Peripheral accumulation of Evans blue dye (EBD) and sodium fluorescein dye (NaF).

<p>(A) Representative images of rats before and after receiving EBD or NaF at equal doses (80 mg/kg i.v. bolus into the femoral vein). Post-injection images were taken at 2 h post-injection. (B) Blood concentration of EBD or NaF prior to and at 5, 30, 60, 90, and 120 min post-injection. Samples were collected from a cannulated femoral artery. (C) Peripheral EBD concentration in the blood and the liver at 120 min post-injection. Data are shown as mean±SEM. **p<0.01, comparison by paired t test. (D) Peripheral NaF concentration in the blood and the liver at 120 min post-injection. Data are shown as mean±SEM. *p<0.05, comparison by paired t test.</p

Central extravasation of Evans blue dye (EBD) following bolus injection and slow infusion.

<p>Regional brain distribution of extravasated EBD in the prefrontal cortex, the motor cortex, the striatum, and the cerebellum following bolus injection (left panel) or slow infusion (right panel) (80 mg/kg i.v. into the femoral vein). Data are shown as mean±SEM. Statistics used are described in the figure.</p

Peripheral accumulation of Evans blue dye (EBD) following bolus injection and slow infusion.

<p>(A) Representative images of rats before and after receiving 4% EBD via bolus injection or slow infusion (1 ml/hr) at equal doses (2 ml/kg i.v. into the femoral vein). (B) Blood concentration of EBD prior to and at 5, 30, 60, 90, and 120 min post-bolus injection or slow infusion. Samples were collected from a cannulated femoral artery. Data are shown as mean±SEM. *p<0.05 and **p<0.01, comparison by TWO-WAY repeated measures ANOVA followed by Sidak’s multiple comparisons test. (C) Peripheral EBD concentration in the liver at 120 min post-bolus injection or slow infusion. Data are shown as mean±SEM. n.s., no significant difference when compared by unpaired t test.</p

Illustration of the brain regions collected for analysis.

<p>Illustration of the brain regions collected for analysis.</p

Central extravasation of sodium fluorescein dye (NaF) in a dose-dependent manner.

<p>Regional brain distribution of extravasated NaF in the prefrontal cortex, the motor cortex, the striatum, and the cerebellum following bolus injection (2 ml/kg i.v. into the femoral vein) of 1%, 2%, or 8% NaF. Data are shown as mean±SEM. **p<0.01 when compared by TWO-WAY repeated measures ANOVA followed by Tukey’s multiple comparisons test.</p

Molecular level activation insights from a NR2A/NR2B agonist

<div><p>N-methyl D-aspartate receptors (NMDARs), a subclass of glutamate receptors have broad actions in neural transmission for major brain functions. Overactivation of NMDARs leading to “excitotoxicity” is the underlying mechanism of neuronal death in a number of neurological diseases, especially stroke. Much research effort has been directed toward developing pharmacological agents to modulate NMDAR actions for treating neurological diseases, in particular stroke. Here, we report that Alliin, a sulfoxide in fresh garlic, exhibits affinity toward NR2A as well as NR2B receptors based on virtual screening. Biological activities of Alliin on these two receptors were confirmed in electrophysiological studies. Ligand-binding site closure, a structural change precluding ion channel opening, was observed with Alliin during 100 ns molecular dynamics simulation. Alliin interactions with NR2A and NR2B suggest that residues E/A413, H485, T690, and Y730 may play important roles in the conformation shift. Activation of NR2A and NR2B by Alliin can be differentiated from that caused by glutamate, the endogenous neurotransmitter. These characteristic molecular features in NR2A and NR2B activation provide insight into structural requirements for future development of novel drugs with selective interaction with NR2A and NR2B for treating neurological diseases, particularly stroke.</p></div

Early infarct volume and blood-brain barrier (BBB) disruption following distal middle cerebral arterial occlusion (dMCAo).

<p>Mice were subjected to 30 min or 120 min of cerebral ischemia induced by dMCAo with or without ipsilateral (ipsi-) or contralateral (contra-) common carotid arterial occlusion (CCAo). (<b>A</b>) Infarct volume was revealed by 2,3,5-triphenyltetrazolium chloride staining 24 h post-ischemia (n = 6 per group). Data are expressed as mean ± SEM, and *p<0.05 and ***p<0.001 indicate a significant infarct volume. (<b>B</b>) BBB permeability was determined by measuring Evans blue dye (EBD) extravasation 6 h post-ischemia (n = 3–5 per group). Data are expressed as mean ± SEM, and *p<0.05 indicates a significant difference compared to the contralateral hemisphere.</p

Hypothermia induced by isoflurane-anesthesia had no effect on early infarct volume following distal middle cerebral arterial occlusion (dMCAo).

<p>To induce brain infarction, mice were subjected to 120 min of dMCAo coupled with ipsilateral common carotid arterial occlusion. At 4 h post-reperfusion (6 h after the onset of ischemia), hypothermia was induced by exposure to 1% or 2% isoflurane for 1 h. Control animals received 0% isoflurane in the same gas chamber and were normothermic. (<b>A</b>) Infarct area per coronal section was revealed by 2,3,5-triphenyltetrazolium chloride staining 24 h post-ischemia (n = 10 per group). Data are expressed as mean ± SEM. The x-axis indicates the location of the coronal section relative to the medial prefrontal cortex (mPFC). No significant difference was detected. (<b>B</b>) Infarct volume derived from (A); n = 10 per group. Data are expressed as mean ± SEM. n.s. indicates no significant difference.</p

Hypothermic but not normothermic isoflurane-anesthesia attenuates early blood-brain barrier (BBB) disruption following distal middle cerebral arterial occlusion (dMCAo).

<p>(<b>A</b>) Body temperature was measured continuously in mice using a rectal probe, and recorded at 0, 30, and 60 min after exposure to 1% or 2% isoflurane (n = 5 per group). Data are expressed as mean ± SEM, and ***p<0.001 indicates a significant change in body temperature over time. (<b>B</b>) To induce ischemic BBB disruption, the mice were subjected to 120 min of dMCAo coupled with ipsilateral common carotid arterial occlusion. At 4 h post-reperfusion (6 h after the onset of ischemia), the mice were placed for 1 h inside a gas chamber filled with 0%, 1%, or 2% isoflurane to induce hypothermia. The severity of BBB disruption was determined by comparing the concentrations of intravascular tracer Evans blue dye (EBD), injected 4 h post-reperfusion and quantified 1 h thereafter, extravasated into the left (control, non-ischemic) and the right (ischemic) hemisphere (n = 13–14 per group). Data are expressed as mean ± SEM. *p<0.05 and ***p<0.001 indicate a significant difference, whereas n.s. indicates no significant difference. (<b>C</b>) Same as (A), except that body temperature was maintained with a heating pad (n = 4 per group). (<b>D</b>) Same as (B), except that body temperature was maintained with a heating pad (n = 4–5 per group).</p