Journal: International Journal of Molecular Sciences

Article Title: Purinergic Receptors P2X7 and P2X4 as Markers of Disease Progression in the rd10 Mouse Model of Inherited Retinal Dystrophy

doi: 10.3390/ijms232314758

Figure Lengend Snippet: P2X7R expression in mouse retina. ( A ) Representative western blot and ( B ) protein quantification of P2X7R expression in C57BL/6J, rd10early, and rd10late mice. ( C ) Mean fluorescence values of P2X7R immunostaining in P2X7R-positive cells analyzed by flow cytometry in rd10early and rd10late mice compared with C57BL/6J mice. Student’s t -test * p < 0.05, ** p < 0.01. C57BL/6J1: postnatal day (P)20; C57BL/6J2: P40–P47; rd10early: P18 and rd10late: P44.

Article Snippet: Membranes were incubated at 4 °C overnight with the appropriate dilution of the following primary antibodies: rabbit anti-P2X7 receptor (extracellular) (1:200; Alomone Labs), rabbit anti-P2X4 receptor (extracellular) (1:200; Alomone Labs), and mouse anti-GAPDH (Sigma-Aldrich).

Techniques: Expressing, Western Blot, Fluorescence, Immunostaining, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Purinergic Receptors P2X7 and P2X4 as Markers of Disease Progression in the rd10 Mouse Model of Inherited Retinal Dystrophy

doi: 10.3390/ijms232314758

Figure Lengend Snippet: P2X7R expression in mouse retinas analyzed by immunohistochemistry. Cross-sectional cryosections of retinas from a representative C57BL/6J mouse ( A , D , G ) and rd10early ( B , E , H ) and rd10late ( C , F , I ) mice, stained with antibodies against P2X7R ( A – C , G – I ). Nuclei were stained with TO-PRO ( D – F ). In C57BL/6J mice, immunopositive fluorescence against P2X7R appears mainly located in the INL and GCL. RPE: retinal pigment epithelial cells; OS: outer segments; IS: inner segments; ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer. Scale bar: 50 µm.

Article Snippet: Membranes were incubated at 4 °C overnight with the appropriate dilution of the following primary antibodies: rabbit anti-P2X7 receptor (extracellular) (1:200; Alomone Labs), rabbit anti-P2X4 receptor (extracellular) (1:200; Alomone Labs), and mouse anti-GAPDH (Sigma-Aldrich).

Techniques: Expressing, Immunohistochemistry, Staining, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Purinergic Receptors P2X7 and P2X4 as Markers of Disease Progression in the rd10 Mouse Model of Inherited Retinal Dystrophy

doi: 10.3390/ijms232314758

Figure Lengend Snippet: P2X4R expression in mouse retina. ( A ) Representative western blot and ( B ) protein quantification of P2X4R expression in C57BL/6J, rd10early, and rd10late mice. ( C ) Mean fluorescence values of P2X4R immunostaining in P2X7R-positive cells analyzed by flow cytometry in rd10early and rd10late mice compared with the control expression in C57BL/6J mice. Student’s t -test, * p < 0.05. C57BL/6J1: postnatal day (P)20; C57BL/6J2: P40-P47; rd10early: P18 and rd10late: P44.

Article Snippet: Membranes were incubated at 4 °C overnight with the appropriate dilution of the following primary antibodies: rabbit anti-P2X7 receptor (extracellular) (1:200; Alomone Labs), rabbit anti-P2X4 receptor (extracellular) (1:200; Alomone Labs), and mouse anti-GAPDH (Sigma-Aldrich).

Techniques: Expressing, Western Blot, Fluorescence, Immunostaining, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Purinergic Receptors P2X7 and P2X4 as Markers of Disease Progression in the rd10 Mouse Model of Inherited Retinal Dystrophy

doi: 10.3390/ijms232314758

Figure Lengend Snippet: P2X7R and P2X4R expression in retinal myeloid cells (CD11b + ). The CD11b immunoreactive cell population was analyzed by flow cytometry. Bar graphs show the number of CD11b + cells expressing P2X7R ( A ) and the mean intensity of P2X7R fluorescence values ( B ). Also, the number of CD11b + cells expressing P2X4R is shown ( C ), and the mean intensity of P2X4R fluorescence values ( D ). C57BL/6J: postnatal day (P)47; rd10early: P18 and rd10late: P44. ** p < 0.01, *** p < 0.001.

Article Snippet: Membranes were incubated at 4 °C overnight with the appropriate dilution of the following primary antibodies: rabbit anti-P2X7 receptor (extracellular) (1:200; Alomone Labs), rabbit anti-P2X4 receptor (extracellular) (1:200; Alomone Labs), and mouse anti-GAPDH (Sigma-Aldrich).

Techniques: Expressing, Flow Cytometry, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Purinergic Receptors P2X7 and P2X4 as Markers of Disease Progression in the rd10 Mouse Model of Inherited Retinal Dystrophy

doi: 10.3390/ijms232314758

Figure Lengend Snippet: P2X7R and P2X4R expression in the CD11b + population, analyzed by flow cytometry. Whole retinal cells from C57BL/6J, rd10early, and rd10late mice were triple-stained with antibodies against CD11b, P2X7R, and P2X4R. After discarding the cell debris and selecting singlets, CD11b + cells were gated ( A ) and the expression of P2X7R and P2X4R was analyzed ( B – D ). ( B ) Double contour plots representing P2X7R and P2X4R expression, showing P2X7R- and P2X4R-positive populations (green line) and P2X7R- and P2X4R-highly immunoreactive populations (blue line). Each plot shows the sum of a minimum of 3 independent replicates. ( C , D ) Bar graphs showing the number of double-positive cells (P2X7R and P2X4R) and mean fluorescence values for P2X7R and P2X4R in the whole double-positive population ( C ) and the highly immunoreactive double-positive population ( D ). Student’s t -test, * p < 0.05, ** p < 0.01, *** p < 0.001, ns: non-statistically significant. C57BL/6J: postnatal day (P)47; rd10early: P18 and rd10late: P44.

Article Snippet: Membranes were incubated at 4 °C overnight with the appropriate dilution of the following primary antibodies: rabbit anti-P2X7 receptor (extracellular) (1:200; Alomone Labs), rabbit anti-P2X4 receptor (extracellular) (1:200; Alomone Labs), and mouse anti-GAPDH (Sigma-Aldrich).

Techniques: Expressing, Flow Cytometry, Staining, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Purinergic Receptors P2X7 and P2X4 as Markers of Disease Progression in the rd10 Mouse Model of Inherited Retinal Dystrophy

doi: 10.3390/ijms232314758

Figure Lengend Snippet: P2X7R and P2X4R expression analysis in CD11b+ cells, assessed by flow cytometry. Whole retinal cells from C57BL/6J, rd10early, and rd10late mice were triple-stained with antibodies against CD11b, P2X7R, and P2X4R. After discarding the cell debris and selecting singlets, CD11b-immunopositive cells were gated and two populations were selected according to medium or high immunoreactivity against the CD11b antibody, as shown in the contour plot (rd10 postnatal day [P]18) ( A ). The dot plot shows the sum of a minimum of 3 independent replicates. The immunoreactivity against P2X7R ( B ) and P2X4R ( C ) was compared in the two CD11b populations of each group of mice. Student’s t -test was performed between the two populations in each condition. Data are presented as mean values ± SD. Student’s t -test, ** p < 0.01. C57BL/6J: P47; rd10early: P18 and rd10late: P44.

Article Snippet: Membranes were incubated at 4 °C overnight with the appropriate dilution of the following primary antibodies: rabbit anti-P2X7 receptor (extracellular) (1:200; Alomone Labs), rabbit anti-P2X4 receptor (extracellular) (1:200; Alomone Labs), and mouse anti-GAPDH (Sigma-Aldrich).

Techniques: Expressing, Flow Cytometry, Staining

Journal: Frontiers in psychiatry

Article Title: Dihydromyricetin Alleviates Diabetic Neuropathic Pain and Depression Comorbidity Symptoms by Inhibiting P2X 7 Receptor.

doi: 10.3389/fpsyt.2019.00770

Figure Lengend Snippet: FIGURE 1 | Molecular docking of dihydromyricetin (DHM) on P2X7 receptor. (A) Simulation modeling of DHM docking on P2X7 receptor was performed by a computer. Molecular docking prediction of DHM on P2X7 receptor was performed by AutoDock 4.2. (B–D) Enlarged view indicating the perfect match enabling DHM to interact with P2X7 receptor.

Article Snippet: Image-Pro Plus6.0 software was used to analyze the immunofluorescence intensity ratio of P2X7 and GFAP coexpression normalized to control values. enzyme-linked Immunosorbent Assay For assessing the levels of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β), we performed enzyme-linked immunosorbent assay (ELISA) using Rat PicoKineTM ELISA Kit (Boster Biological Technology), according to the manufacturer’s instructions.

Techniques:

Journal: Frontiers in psychiatry

Article Title: Dihydromyricetin Alleviates Diabetic Neuropathic Pain and Depression Comorbidity Symptoms by Inhibiting P2X 7 Receptor.

doi: 10.3389/fpsyt.2019.00770

Figure Lengend Snippet: FIGURE 4 | Effects of dihydromyricetin (DHM) on the expression of P2X7 mRNA in the dorsal root ganglia (DRGs; A), spinal cord (B), and hippocampus (C) of rats with diabetic neuropathic pain (DNP) and major depressive disorder (MDD) (model). Effects of DHM on the expression of P2X7 protein in the dorsal root ganglia (DRGs; D), spinal cord (E), and hippocampus (F) of rats with model. Data are displayed as the means ± standard error of the mean. **p<0.01 vs. control group;

Article Snippet: Image-Pro Plus6.0 software was used to analyze the immunofluorescence intensity ratio of P2X7 and GFAP coexpression normalized to control values. enzyme-linked Immunosorbent Assay For assessing the levels of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β), we performed enzyme-linked immunosorbent assay (ELISA) using Rat PicoKineTM ELISA Kit (Boster Biological Technology), according to the manufacturer’s instructions.

Techniques: Expressing, Control

Journal: Frontiers in psychiatry

Article Title: Dihydromyricetin Alleviates Diabetic Neuropathic Pain and Depression Comorbidity Symptoms by Inhibiting P2X 7 Receptor.

doi: 10.3389/fpsyt.2019.00770

Figure Lengend Snippet: FIGURE 5 | Effects of dihydromyricetin (DHM) on the coexpression of P2X7 and glial fibrillary acidic protein (GFAP) in the dorsal root ganglia (DRGs) of rats with diabetic neuropathic pain (DNP) and major depressive disorder (MDD) (model). Data are displayed as the means ± standard error of the mean. **p < 0.01 vs. control group; ##p < 0.01 vs. model group. Scale bars, 100 µm.

Article Snippet: Image-Pro Plus6.0 software was used to analyze the immunofluorescence intensity ratio of P2X7 and GFAP coexpression normalized to control values. enzyme-linked Immunosorbent Assay For assessing the levels of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β), we performed enzyme-linked immunosorbent assay (ELISA) using Rat PicoKineTM ELISA Kit (Boster Biological Technology), according to the manufacturer’s instructions.

Techniques: Control

Journal: Frontiers in psychiatry

Article Title: Dihydromyricetin Alleviates Diabetic Neuropathic Pain and Depression Comorbidity Symptoms by Inhibiting P2X 7 Receptor.

doi: 10.3389/fpsyt.2019.00770

Figure Lengend Snippet: FIGURE 6 | Effects of dihydromyricetin (DHM) on the coexpression of P2X7 and glial fibrillary acidic protein (GFAP) in the spinal cord of rats with diabetic neuropathic pain (DNP) and major depressive disorder (MDD) (model). Data are displayed as the means ± standard error of the mean. **p < 0.01 vs. control group; ##p < 0.01 vs. model group. Scale bars, 100 µm.

Article Snippet: Image-Pro Plus6.0 software was used to analyze the immunofluorescence intensity ratio of P2X7 and GFAP coexpression normalized to control values. enzyme-linked Immunosorbent Assay For assessing the levels of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β), we performed enzyme-linked immunosorbent assay (ELISA) using Rat PicoKineTM ELISA Kit (Boster Biological Technology), according to the manufacturer’s instructions.

Techniques: Control

Journal: Frontiers in psychiatry

Article Title: Dihydromyricetin Alleviates Diabetic Neuropathic Pain and Depression Comorbidity Symptoms by Inhibiting P2X 7 Receptor.

doi: 10.3389/fpsyt.2019.00770

Figure Lengend Snippet: FIGURE 7 | Effects of dihydromyricetin (DHM) on the coexpression of P2X7 and glial fibrillary acidic protein (GFAP) in the hippocampus of rats with diabetic neuropathic pain (DNP) and major depressive disorder (MDD) (model). Data are displayed as the means ± standard error of the mean. **p < 0.01 vs. control group; ##p < 0.01 vs. model group. Scale bars, 200 µm.

Article Snippet: Image-Pro Plus6.0 software was used to analyze the immunofluorescence intensity ratio of P2X7 and GFAP coexpression normalized to control values. enzyme-linked Immunosorbent Assay For assessing the levels of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β), we performed enzyme-linked immunosorbent assay (ELISA) using Rat PicoKineTM ELISA Kit (Boster Biological Technology), according to the manufacturer’s instructions.

Techniques: Control