Nine-min OGD induces an increase of DCX immunofluorescence in the SGZ of DG; this effect is antagonized by MRS2179.

<p>Immunohistochemical staining of BrdU⁺ and DCX⁺ cells in the SGZ of DG. Upper left panel: a schematic hippocampal slice. The region in the box (black arrow) represents the SGZ shown in a-d. Panels a-d: Confocal images of the double-staining immunohistochemical analysis of BrdU⁺ (green) and DCX⁺ (red) cells in the SGZ. Double-stained cells are clearly visible in the SGZ of DG in control slices 24 hours after slice preparation (a), and 24 hours from the end of OGD carried out in the absence (b) or in the presence of 10 µM BBG (c) or 10 µM MRS2179 (d). DCX⁺ neuroblasts show higher DCX immunofluorescence, if compared to control slice, both after OGD alone or OGD+BBG. This effect was not evident in the slice in which OGD was applied in combination with MRS2179 (d). The vast majority of BrdU⁺ nuclei belong to DCX⁺ cells, as shown at a higher magnification in the upper right panel (b1). SGZ: subgranular zone; GCL: granule cell layer; BrdU, 5′-Bromo-2′-deoxyuridine; DCX, doublecortin. Scale bar: 50 µm (a-d); 10 µm (b1). e: quantification of DCX immunofluorescence in the SGZ of DG in different experimental conditions: control slices (n = 8) or in OGD slices in the absence (n = 8) or in the presence of BBG (10 µM, n = 4) or MRS2179 (10 µM, n = 6). Analyses were performed 24 hours from the end of OGD and at the corresponding times in control conditions. Each column represents the area of DCX immunofluorescence calculated using ImageJ on confocal acquisitions (number of pixels in thresholded images). Bars represent the mean±SEM. In parentheses is reported the number of slices investigated. **<i>P</i><0.01 <i>vs</i> control and MRS2179 groups, One-way ANOVA followed by Newman–Keuls post hoc test.</p

Temporal profile of cell proliferation in the SGZ of DG.

<p>Quantification of BrdU⁺ cells in the SGZ of the DG at 3, 6 and 24 hours after the end of OGD. Each column shows the total number of BrdU⁺ cells in the SGZ. Bars represent the mean±SEM. In parentheses is the number of slices investigated. *<i>P</i><0.05 and **<i>P</i><0.01 <i>vs</i> control, One-way ANOVA followed by Newman–Keuls post hoc test.</p

The Selective Antagonism of P2X₇ and P2Y₁ Receptors Prevents Synaptic Failure and Affects Cell Proliferation Induced by Oxygen and Glucose Deprivation in Rat Dentate Gyrus

<div><p>Purinergic P2X and P2Y receptors are broadly expressed on both neurons and glial cells in the central nervous system (CNS), including dentate gyrus (DG). The aim of this research was to determine the synaptic and proliferative response of the DG to severe oxygen and glucose deprivation (OGD) in acute rat hippocampal slices and to investigate the contribution of P2X₇ and P2Y₁ receptor antagonism to recovery of synaptic activity after OGD. Extracellular field excitatory post-synaptic potentials (fEPSPs) in granule cells of the DG were recorded from rat hippocampal slices. Nine-min OGD elicited an irreversible loss of fEPSP and was invariably followed by the appearance of anoxic depolarization (AD). Application of MRS2179 (selective antagonist of P2Y₁ receptor) and BBG (selective antagonist of P2X₇ receptor), before and during OGD, prevented AD appearance and allowed a significant recovery of neurotransmission after 9-min OGD. The effects of 9-min OGD on proliferation and maturation of cells localized in the subgranular zone (SGZ) of slices prepared from rats treated with 5-Bromo-2′-deoxyuridine (BrdU) were investigated. Slices were further incubated with an immature neuron marker, doublecortin (DCX). The number of BrdU⁺ cells in the SGZ was significantly decreased 6 hours after OGD. This effect was antagonized by BBG, but not by MRS2179. Twenty-four hours after 9-min OGD, the number of BrdU⁺ cells returned to control values and a significant increase of DCX immunofluorescence was observed. This phenomenon was still evident when BBG, but not MRS2179, was applied during OGD. Furthermore, the P2Y₁ antagonist reduced the number of BrdU⁺ cells at this time. The data demonstrate that P2X₇ and P2Y₁ activation contributes to early damage induced by OGD in the DG. At later stages after the insult, P2Y₁ receptors might play an additional and different role in promoting cell proliferation and maturation in the DG.</p></div

The block of P2Y1R counteracts the synaptic failure induced by severe OGD in the DG.

<p>AD was recorded as a negative d.c. shift in response to 9-min OGD in control (<b>a</b>, n = 26). MRS2179 (10 µM) completely prevented the appearance of AD in 23 out of 28 slices during 9-min OGD (<b>b</b>). MRS2179 (10 µM) had no effect in 5 out of 28 slices after 9-min OGD (<b>c</b>, n = 6), in which AD was recorded as a negative d.c. shift. <b>d</b>. example traces taken at the time-points indicated on the graph, immediately before (1), 10 min after the beginning of MRS2179 application (2), 9 minutes after OGD+MRS2179 (3) or 50 min after washout in oxygenated aCSF (4). Inset: example trace taken from the same hippocampal slice 24 hours from the end of OGD applied in the presence of 10 µM MRS2179. In each graph, traces are averages of 3 consecutive responses. Scale bars: 10 ms, 0.5 mV. <b>f</b>. The graph shows the time-course of the effect caused by 9-min OGD on fEPSP amplitude (mean±SEM) in untreated OGD slices (n = 26) and in OGD slices treated with 10 µM MRS2179, in which AD was absent (n = 23) or present (n = 5). fEPSPs amplitude is expressed as percent of the respective pre-ischemic baseline. Open bar: time of drug application.</p

Antagonism of P2X7R or P2Y1R does not affect basal synaptic transmission, but significantly delays the AD appearance during prolonged OGD.

<p>a. Bars in the graph represent the average fEPSP amplitude (mean±SEM) recorded immediately before (control) and at the end of 10 min application of 10 µM BBG or 10 µM MRS2179. b. Each column represents the mean ± SEM of AD latency (left panel) and AD amplitude (right panel) recorded in the DG during 30 min OGD in the absence (n = 11) or in the presence of BBG (10 µM, n = 4) or MRS2179 (10 µM, n = 4) alone or in combination (n = 4). Note that both P2 antagonists significantly delayed AD development and that this effect is not modified by the combination of the two drugs (**<i>P</i><0.01, One-way ANOVA followed by Newman–Keuls post hoc test, compared to untreated OGD slices). AD latency was measured from the beginning of OGD insult. The number (n) of slices tested is reported inside columns.</p

AD development and synaptic failure in the DG of rat hippocampal slices after 9-min OGD.

<p><b>a</b>. Upper panel: fEPSP taken from a typical experiment at the time-points indicated on the graph in a control hippocampal slice (Ctr) continuously superfused with oxygenated aCSF. Lower panel: example traces taken at the time-points indicated on the graph recorded from a hippocampal slice before (4), at the end of 9-min OGD (5) or 50 min after washout in oxygenated aCSF (6). <b>b</b>. The graph shows the time-course of the fEPSP amplitude, expressed as percent of pre-ischemic baseline in the DG (mean±SEM) in a group of control slices (Ctr, n = 7) or in a group of slices subjected to 9-min OGD (OGD, n = 34). Note that, while a stable fEPSP was recorded in control slices, the ischemic insult elicited a gradual reduction of fEPSPs amplitude, which completely disappears and does not recover even after prolonged washing in oxygenated aCSF. Inset: example traces taken from the same respective hippocampal slices shown in panel <b>a</b>, in control (Ctr, recorded 24 hours from slice preparation) or 24 hours from the end of OGD. In each graph, traces are averages of 3 consecutive responses. Scale bars: 10 ms, 0.5 mV. <b>c</b>. Anoxic depolarization (AD) was recorded as a negative d.c. shift in response to 9-min OGD. The d.c. shift was always recorded (n = 34) during 9-min OGD. <b>d</b>. Each column represents the mean±SEM of AD latency or AD amplitude recorded in the DG during 9-min OGD. AD latency was measured from the beginning of OGD insult.</p

The P2X7R antagonist, BBG, prevents the synaptic failure induced by 9-min OGD in the DG.

<p>AD was recorded as a negative d.c. shift in response to 9-min OGD in control conditions (<b>a</b>, n = 21). BBG (10 µM) completely prevented the appearance of AD in 22 out of 28 slices during 9-min OGD (<b>b</b>). BBG (10 µM) had no effect in 6 out of 28 slices after 9-min OGD (<b>c</b>, n = 6), in which AD was recorded as a negative d.c. shift. <b>d</b>. example traces taken at the time-points indicated on the graph, immediately before (1), 10 min after the beginning of BBG application (2), 9 minutes after OGD+BBG (3) or 50 min after washout in oxygenated aCSF (4). Inset: example trace taken from the same hippocampal slice 24 hours from the end of OGD applied in the presence of 10 µM BBG. In each graph, traces are averages of 3 consecutive responses. Scale bars: 10 ms, 0.5 mV. <b>f</b>. The graph shows the time-course of the effect caused by 9-min OGD on fEPSP amplitude (mean ± SEM) in untreated OGD slices (n = 21) and in OGD slices treated with 10 µM BBG, in which AD was absent (n = 22) or present (n = 6). fEPSPs amplitude is expressed as percent of the respective pre-ischemic baseline. Open bar: time of drug application.</p

Additional file 1 of VEGF-A/VEGFR-1 signalling and chemotherapy-induced neuropathic pain: therapeutic potential of a novel anti-VEGFR-1 monoclonal antibody

Additional file 1: Table S1. List of antibodies used for immunohistochemistry and western blot assays

Additional file 3 of VEGF-A/VEGFR-1 signalling and chemotherapy-induced neuropathic pain: therapeutic potential of a novel anti-VEGFR-1 monoclonal antibody

Additional file 3: Figure S2. Hypersensitivity-induced by VEGF-A family members is not due to the interaction with VEGFR-2. The response to a thermal stimulus (Cold plate test) was recorded after intrathecal infusion (30 ng) of (a) VEGF165b (n=5), (b) PlGF-2 (n=5), (c) VEGF-E (n=5), following pre-treatment (15 min before) with vehicle or anti-VEGFR-2 mAb DC101 (100 pg). Each value represents the mean ± SEM. **P<0.01 vs vehicle + vehicle-treated animals. The analysis of variance was performed by one-way ANOVA. A Bonferroni’s significant difference procedure was used as post-hoc comparison

Additional file 10 of VEGF-A/VEGFR-1 signalling and chemotherapy-induced neuropathic pain: therapeutic potential of a novel anti-VEGFR-1 monoclonal antibody

Additional file 10: Figure S8. D16F7 mAb reduces oxaliplatin-induced pain after systemic administration. Effect of D16F7 mAb evaluated by (a) Cold plate and (b) Paw pressure tests in a mouse model of oxaliplatin-induced neuropathy after i.p. injection (a, b, n = 6). Each value represents the mean ± SEM. **P < 0.01 vs vehicle + vehicle-treated animals; ^^P < 0.01 vs oxaliplatin + vehicle-treated animals. The analysis of variance was performed by one-way ANOVA. A Bonferroni’s significant difference procedure was used as post-hoc comparison