Figure 1.
P188 reduces ischemia/reperfusion-induced brain injury.
(A) TTC staining of infarct brain regions. (B) Quantitative analysis of brain infarct volume, presented as percentage of the ipsilateral hemisphere. (C) Motor deficits and (D) brain edema. Data are represented as mean ± SD (n = 10 ). *, p<0.05 vs the saline-treated group. ##, p<0.01 vs the sham-operated group. P188 (S) = 0.2 g/kg; P188 (M) = 0.4 g/kg; P188 (L) = 0.8 g/kg.
Figure 2.
P188 improves long-term functional recovery after ischemia/reperfusion.
(A) Post-stroke survival rate three weeks after stroke. *P<0.05, analyzed using log-rank test. (B) Wire hanging test. Time to stay hanging on the wire (T/hang). (C and D) Pole test. Time to turn the head downwards (T/turn). Time to reach the floor (T/floor). #, p<0.05 vs. sham-operated group; *, P<0.05 vs. saline-treated group. (E-G) Brain atrophy 3 weeks after tMCAO. Brain atrophy was determined using cresyl violet staining. The loss of brain volume was calculated as percentage of brain loss over total brain area of sham-operated mice.
Figure 3.
Effects of P188 on the membrane integrity.
Cells labeled with PI red fluorescence indicated cells with disrupted membranes (A, D, G and J). Blue fluorescence counterstaining with DAPI indicated total cells (B E H and K). (A) PI-positive cells were abundantly scattered in saline-treated hippocampus, including CA1, CA2, CA3 and DG, while less number of PI-positive cells was found in P188-treated group (D). In the striatal sections, numerous cells were labeled with PI in saline-treated group (G), whereas much less number of PI-positive cells in p188-treated group (J). (M) Quantitative analyses of PI-positive cells in control and P188-treated groups. Data were expressed as percentage of DAPI+ cells. *, P<0.05 vs. saline-treated group. Bar = 250 µm.
Figure 4.
P188 protectes HT22 hippocampal cells against OGD/R-induced cytotoxicity.
(A) Cell viability with different concentrations of p188 following OGD 18 h/R 24 h injury or under normal condition (−3, −4, −5 and −6 denoted 10−3 M, 10−4 M, 10−5 M and 10−6 M, respectively, ##, P<0.01 and ###, P<0.001 vs. without P188 in control groups, **, P<0.01; ***, P<0.001 vs. without P188 in OGD/R condition). (B) LDH leakage induced by OGD18 h/R 24 h injury or in normal condition with different concentrations of p188. #, P<0.05; ##, P<0.01 vs. without P188 in control groups. *, P<0.05; ***, P<0.001 vs. without P188 in OGD/R condition). (C) Representative microphotographs of propidium iodide (PI) fluorescence merged with white bright-light. HT22 cells were under normal condition without P188 or treated with OGD 18 h/R 24 h with or without P188.
Figure 5.
P188 seals the membrane rupture of HT22 cell.
Cells stained with PI indicated cells with disrupted membranes (PI+, A and D). Cells that remained permeable after P188 or PBS treatment showed green fluorescence (SYTOX® Green+, B and E ) or yellow in merged photograph (C and F ). Cell membrane resealed by P188 still showed red fluorescence in merged photograph but no green fluorescence (F).
Figure 6.
P188 attenuates BBB damage and inhibited protein levels and activity of MMP-9.
(A) Evans blue dye extravasation 24 h after tMCAO. The blue staining denoted the areas with increased BBB permeability. (B) Quantification of Evans blue dye extravasation after tMCAO. Bars represent mean ± SE. *, p<0.05 vs saline-treated mice (n = 4). (C) MMP-9 activity analyzed by gelatin zymograms. (D) Western blots analysis of MMP-9 protein levels. *, p<0.05 vs saline-treated brains (n = 4).