Journal: The Journal of Neuroscience

Article Title: Activation of Spinal Glucagon-Like Peptide-1 Receptors Specifically Suppresses Pain Hypersensitivity

doi: 10.1523/JNEUROSCI.4703-13.2014

Figure Lengend Snippet: Representative photomicrographs of GLP-1R expression in HEK293 cells stably expressing human GLP-1Rs (A, B), HEK293T cells that do not express GLP-1Rs (C, D), PC12 cells expressing rat GLP-1Rs (E, F), and their corresponding negative controls of the secondary antibody (B, D, F), as well as the superficial spinal dorsal horn (laminae I-III; G), cortex (H), hippocampus (I), dorsal root ganglia (J), pancreatic islets (K), and skeletal muscle (L) from 6 normal rats. Tissues were doubly labeled with the GLP-1R and GAPDH antibodies. DAPI staining was also used to determine cell nuclei. Representative photomicrographs for the preventive effect of the GLP-1R antigenic peptide EQ14 on GLP-1R immunoreactive fluorescence staining in the spinal cords from 6 spinal nerve ligation-induced neuropathic rats. M, Specific fluorescence staining with the GLP-1R antibody alone. N, Fluorescence staining with the GLP-1R antibody in the presence of EQ14. O, Fluorescence staining with EQ14 alone. P, Negative control of the secondary antibody. Scale bars: A–F, 150 μm; G–L, 100 μm; M–P, 300 μm.

Article Snippet: Both frozen sections and flasks were incubated in 10% goat serum (v/v) and 0.5% Triton X-100 (v/v) in PBS for 1 h. Frozen sections and flasks were incubated with the human and rat GLP-1R antibody (ab119287, rabbit polyclonal, 1:100 for tissue sections, 1:200 for cell flasks, Abcam) and/or other appropriate primary antibodies for 24 h at 4°C.

Techniques: Expressing, Stable Transfection, Labeling, Staining, Fluorescence, Ligation, Negative Control

Journal: The Journal of Neuroscience

Article Title: Activation of Spinal Glucagon-Like Peptide-1 Receptors Specifically Suppresses Pain Hypersensitivity

doi: 10.1523/JNEUROSCI.4703-13.2014

Figure Lengend Snippet: Expression of GLP-1Rs in the spinal cord, cortex, hippocampus, dorsal root ganglia, and skeletal muscle of normal rats by Western blotting where the GAPDH protein was used as loading control: representative gel (A) and GLP-1R/GAPDH band intensity ratio (B). Upregulation of GLP-1R immunostaining specifically expressed on microglia in the spinal dorsal horn after peripheral nerve injury. Photomicrographs were taken from the spinal cords in sham and neuropathic rats. Peripheral neuropathy was induced by unilateral L5–L6 spinal nerve ligation, and frozen sections were obtained from the spinal lumbar enlargements 2 weeks after surgery. Immunofluorescence was labeled with the GLP-1R antibody (C, G), microglial marker CD11b (OX42) (D, H), astrocytic marker GFAP (E, I), and mature neuronal marker NeuN (F, J). GLP-1R-(K) and OX42 (L)/GFAP (M)/NeuN (N)-immunolabeled surface areas were quantified from the spinal dorsal horn (laminae I-V; as indicated in C) using the ImageJ computer program. The averaged percentage immunolabeled surface area was the fraction of the positive immunofluorescent surface area of total measured area in the picture from 3 sections of each spinal cord. Scale bars: 500 μm. Data are mean ± SEM (n = 5 or 6 in each group). aStatistically significant difference from the spinal cord group in B (p < 0.05 by one-way ANOVA followed by post hoc Student-Newman-Keuls test) or the contralateral dorsal horns in K–N (p < 0.05 by paired Student's t test).

Article Snippet: Both frozen sections and flasks were incubated in 10% goat serum (v/v) and 0.5% Triton X-100 (v/v) in PBS for 1 h. Frozen sections and flasks were incubated with the human and rat GLP-1R antibody (ab119287, rabbit polyclonal, 1:100 for tissue sections, 1:200 for cell flasks, Abcam) and/or other appropriate primary antibodies for 24 h at 4°C.

Techniques: Expressing, Western Blot, Immunostaining, Ligation, Immunofluorescence, Labeling, Marker, Immunolabeling

Journal: The Journal of Neuroscience

Article Title: Activation of Spinal Glucagon-Like Peptide-1 Receptors Specifically Suppresses Pain Hypersensitivity

doi: 10.1523/JNEUROSCI.4703-13.2014

Figure Lengend Snippet: Representative photomicrographs of GLP-1R double fluorescence labeling with the microglial marker OX42 (A–C), astrocytic marker GFAP (D–F), and mature neuronal marker neuronal nuclei (NeuN; G–I) in the spinal cord. Photomicrographs were taken from the entire spinal cords (A, B, D, E, G, H) and superficial dorsal horns (laminae I-III; C, F, I) in sham and neuropathic rats (n = 5 or 6 in each group), respectively. Peripheral neuropathy was induced by unilateral L5–L6 spinal nerve ligation, and frozen sections were obtained from the spinal lumbar enlargements 2 weeks after surgery. Arrows indicate double-labeling of GLP-1Rs in microglia (C), but not in astrocytes (F) or neurons (I). There is increased fluorescence intensity of labeling of GLP-1R/OX42 in the areas of in the I-V laminae after peripheral nerve injury. Specific expression of GLP-1Rs on all primarily cultured spinal microglial cells (J), but not on astrocytes (K) or neurons (L) from the spinal dorsal horn of neonatal rats. DAPI staining was also used to determine cell nuclei. Scale bars: A, B, D, E, G, H, 500 μm; C, F, I, 50 μm; J–L, 25 μm.

Article Snippet: Both frozen sections and flasks were incubated in 10% goat serum (v/v) and 0.5% Triton X-100 (v/v) in PBS for 1 h. Frozen sections and flasks were incubated with the human and rat GLP-1R antibody (ab119287, rabbit polyclonal, 1:100 for tissue sections, 1:200 for cell flasks, Abcam) and/or other appropriate primary antibodies for 24 h at 4°C.

Techniques: Fluorescence, Labeling, Marker, Ligation, Expressing, Cell Culture, Staining

Journal: The Journal of Neuroscience

Article Title: Activation of Spinal Glucagon-Like Peptide-1 Receptors Specifically Suppresses Pain Hypersensitivity

doi: 10.1523/JNEUROSCI.4703-13.2014

Figure Lengend Snippet: Effects of 7 day intrathecal injections of the GLP-1R gene silencer siRNA/GLP-1R on spinal GLP-1R gene expression (relative to gapdh) (A), spinal (B, C), and dorsal root ganglial (D, with representative gels as in the inset) GLP-1R protein expression (relative to β-actin) and formalin-induced flinching responses (E, F) in rats. The vehicle PEI (7.5 μg) control group, nonspecific oligoneucleotides (oligo, 5 μg) control group, and siRNA/GLP-1R (5 μg) group were multidaily intrathecally injected for 7 d in rats. On the eight day, rats received a single intrathecal injection of saline (10 μl) or exenatide (30 ng) before 5% formalin challenge. Nociceptive behavior was quantified by counting the number of paw flinches in 1 min epochs. For the GLP-1R expression study, homogenates were obtained from spinal lumbar enlargements or dorsal rood ganglia immediately after the completion of the behavior tests. Blockade effects of intrathecal injection of the specific GLP-1R antagonist exendin(9–39) on antinociceptive effects of GLP-1(7–36) and exenatide in the rat and mouse formalin test (G–L). Rats and mice received two intrathecal treatments 30 min before subcutaneous injection of 10 or 50 μl of 1% or 5% formalin. The cumulated licking/biting duration from 0 to 5 min and 20 to 40 min in mice after formalin injection represented acute nociception and tonic hyperalgesia, respectively. Data are mean ± SEM (n = 6 in each group). a,bStatistical significance compared with the vehicle control and the exenatide or GLP-1(7–36) group, respectively (p < 0.05 by one-way ANOVA followed by post hoc Student-Newman-Keuls test).

Article Snippet: Both frozen sections and flasks were incubated in 10% goat serum (v/v) and 0.5% Triton X-100 (v/v) in PBS for 1 h. Frozen sections and flasks were incubated with the human and rat GLP-1R antibody (ab119287, rabbit polyclonal, 1:100 for tissue sections, 1:200 for cell flasks, Abcam) and/or other appropriate primary antibodies for 24 h at 4°C.

Techniques: Expressing, Injection