A: Seizure occurrence after perfusion of the <i>in</i><i>vitro</i> isolated guinea pig brain with different K⁺ salt solutions.

<p>The white columns represent the total number of experiments. The light grey, dark grey and black columns mark the effects of the perfusion of K⁺ salts at 8, 14.2m 20 mM, respectively. <b>B</b>: Time at onset (black columns) and duration (white columns) of seizure activity after perfusion with 14.2 KPF₆, 14.2 mM KClO₄ and 20 mM KBF₄. <b>C</b>: Brain parenchyma concentration of two salts (KPF₆ and KBF₄) estimated by ¹⁹F MR spectroscopy on CA1-EC specimens, collected after arterial perfusion with 14.2 mM KPF₆ (n= 4; black column) and 20 mM KBF₄ (n= 3; grey column). </p

Effects of 4.2 mM KPF₆ (A), 14.2 mM KClO₄ (B) and 20 mM KCl (C) on the field potentials evoked in the piriform cortex by lateral olfactory tract (LOT) stimulation.

<p>Left, middle and right traces: before, during and after washout of the K⁺ salts, respectively.</p

A. Schematic protocol of the experiments. Electrophysiological recordings are performed for the entire experimental period

<p>Each perfusion of salts lasted 15 min. and was followed by 30 min. wash-out. Brain tissue samples were than collected and processed for the ¹⁹F MR spectroscopy. <b>B:</b> Structures of KX salts (X= PF₆^-, BF₄^-, ClO₄^-, Br^-, AcO^- and Cl^-) and electrostatic potential surfaces (B3LYP/6-311+G**) of individuals ions and ion pairs. Blue regions indicate positive potentials and red regions negative ones. Maximum values of the electrostatic potential, at K atom, and minimum values, at O, F, Br and Cl atoms are indicated besides each structure. Molecular volumes are also reported (ų).</p

KPF₆ 14.2 mM perfusion induced large amplitude depolarization events that propagated through the cortical mantel, from the hippocampus to the entorhinal (EC) and piriform cortex (PC).

<p>These events were interpreted as spreading depolarization phenomena (Somjen 2001). </p

Effects of perfusion of K⁺ salts in the <i>in</i><i>vitro</i> isolated guinea pig brain.

<p>A scheme of the position of the recording and stimulating (S1, on the lateral olfactory tract) electrodes is shown in the upper part of the figure. The upper set of traces illustrates the effect of 14.2 mM KPF₆ perfusion (right) compared to control (left); seizure activity was induced by this salt. In the lower part of the figure the effect of perfusion with 14.2 mM KClO₄ is shown. PC: piriform cortex; EC: entorhinal cortex; HIP: CA1 region of the hippocampus.</p

Figure 2

<p>A. Recostrution of the distribution of NCs superimposed on a coronal section immunoreacted with anti-MAP-2 antibody (immunoperoxydase staining) to identify the ischemic region. Green fluorescent CFDA-stained stem cells were counted and plotted on the adjacent section stained with the MAP-2 fluorescent antibody (calibration bar = 1 cm). In B and C, microphotographs of green fluorescent CFDA-stained stem cells on sections counterstained with the fluorescent anti-MAP-2 antibody are shown at ×10 (B) and ×40 (C) magnifications. The pictures were taken in the transition region between the MAP-2⁺ and the MAP-2⁻ areas in the olfactory region. Calibration bars: 150 µm and 16 µm for B and C, respectively.</p

Mean NCs density (cells/cm²) in the MAP-2 positive zone contralateral (left column) and ipsilateral (middle column) to the ischemic side and in the MAP-2 negative zone of the ischemic hemisphere (right column) in 6 different experiments.

<p>On the left, the number of slices on which counts were made for each experiments is indicated.</p

Confocal fluorescence microscope images of PECAM-1 stained sections (50 µm thick) cut from the piriform region of brains fixed at the end of the electrophysiological experiment.

<p>CFDA green fluorescent NCs and brain vessels are shown at different magnifications. Calibration bar = 100 µm. NCs were observed in close proximity to cerebral vessels.</p

Experimental protocols.

<p>Ischemia was induced for 30 minutes, 2 hours after the <i>in vitro</i> placement of the isolated brain. NC were perfused for 1 h immediately either after the reopening of the vessel or 1 our later (protocol 2). The perfusion was followed by a wash-out period with a solution without NCs. At 5 hours <i>in vitro</i> the brains were fixed for immunohistochemistry. The bottom of the panel shows an example of simultaneous DC recordings from 4 different sites in an isolated guinea pig brain. Hypoxic depressions (HD) were recorded in the electrodes located in the regions vascularized by the occluded MCA. Potentials evoked by LOT stimulation before and during the first part of ischemia (arrowhead) disappeared when HD occurred, and recovered during MCA reperfusion. Evoked potentials in the hemisphere contralateral to MCA occlusion were not altered.</p

Figure 4

<p>(A) Changes in extracellular pH <u>(pH_e)</u> in the PC and in mOT induced by ipsilateral MCA occlusion and reperfusion. Occlusion induced a rapid metabolic acidification of the extracellular microenviroment in PC, interrupted by a transient and mild basification (arrow) associated to the hypoxic spreading depression (HD, asterisk). No changes in extracellular [H⁺] were recorded in the mOT, that is not served by the MCA. (B) Simultaneous changes in extracellular potassium concentration ([K⁺]_o)and extracellular pH in the PC after MCA occlusion and reperfusion. An initial enhancement in [K⁺] was followed by a fast and large increase in [K⁺]_o. associated to the HD. The schematic drawing on the right illustrates the position of the two-barrel recording electrodes. The field responses (FP) recorded with the conventional extracellular barrel are also shown. The period of MCA occlusion is marked by the shaded area.</p