Journal: Scientific Reports

Article Title: Involvement of P2X7 receptor in neuronal degeneration triggered by traumatic injury

doi: 10.1038/srep38499

Figure Lengend Snippet: ( A ) Retinal cross-sections stained for P2X7 receptor (green), Brn3a (red, detecting RGC nuclei), and nuclei (DAPI, blue), from intact or 5 days after injured (ONC) from wild type or P2rx7 −/− mice. The images show that the P2X7 receptor (green signal) in intact wild type retinas is found in the inner plexiform layer (asterisk), blood vessels (arrowheads) and weakly in the somas of RGCs (arrows, identified with Brn3a, red signal). Upon optic nerve crush, P2X7 receptor signal is stronger in the ganglion cell layer, mainly in the axons and somas of RGCs (arrows). In the P2rx7 −/− mice, some P2X7 receptor staining is observed in the inner plexiform layer and in blood vessels (arrowhead), but none in the GCL from intact or injured retinas. ( B ) Western blotting for P2X7 receptor or β-actin from cellular protein extract from mouse bone marrow derived macrophages (BMDM) or from retina pools ( n = 3 retinas/pool) of intact retinas, injured retinas (ONC) or contralateral fellow retinas (F) at different days after ONC. Asterisk denotes unspecific protein signal found in retinas. The right panel shows densitometry quantification of two Western blots for P2X7 receptor signal normalized to β-actin and to the expression on intact retinas (dashed line). d: days. Full-length blots are presented in .

Article Snippet: The P2X7 receptor was identified with rabbit anti-P2X7 receptor C-terminal (1:50, Alomone Labs).

Techniques: Staining, Western Blot, Derivative Assay, Expressing

Journal: Scientific Reports

Article Title: Involvement of P2X7 receptor in neuronal degeneration triggered by traumatic injury

doi: 10.1038/srep38499

Figure Lengend Snippet: Magnifications from flat mounted retinas showing Brn3a + RGCs (red) and OHSt traced-RGCs (white-blue) in the same frame from an intact P2rx7 −/− mice ( A ) and from wild type and P2rx7 −/− mice at increasing times after ONC ( B ). Green arrows point to phagocytic microglial cells. d: days.

Article Snippet: The P2X7 receptor was identified with rabbit anti-P2X7 receptor C-terminal (1:50, Alomone Labs).

Techniques:

Journal: Scientific Reports

Article Title: Involvement of P2X7 receptor in neuronal degeneration triggered by traumatic injury

doi: 10.1038/srep38499

Figure Lengend Snippet: ( A ) Isodensity maps showing the topography of Brn3a + RGCs in wild type (two left columns) and P2rx7 −/− (two right columns) mice. As the time post-lesion increases, the density of Brn3a + RGCs decreases across the retina, but this loss is delayed in the P2rx7 −/− mouse, in accordance with the quantitative data. Colour scale for these maps is shown in the first row, second map from the left and goes from 0 (purple) RGCs/mm 2 to 4,800 or more (red) RGCs/mm 2 . RE: right eye, LE: left injured eye. D: dorsal, N: nasal, T: temporal, V: ventral. ( B ) In the left graph the mean ± SD of Brn3a + RGCs in intact control (C) and injured retinas in both mice strains (Y axis) has been plotted against time post lesion (X axis). Up to 9 days after ONC, the loss of RGCs is linear and the slope of the regression line (m) is more steeper in wild type than in P2rx7 −/− mice because RGC loss is slower in the latter. The right panel shows the percentage of Brn3a + RGCs in the P2rx7 −/− considering 100% the number of Brn3a + RGCs in the wild type strain at the same time points (dashed line). *** Significantly greater number of Brn3a + RGCs compared to wild type at the same time point (Two way ANOVA; Bonferroni’s post-hoc test p < 0.001). d:days.

Article Snippet: The P2X7 receptor was identified with rabbit anti-P2X7 receptor C-terminal (1:50, Alomone Labs).

Techniques:

Journal: Scientific Reports

Article Title: Involvement of P2X7 receptor in neuronal degeneration triggered by traumatic injury

doi: 10.1038/srep38499

Figure Lengend Snippet: ( A ) Magnifications from flat mounted retinas showing Brn3a (red) and melanopsin (green) immunodetection in intact and injured wild type and P2rx7 −/− mice. ( B ) Neighbour maps showing the topography of m + RGCs in wild type (two left columns) and P2rx7 −/− (two right columns) mice. As the time post-lesion increases, the density of m + RGCs decreases mainly in the dorsal retina. In these maps each dot represents a single m + RGC and its colour indicates the number of m + RGCs around it (neighbours) in a radius of 0.16 mm and goes from 0–1 (purple) to 12–13 or more (red) neighbours. RE: right eye, LE: left injured eye. D: dorsal, N: nasal, T: temporal, V: ventral. ( C ) In the left graph the mean ± SD of m + RGCs in intact control ( C ) and injured retinas in both mice strains (Y axis) has been plotted against time post lesion (X axis). Up to 9 days after ONC, the loss of m + RGCs is linear but slower than the loss of RGCs. The right panel shows the percentage of m + RGCs in P2rx7 −/− mice considering 100% the number of m + RGCs in the wild type strain at the same time points (dashed line). ** Significantly greater number of m + RGCs compared to wild type at the same time point (Two way ANOVA; Bonferroni’s post-hoc test p < 0.01). d:days.

Article Snippet: The P2X7 receptor was identified with rabbit anti-P2X7 receptor C-terminal (1:50, Alomone Labs).

Techniques: Immunodetection

Journal: Scientific Reports

Article Title: Involvement of P2X7 receptor in neuronal degeneration triggered by traumatic injury

doi: 10.1038/srep38499

Figure Lengend Snippet: Histograms showing the total number (mean ± standard deviation) of Brn3a + (left) or melanopsin + (right) RGCs at increasing times after ONC and a single itravitreal administration of vehicle or 300 ng of the P2X7 receptor antagonist A438079. The number inside the bars is the percentage of survival considering intact retinas as 100%. One way ANOVA with Tukey post-hoc test to compare ONC + vehicle or ONC + antagonist vs . intact retinas or Two way ANOVA (variables time and treatment) with Bonferroni post hoc test to compare vehicle- vs . antagonist- treated retinas at the same time point. d:days.

Article Snippet: The P2X7 receptor was identified with rabbit anti-P2X7 receptor C-terminal (1:50, Alomone Labs).

Techniques: Standard Deviation

Journal: Scientific Reports

Article Title: Involvement of P2X7 receptor in neuronal degeneration triggered by traumatic injury

doi: 10.1038/srep38499

Figure Lengend Snippet: ( A,B ) Neighbour maps depicting the distribution of phagocytic microglial cells (PMCs) in injured retinas (left retinas, two left columns) and contralateral to the injured retinas (right retinas, two right columns) in wild type ( A ) and P2rx7 −/− ( B ) mice. In these maps each dot represents a single PMC and its colour indicates the number of PMCs around it (neighbours) in a radius of 0.165 mm and goes from 0–1 (purple) to 12–13 or more (red) neighbours. D: dorsal, N: nasal, T: temporal, V: ventral. ( C ) X,Y scatter plots showing the mean ± SD of PMCs in the injured (left panel) and contralateral to the lesion (right panel) retinas of both mice strains vs. time post-lesion. In the injured retinas, the increase of PMCs is linear. *Significantly greater number of PMCs compared to wild type at the same time point (Two way ANOVA; Bonferroni’s post-hoc test * p < 0.05, **p < 0.01, ***p < 0.001). d:days.

Article Snippet: The P2X7 receptor was identified with rabbit anti-P2X7 receptor C-terminal (1:50, Alomone Labs).

Techniques:

Journal: Scientific Reports

Article Title: Involvement of P2X7 receptor in neuronal degeneration triggered by traumatic injury

doi: 10.1038/srep38499

Figure Lengend Snippet: Number of retinas analyzed in this study.

Article Snippet: The P2X7 receptor was identified with rabbit anti-P2X7 receptor C-terminal (1:50, Alomone Labs).

Techniques: