The P2 Receptor Antagonist PPADS Supports Recovery from Experimental Stroke In Vivo

BACKGROUND: After ischemia of the CNS, extracellular adenosine 5'-triphosphate (ATP) can reach high concentrations due to cell damage and subsequent increase of membrane permeability. ATP may cause cellular degeneration and death, mediated by P2X and P2Y receptors. METHODOLOGY/PRINCIPAL FINDINGS: The effects of inhibition of P2 receptors by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) on electrophysiological, functional and morphological alterations in an ischemia model with permanent middle cerebral artery occlusion (MCAO) were investigated up to day 28. Spontaneously hypertensive rats received PPADS or vehicle intracerebroventricularly 15 minutes prior MCAO for up to 7 days. The functional recovery monitored by qEEG was improved by PPADS indicated by an accelerated recovery of ischemia-induced qEEG changes in the delta and alpha frequency bands along with a faster and sustained recovery of motor impairments. Whereas the functional improvements by PPADS were persistent at day 28, the infarct volume measured by magnetic resonance imaging and the amount of TUNEL-positive cells were significantly reduced by PPADS only until day 7. Further, by immunohistochemistry and confocal laser scanning microscopy, we identified both neurons and astrocytes as TUNEL-positive after MCAO. CONCLUSION: The persistent beneficial effect of PPADS on the functional parameters without differences in the late (day 28) infarct size and apoptosis suggests that the early inhibition of P2 receptors might be favourable for the maintenance or early reconstruction of neuronal connectivity in the periinfarct area after ischemic incidents

Effect of PPADS on the motor deficit in the beam balance test and the rotarod test up to day 28 after permanent MCAO.

<p>Values are expressed as means ± SEM. (n = 8 each group) of sideslips on the beam (<b>A</b>) and the percentage time of baseline the animals kept on the rotarod (<b>B</b>), * <i>P</i><0.05, ** p<0.001 <i>vs.</i> MCAO/ACSF, + <i>P</i><0.05 <i>vs.</i> day 1, # <i>P</i><0.01 <i>vs.</i> basal.</p

Effect of PPADS on the time course of EEG changes in the delta- and alpha-frequency bands caused by permanent MCAO of rats in four cortical regions up to day 28 after permanent MCAO.

<p>The delta-frequency band is shown in the left panel (<b>A</b>, <b>C</b>, <b>E</b>, <b>G</b>) and the alpha-frequency band in the right panel (<b>B</b>, <b>D</b>, <b>F</b>, <b>H</b>). The inset shows the placement of the four cortical electrodes and the reference electrode. The electrode P2 is located above the expected ischemic area indicated by grey shading, whereas the other electrodes are located above the contralateral parietal cortex (P1) and the respective frontal cortices (F2 and F1). Data are calculated as percental changes of the relative power from baseline measurements and expressed as means ± SEM. (ACSF: n = 7; PPADS: n = 8), * <i>P</i><0.05, ** <i>P</i><0.001 <i>vs.</i> MCAO/ACSF; + <i>P</i><0.05 <i>vs.</i> day 1, # <i>P</i><0.01 <i>vs.</i> basal.</p

Effect of PPADS on the extent of cell death measured at TUNEL-positive cells.

<p>(<b>Aa</b>) The brain slice shows the infarct area at the striatal level of the brain and the caudal and ventral areas in the penumbra counted for TUNEL-positive cells. An example for a cell with deep brown-labelled apoptotic bodies (arrow) is given (<b>Ab</b>; scale bar: 20 µm). Images of caudal areas at day seven after MCAO of TUNEL-immunostained brain slices from animals treated either with ACSF (<b>Ac</b>) or with PPADS (<b>Ad</b>). TUNEL-positive cells are indicated by arrows. (<b>B</b>) The pooled number of TUNEL-positive cells counted on striatal and hippocampal brain slices from animals treated either with ACSF or with PPADS is shown for day 1, day 7 and day 28. * <i>P</i><0.05 <i>vs.</i> MCAO/ACSF treated animals, + <i>P</i><0.05 <i>vs.</i> day 7. (<b>Ca–d</b>) Confocal images of double immunofluorescence to characterize TUNEL-positive cell bodies (yellow-green immunofluorescence) in degenerating neurons (<b>b</b>, red Cy3-immunofluorescence, MAP2-positive), and in astrocytes (<b>d</b>, red Cy3-immunofluorescence, GFAP-positive) in the periinfarct area, seven days after MCAO. The arrows indicate co-expression in the same cell (Scale bars: (a,b) = 20 µm; (c,d) = 10 µm).</p

Effect of PPADS on the infarct volume calculated at days one, seven and 28 after MCAO from volumetric image analysis of T2W MRI.

<p>Examples for the extension of the infarct area at day 7 after MCAO with ACSF and PPADS by T2W MRI (<b>A</b>). Volumes are expressed as means ± SEM (n = 8); * <i>P</i><0.05 <i>vs.</i> MCAO/ACSF (<b>B</b>). The infarct volumes are expressed as means ± SEM (n = 8 each), * <i>P</i><0.05 <i>vs.</i> MCAO/ACSF treated animals, + <i>P</i><0.05 <i>vs.</i> day 1.</p

Time schedule of the treatments and investigations in the three types of experiments performed.

<p>OP, operation; MCAO, medial cerebral artery occlusion.</p