonin gene‐related peptide (CGRP)–expressing neurons in lumbar DRGs. A, Quantification of CGRP+FG+ L4 and L5 DRG neurons during CIA development (n = 4–6 rats per group). B, CGRP+ neurons in L5 DRGs retrograde‐labeled with FG in control rats and rats with CIA at 18 days. Arrows indicate FG+CGRP+ neurons; arrowheads indicate FG+ neurons; chevrons indicate CGRP+ neurons. C, Quantification of CGRP+ neurons in whole L4 and L5 DRGs during CIA development (n = 4 rats per group). D, CGRP+ neurons in control rats and rats with CIA at 18 days. Values are the mean ± SEM. ** = P < 0.01; *** = P < 0.001 versus controls, by two‐way analysis of variance followed by Tukey's test. Bars = 100 μm. See Figure 2 for other definitions.

Article Snippet: CGRP receptor antagonist CGRP 8–37 (1 mg/ml; Bachem) was delivered at 24 μl/day for 7 days (from day 11 to day 18 postimmunization), resulting in a dose of 24 μg/day as reported previously .

Techniques: Expressing, Labeling, Control

Journal: Arthritis & Rheumatology (Hoboken, N.j.)

Article Title: Calcitonin Gene‐Related Peptide–Expressing Sensory Neurons and Spinal Microglial Reactivity Contribute to Pain States in Collagen‐Induced Arthritis

doi: 10.1002/art.39082

Figure Lengend Snippet: CIA development is associated with increased p‐ERK in lumbar DRGs. A, Quantification of FG+p‐ERK+ neurons and FG+ calcitonin gene‐related peptide–positive (CGRP+) p‐ERK+ neurons in L4 and L5 DRGs during CIA development. B, Quantification of p‐ERK+ neurons and p‐ERK+CGRP+ neurons in the whole neuronal population in L4 and L5 DRGs during CIA development. In A and B, n = 4–6 rats per group. C, Phospho‐ERK+ and CGRP+ neurons in L5 DRGs retrograde‐labeled with FG in control rats and rats with CIA at 18 days. Arrows indicate FG+p‐ERK+CGRP+ neurons; arrowheads indicate FG+CGRP+ neurons; chevrons indicate CGRP+p‐ERK+ neurons. D, Phospho‐ERK+ and CGRP+ neurons in the whole neuronal population in L4 and L5 DRGs in control rats and rats with CIA at 18 days. Values are the mean ± SEM. ** = P < 0.01; *** = P < 0.001 versus controls, by two‐way analysis of variance followed by Tukey's test. Bars = 100 μm. See Figure 2 for other definitions.

Article Snippet: CGRP receptor antagonist CGRP 8–37 (1 mg/ml; Bachem) was delivered at 24 μl/day for 7 days (from day 11 to day 18 postimmunization), resulting in a dose of 24 μg/day as reported previously .

Techniques: Labeling, Control

Journal: Arthritis & Rheumatology (Hoboken, N.j.)

Article Title: Calcitonin Gene‐Related Peptide–Expressing Sensory Neurons and Spinal Microglial Reactivity Contribute to Pain States in Collagen‐Induced Arthritis

doi: 10.1002/art.39082

Figure Lengend Snippet: Enhanced release of calcitonin gene‐related peptide (CGRP) and significant microgliosis in the dorsal horn during collagen‐induced arthritis (CIA). A, Activity‐evoked release of CGRP from primary afferent fibers in the dorsal horn of control rats at 18 days and rats with CIA at 7 days and 18 days (n = 7 rats per group). Mean ± SEM basal peptide content was 7.8 ± 0.27 pg/8 ml (n = 9 rats). R1–R4 = recovery fractions. B, Left and top, Ionized calcium–binding adapter molecule 1 (IBA‐1)–positive profiles in control rats at 18 days and in rats with CIA at 7 days and 18 days. Bottom right, Quantification of IBA‐1+ profiles in the dorsal horn of control rats and rats with CIA during CIA development (n = 4–6 rats per group). Values are the mean ± SEM. * = P < 0.05; ** = P < 0.01; *** = P < 0.001 versus controls, by two‐way analysis of variance followed by Tukey's test. ### = P < 0.001 versus basal levels, by two‐way analysis of variance followed by Tukey's test. Bar = 100 μm.

Article Snippet: CGRP receptor antagonist CGRP 8–37 (1 mg/ml; Bachem) was delivered at 24 μl/day for 7 days (from day 11 to day 18 postimmunization), resulting in a dose of 24 μg/day as reported previously .

Techniques: Activity Assay, Control, Binding Assay

Journal: Arthritis & Rheumatology (Hoboken, N.j.)

Article Title: Calcitonin Gene‐Related Peptide–Expressing Sensory Neurons and Spinal Microglial Reactivity Contribute to Pain States in Collagen‐Induced Arthritis

doi: 10.1002/art.39082

Figure Lengend Snippet: Intrathecal (IT) administration of a CGRP antagonist attenuates established mechanical hypersensitivity in CIA. A and B, Established mechanical hypersensitivity ( A ) but not heat hypersensitivity ( B ) attenuated by 7‐day intrathecal administration of CGRP 8–37 (24 μg/day) (n = 9 rats per group). C, Quantification of IBA‐1+ profiles in the L4 and L5 dorsal horns at 18 days (n = 4 rats per group). Values are the mean ± SEM. ** = P < 0.01; *** = P < 0.001 versus vehicle (Veh)–treated controls, by two‐way repeated‐measures analysis of variance followed by Tukey's test ( A and B ) or by Student's t‐ test ( C ). PWT = paw withdrawal threshold; PWLT = paw withdrawal latency time (see Figure 5 for other definitions).

Article Snippet: CGRP receptor antagonist CGRP 8–37 (1 mg/ml; Bachem) was delivered at 24 μl/day for 7 days (from day 11 to day 18 postimmunization), resulting in a dose of 24 μg/day as reported previously .

Techniques:

Journal: Cell

Article Title: Blocking neuronal signaling to immune cells treats streptococcal invasive infection

doi: 10.1016/j.cell.2018.04.006

Figure Lengend Snippet: (A) Representative Fura-2 ratiometric fields (left) and calcium traces (center) of DRG neurons responding to filtered supernatant from S. pyogenes M1 (5×109 cfu/mL), capsaicin (1 µm), and KCl (40 mM). Proportions (Right) of capsaicin non-responsive (Cap−) and capsaicin responsive (Cap+) neurons that responded to M1 supernatant (n=3–4 fields/condition). (B–D) Representative Fura-2 ratiometric fields (B) and calcium traces (C) of DRG neurons stimulated with filtered supernatant from S. pyogenes M1 (wt) or isogenic mutants lacking SLS (ΔsagA), both SLO and SLS (ΔsloΔsagA), or double mutant bacteria in which sagA expression was restored (ΔsloΔsagA+pDL:sagA). (D) Proportions of responding DRG neurons to bacterial supernatant from S. pyogenes M1 (wt) or isogenic mutant strains (n=3 fields/condition). (E) DRG neurons stimulated for 30 min with supernatant from S. pyogenes M1 (wt), isogenic mutants, or medium, analyzed for in vitro release of CGRP (n=5 samples/group). Statistical analysis: (A) Two-way ANOVA, Bonferroni post-tests. (D,E) One-way ANOVA, Tukey post-tests. *p<0.05 ***p<0.001 ****p<0.0001. ns=not significant. Scale bars, 50 µm. Mean±SEM. See Figure S2 for related data.

Article Snippet: CGRP (1 μM, GenScript) or the antagonists CGRP 8–37 (1 μM, GenScript) or BIBN4096 (1 μM, Tocris) were added to the cultures immediately before S. pyogenes .

Techniques: Mutagenesis, Bacteria, Expressing, In Vitro

Journal: Cell

Article Title: Blocking neuronal signaling to immune cells treats streptococcal invasive infection

doi: 10.1016/j.cell.2018.04.006

Figure Lengend Snippet: (A) Histopathology of flank biopsies from vehicle or RTX-treated mice 3 days after injection of S. pyogenes M1 (5×106 cfu). Scale bars, 50 µm. (B) Bacterial load recovery (log10 cfu) from flank lesions and spleens in RTX or vehicle-treated mice after S. pyogenes M1 injection (5×106 cfu, n=4/group). (C–E) Flow cytometry of leukocyte recruitment in necrotizing lesions 1 day after S. pyogenes M1 injection (5×106 cfu): (C) Representative FACS plots showing neutrophils (CD11b+Ly6G+ gates) in lesion samples. (D–E) Quantification of immune cell populations by flow cytometry in flank biopsies from infected Trpv1-Cre/Dta mice or control littermates (n=4/group), or from uninfected mice, infected vehicle-treated mice, or infected RTX-treated mice (n=4–5/group). (F–H) Measurement of CGRP release ex vivo from flank skin punch biopsies. (G) CGRP release from uninfected skin (0 h), 7 h, or 24 h after S. pyogenes M1 injection (5×106 cfu) (n=3/group). (H) CGRP release from uninfected skin or 7 h after S. pyogenes M1 (5×106 cfu) injection of Trpv1-Cre/Dta mice or control littermates, or Vehicle or RTX-treated mice (n=3/group). Statistical analysis: (B,D,E,H) Two-way ANOVA, Bonferroni post-tests. (G) One-way ANOVA, Tukey post-tests. *p<0.05 **p<0.01 ***p<0.001 ****p<0.0001. ns=not significant. nd=none detected. Mean±SEM. See Figure S5 for related data.

Article Snippet: CGRP (1 μM, GenScript) or the antagonists CGRP 8–37 (1 μM, GenScript) or BIBN4096 (1 μM, Tocris) were added to the cultures immediately before S. pyogenes .

Techniques: Histopathology, Injection, Flow Cytometry, Infection, Control, Ex Vivo

Journal: Cell

Article Title: Blocking neuronal signaling to immune cells treats streptococcal invasive infection

doi: 10.1016/j.cell.2018.04.006

Figure Lengend Snippet: (A–D) Subcutaneous administration of BoNT/A (25 pg/100 µL) or vehicle 6 days prior to S. pyogenes M1 injection in flank skin (5×106 cfu). (B) Representative images of lesions (day 8), (C) Dermonecrosis size measurements, and (D) Weight loss over time after injection of S. pyogenes (n=5–10/group). (E–H) Intrathecal administration of BoNT/A or vehicle 1 day prior to S. pyogenes M1 injection in flank skin (5×106 cfu). (F) Representative images of lesions (day 8), (G) Dermonecrosis size measurements, and (H) Weight loss over time after injection of S. pyogenes (n=6/group). (I) DRG neurons exposed to BoNT/A (25 pg/200 µL) or medium for 24 h were stimulated with S. pyogenes supernatant (5×109 cfu/mL) for 30 min, and CGRP was measured in neuronal supernatant (n=5/group). (J) CGRP release from skin punch biopsies of mice treated intrathecally or locally with BoNT/A, 7 h after S. pyogenes M1 (5×106 cfu) injection (n=3/group). Statistical analysis: (C,D,G,H) Two-way ANOVA, Bonferroni post-tests. (I,J) One-way ANOVA, Tukey post-tests. *p<0.05 **p<0.01 ***p<0.001 ****p<0.0001. ns=not significant. Mean±SEM. See Figure S6 for related data.

Article Snippet: CGRP (1 μM, GenScript) or the antagonists CGRP 8–37 (1 μM, GenScript) or BIBN4096 (1 μM, Tocris) were added to the cultures immediately before S. pyogenes .

Techniques: Injection

Journal: Cell

Article Title: Blocking neuronal signaling to immune cells treats streptococcal invasive infection

doi: 10.1016/j.cell.2018.04.006

Figure Lengend Snippet: (A) DRG neurons were pretreated with BoNT/A for 24 h, or with CGRP antagonists (CGRP8–37 or BIBN4096) immediately before co-incubation with mouse neutrophils and S. pyogenes M1 for 1 h. Bacterial survival was measured as the multiplication factor of surviving colonies/starting inoculum (n=3–4 replicates/group). (B) Mouse neutrophils were incubated with S. pyogenes M1 in presence of CGRP or vehicle for 1 h, and bacterial survival measured (n=4/group). (C) Human whole blood was incubated with S. pyogenes M1 in presence of CGRP or vehicle for 3 h, and bacterial survival measured (n=3/group). (D) Representative images of lesions at day 8 (left) and dermonecrosis size (right) of mice treated 2 h after S. pyogenes M1 injection (5×106 cfu) with vehicle, BoNT/A, or BIBN4096 (n=6–7/group). (E–G) Mice were treated subcutaneously with BoNT/A or vehicle at day 2 and day 9 following flank injection of S. pyogenes M1 (5×106 cfu). Representative images show lesions before and after treatment (E). Dermonecrotic lesions (F) and abscess sizes (G) were measured over time (n=10/group). Blue dots show injection sites at day 2 and day 9. Arrows show BoNT/A treatments. Statistical analysis: (A–C) One-way ANOVA, Tukey post-tests. (D–G) Two-way ANOVA, Bonferroni post-tests. (A–C,F–G) *p<0.05 **p<0.01 ***p<0.01 ****p<0.0001. (D) BIBN4096 vs veh: *p<0.05 **p<0.01 ***p<0.001 ****p<0.0001, BoNT/A vs veh: †p<0.05 ††p<0.01 †††p<0.001 ††††p<0.0001. ns=not significant. Mean±SEM. See Figure S7 for related data.

Article Snippet: CGRP (1 μM, GenScript) or the antagonists CGRP 8–37 (1 μM, GenScript) or BIBN4096 (1 μM, Tocris) were added to the cultures immediately before S. pyogenes .

Techniques: Incubation, Injection