ephb2 fc  (R&D Systems)

Journal: Frontiers in Pharmacology

Article Title: Activation of EphrinB2/EphB2 signaling in the spine cord alters glia-neuron interactions in mice with visceral hyperalgesia following maternal separation

doi: 10.3389/fphar.2024.1463339

Figure Lengend Snippet: Maternal separation early in development causes visceral hyperalgesia in response to colorectal distension. (A) Schematic illustration of the experimental protocol. CRD, colorectal distension; MS, maternal separation (B) Score on abdominal withdrawal reflex after colorectal distension. Quantification is shown for eight animals per condition. CON, control. *P < 0.05, ****P < 0.0001 vs. CON, based on two-way repeated-measures ANOVA, followed by the Bonferroni multiple-comparisons test. (C) Representative thin sections of spinal cord after immunostaining against c-fos and quantification of cells expressing c-fos. CRD induced Fos protein expression in superficial lamine of spinal cord, we count the c-fos + neurons in lamine I-V of spinal cord of view at magnification ×10 and average the results. Results are shown for five animals per condition. Scale bar, 50 µm. Based on one-way ANOVA and the Bonferroni multiple-comparisons test: **P < 0.01, ****P < 0.0001 between CON and CON + CRD or between MS and MS +CRD; #### P < 0.0001 between CON + CRD and MS + CRD. (D) Representative Western blotting of total lysate of spine tissue and quantification of EphB2 and EphrinB2. Protein levels were normalized to those of Actin in the same sample, and the relative protein level in the control group without CRD was defined as 1. Quantification is shown for four animals per treatment. Based on one-way ANOVA and the Bonferroni multiple-comparisons test: *P < 0.05, ****P < 0.0001 between CON and CON + CRD or between MS and MS +CRD; #### P < 0.0001 between CON + CRD and MS + CRD. (E) Correlation between levels of c-fos and levels of EphB2 or EphrinB2 based on Western blotting of total lysates of spine tissue.

Article Snippet: Mice that experienced maternal separation or not were anesthetized intraperitoneally using sodium pentobarbital (40 mg/kg), then injected intrathecally with one of the following: a chimera of EphB2 with Fc (catalog no. 467-B2, R&D Systems, Minnesota, United States), which binds to EphrinB2 and prevents it from stimulating endogenous EphB2; a chimera of EphrinB2 and Fc (catalog no. 496-EB, R&D Systems, Minnesota, United States), which binds and activates endogenous EphB2; or, as a negative control, recombinant Fc from human IgG1 (catalog no. 110-HG, R&D Systems, Minnesota, United States).

Techniques: Control, Immunostaining, Expressing, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Activation of EphrinB2/EphB2 signaling in the spine cord alters glia-neuron interactions in mice with visceral hyperalgesia following maternal separation

doi: 10.3389/fphar.2024.1463339

Figure Lengend Snippet: Visceral hyperalgesia in response to colorectal distension activates EphrinB2/EphB2 signaling and downstream MAP kinases. Mice were subjected to maternal separation (MS) early in development or not (CON), then later subjected to colorectal distension (CRD) or not. (A) Representative western blots of total lysate from spinal cord and quantification of the active, phosphorylated forms of the MAP kinases ERK, p38 and JNK. Levels of phosphorylated protein were normalized to those of the corresponding total protein, and the relative level of phosphorylated protein in the control group without CRD was defined as 1. Quantification is shown for four animals per condition. Based on one-way ANOVA and the Bonferroni multiple-comparisons test: *P < 0.05, ****P < 0.0001 between CON and CON + CRD or between MS and MS +CRD; #### P < 0.0001 between CON + CRD and MS + CRD. (B–D) Immunostaining of thin sections of spinal cord against EphB2 (red) and the phosphorylated forms of p38, ERK, or JNK (green). Scale bar, 50 µm. The boxed regions in the large images are shown at higher magnification on the far right (“Zoom”). (E) Co-expression of spinal EphB2 and MAPKs. Based on one-way ANOVA and the Bonferroni multiple-comparisons test: *P < 0.05, ***P < 0.001 between CON and MS. (F) Proportion of spinal cord cells expressing each activated MAP kinase that also expressed EphB2 ( left plot ) or proportion of spinal cord cells expressing EphB2 that also expressed each of the phosphorylated MAP kinases ( right plot ).

Article Snippet: Mice that experienced maternal separation or not were anesthetized intraperitoneally using sodium pentobarbital (40 mg/kg), then injected intrathecally with one of the following: a chimera of EphB2 with Fc (catalog no. 467-B2, R&D Systems, Minnesota, United States), which binds to EphrinB2 and prevents it from stimulating endogenous EphB2; a chimera of EphrinB2 and Fc (catalog no. 496-EB, R&D Systems, Minnesota, United States), which binds and activates endogenous EphB2; or, as a negative control, recombinant Fc from human IgG1 (catalog no. 110-HG, R&D Systems, Minnesota, United States).

Techniques: Western Blot, Control, Immunostaining, Expressing

Journal: Frontiers in Pharmacology

Article Title: Activation of EphrinB2/EphB2 signaling in the spine cord alters glia-neuron interactions in mice with visceral hyperalgesia following maternal separation

doi: 10.3389/fphar.2024.1463339

Figure Lengend Snippet: Expression of EphrinB2 and EphB2 in neurons, astrocytes and microglia in spinal cord. (A, B) Mice that with (MS) or without (Con) experienced maternal separation were subjected to colorectal distension, then the spinal cord was thin-sectioned and immunostained against EphB2 (A) or EphrinB2 (B) and a marker for neurons (NeuN), activated microglia (Iba1), or astrocytes (GFAP). Scale bar, 50 µm. The boxed regions in the large images are shown at higher magnification on the far right (“Zoom”). (C, D) Co-expression of spinal EphB2 or EphrinB2 and NeuN, Iba1or GFAP. Based on one-way ANOVA and the Bonferroni multiple-comparisons test: *P < 0.05, **P < 0.01 between CON and MS. (E, F) The proportion of EphB2 (E) or EphrinB2 (F) activation in neurons (NeuN), microglia (Iba1), or astrocytes (GFAP).

Article Snippet: Mice that experienced maternal separation or not were anesthetized intraperitoneally using sodium pentobarbital (40 mg/kg), then injected intrathecally with one of the following: a chimera of EphB2 with Fc (catalog no. 467-B2, R&D Systems, Minnesota, United States), which binds to EphrinB2 and prevents it from stimulating endogenous EphB2; a chimera of EphrinB2 and Fc (catalog no. 496-EB, R&D Systems, Minnesota, United States), which binds and activates endogenous EphB2; or, as a negative control, recombinant Fc from human IgG1 (catalog no. 110-HG, R&D Systems, Minnesota, United States).

Techniques: Expressing, Marker, Activation Assay

Journal: Frontiers in Pharmacology

Article Title: Activation of EphrinB2/EphB2 signaling in the spine cord alters glia-neuron interactions in mice with visceral hyperalgesia following maternal separation

doi: 10.3389/fphar.2024.1463339

Figure Lengend Snippet: Inhibition of EphrinB2/EphB2 signaling mitigates the effects of maternal separation. (A) Schematic illustration of the experimental protocol. Mice were subjected to maternal separation (MS) early in development or not (CON), intrathecally injected with a chimera of EphB2 and Fc (EphB2-Fc) that inhibits EphB2 signaling or a negative-control chimera (IgG-Fc), then subjected to colorectal distension (CRD), during which the abdominal withdrawal reflex (AWR) was measured. Subsequently, the spinal cord was analyzed by Western blotting and immunohistochemistry to observe expression of key proteins. (B) AWR score during CRD. Quantification is shown for eight animals per condition. Based on two-way repeated-measure ANOVA and the Bonferroni multiple-comparisons test: **P < 0.01, ****P < 0.0001 between CON + IgG-Fc and MS + IgG-Fc; # p < 0.05, ### p < 0.001 between MS + IgG-Fc and MS + EphB2-Fc. (C) Representative micrographs of spinal cord tissue after immunostaining against c-fos and quantification of the number of cells expressing c-fos. We count the c-fos + neurons in lamine I-V of spinal cord of view at magnification ×10 and average the results. Results are shown for five animals per condition. Scale bar, 50 µm. Based on one-way ANOVA and the Bonferroni multiple-comparisons test: ****P < 0.0001 between MS + IgG-Fc and MS + IgG-Fc + CRD; ## P < 0.01 between MS + IgG-Fc + CRD and MS + EphB2-Fc + CRD. (D–F) Representative western blots of total lysate from spinal cord and quantification of (D) the active, phosphorylated forms of the MAP kinases ERK, p38 and JNK; (E) the cell type markers NeuN, GFAP and Iba1; or (F) NR2B and NR2B phosphorylated on Tyr1472. Levels of phosphorylated protein were normalized to those of the corresponding total protein, while levels of markers, NR2B or phospho-NR2B were normalized to those of Actin; the resulting relative protein levels in the MS+IgG-Fc group were defined as 1. Quantification is shown for four animals per condition. Based on unpaired t-test, ****P < 0.0001 compared with MS + IgG-Fc group.

Article Snippet: Mice that experienced maternal separation or not were anesthetized intraperitoneally using sodium pentobarbital (40 mg/kg), then injected intrathecally with one of the following: a chimera of EphB2 with Fc (catalog no. 467-B2, R&D Systems, Minnesota, United States), which binds to EphrinB2 and prevents it from stimulating endogenous EphB2; a chimera of EphrinB2 and Fc (catalog no. 496-EB, R&D Systems, Minnesota, United States), which binds and activates endogenous EphB2; or, as a negative control, recombinant Fc from human IgG1 (catalog no. 110-HG, R&D Systems, Minnesota, United States).

Techniques: Inhibition, Injection, Negative Control, Western Blot, Immunohistochemistry, Expressing, Immunostaining

Journal: Frontiers in Pharmacology

Article Title: Activation of EphrinB2/EphB2 signaling in the spine cord alters glia-neuron interactions in mice with visceral hyperalgesia following maternal separation

doi: 10.3389/fphar.2024.1463339

Figure Lengend Snippet: Activation of EphrinB2/EphB2 signaling reproduces the effects of maternal separation in naïve mice. (A) Schematic illustration of the experimental protocol. Mice that had not experienced maternal separation early in development were intrathecally injected with a chimera of EphrinB2 and Fc (EphB2-Fc) that activates EphB2 signaling or a negative-control chimera (IgG-Fc), then subjected to colorectal distension (CRD), during which the abdominal withdrawal reflex (AWR) was measured. Subsequently, the spinal cord was analyzed by Western blotting and immunohistochemistry to observe expression of key proteins. (B) AWR score during CRD. Quantification is shown for eight animals per condition. Based on two-way repeated-measure ANOVA and the Bonferroni multiple-comparisons test: *P < 0.05, **P < 0.01, ****P < 0.0001 vs IgG-Fc group. (C) Representative western blots of total lysate from spinal cord and quantification of the active, phosphorylated forms of the MAP kinases ERK, p38 and JNK. Levels of phosphorylated protein were normalized to those of the corresponding total protein, and the relative level of phosphorylated protein in the IgG-Fc group was defined as 1. Quantification is shown for four animals per condition. Based on one-way ANOVA and Bonferroni multiple comparisons test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. IgG-Fc group, n = 4 mice in each group. (D, E) Representative western blots of total lysate from spinal cord and quantification of (D) the cell type markers NeuN, GFAP and Iba1; or (E) c-fos, NR2B, and NR2B phosphorylated on Tyr1472. Protein levels were normalized to those of Actin, and the relative protein level in the IgG-Fc group was defined as 1. Quantification is shown for four animals per condition. Based on one-way ANOVA and Bonferroni multiple comparisons test, *P < 0.05, ***P < 0.001, ****P < 0.0001 vs. IgG-Fc group.

Article Snippet: Mice that experienced maternal separation or not were anesthetized intraperitoneally using sodium pentobarbital (40 mg/kg), then injected intrathecally with one of the following: a chimera of EphB2 with Fc (catalog no. 467-B2, R&D Systems, Minnesota, United States), which binds to EphrinB2 and prevents it from stimulating endogenous EphB2; a chimera of EphrinB2 and Fc (catalog no. 496-EB, R&D Systems, Minnesota, United States), which binds and activates endogenous EphB2; or, as a negative control, recombinant Fc from human IgG1 (catalog no. 110-HG, R&D Systems, Minnesota, United States).

Techniques: Activation Assay, Injection, Negative Control, Western Blot, Immunohistochemistry, Expressing

Journal: Frontiers in Pharmacology

Article Title: Activation of EphrinB2/EphB2 signaling in the spine cord alters glia-neuron interactions in mice with visceral hyperalgesia following maternal separation

doi: 10.3389/fphar.2024.1463339

Figure Lengend Snippet: Proposed model for the role of EphrinB2/EphB2 signaling in visceral hyperalgesia due to stress in early life. Activation of spinal EphrinB2/EphB2 signaling alters glia-neuron interactions, which contributes to visceral hyperalgesia arising from the stress of earlier maternal separation. Upon activation of EphrinB2/EphB2 signaling, spinal astrocytes and microglia interact with neurons via synapses, activated glia release inflammatory mediators that induce excessive excitatory neuronal activity involving the NMDA receptor. Figure Created with BioRender.com .

Article Snippet: Mice that experienced maternal separation or not were anesthetized intraperitoneally using sodium pentobarbital (40 mg/kg), then injected intrathecally with one of the following: a chimera of EphB2 with Fc (catalog no. 467-B2, R&D Systems, Minnesota, United States), which binds to EphrinB2 and prevents it from stimulating endogenous EphB2; a chimera of EphrinB2 and Fc (catalog no. 496-EB, R&D Systems, Minnesota, United States), which binds and activates endogenous EphB2; or, as a negative control, recombinant Fc from human IgG1 (catalog no. 110-HG, R&D Systems, Minnesota, United States).

Techniques: Activation Assay, Activity Assay