Neuroprotective effects of hHSP27 against ischemic/reperfusion injury 72 h after reperfusion.

<p><b><i>A,</i></b> Photomicrographs of infarct areas stained with cresyl violet in control and hHSP27 groups 72 h after reperfusion. Infarct areas are circumscribed with dotted lines. Scale bar = 1 mm. <b><i>B,</i></b> Infarct volumes in control and hHSP27 groups. <b><i>C,</i></b> Neurological deficit scores in control and hHSP27 groups. Data are presented as mean±SEM of 3 mice (<b><i>B</i></b>) and 5 mice (<b><i>C</i></b>) in each group. *<i>P</i><0.05, **<i>P</i><0.001 vs. controls.</p

Characterization of hHSP27.

<p><b><i>A–B,</i></b> Isolated human heat shock protein 27 (hHSP27) or recombinant HSP27 (rHSP27) proteins (1 µg or 10 µg) were separated by SDS-PAGE (<b><i>A</i></b>) and native-PAGE (<b><i>B</i></b>) and stained with Coomassie brilliant blue. <b><i>C,</i></b> hHSP27 proteins were immunoblotted with antibodies against HSP27, phosphorylated S15 HSP27, S78 HSP27, and S82 HSP27. <b><i>D,</i></b> hHSP27 proteins were separated by native-PAGE and immunoblotted with antibodies against αβ-crystallin and HSP20. <b><i>E,</i></b> rHSP27 (10 ng), hHSP27 (10 ng), αβ-crystallin (5 ng), and HSP20 (5 ng) were separated by SDS-PAGE followed by immunoblotting with antibodies against HSP27, αβ-crystallin, and HSP20.</p

hHSP27 attenuates ischemic brain damage.

<p><b><i>A,</i></b> Schematic of hHSP27 injection schedules for determining the HSP27 treatment protocol. <b><i>B,</i></b> Photomicrographs of cresyl violet-stained infarcts in controls, the 5-µg hHSP27 group (0 h), 50-µg hHSP27 group (0 h), and 50-µg hHSP27 group (1 h), 24 h after reperfusion. Infarct areas are circumscribed with dotted lines. Scale bar = 1 mm. <b><i>C,</i></b> Infarct volumes at various doses (given at 0 h) and schedules (50 µg doses) of hHSP27. <b><i>D,</i></b> Neurological deficit scores in controls, the 50-µg hHSP27 group (0 h), and the 50-µg hHSP27 group (1 h) immediately (Post) and 24 h following reperfusion. <b><i>E,</i></b> Temporal changes in regional cerebral blood flow (rCBF), before (Pre), during, and 24 h after MCAO. <b><i>F,</i></b> Photomicrographs of infarct areas stained with 2,3,5-triphenyltetrazolium chloride in controls and the 50-µg hHSP27 group (1 h) 24 h after reperfusion. Data are presented as mean±SEM of 3 mice (<b><i>B,E</i></b>) and 5 mice (<b><i>D</i></b>) in each group. *<i>P</i><0.05, **<i>P</i><0.001 vs. controls. hHSP27, human heat shock protein; MCAO, middle cerebral artery occlusion.</p

Anti-HSP27 antibody and dephosphorylation inhibit hHSP27 neuroprotective effects.

<p><b><i>A,</i></b> Infarct volumes in control, hHSP27 (50 µg), hHSP27 plus HSP27 antibody cocktails, HSP27 elution peptide, recombinant HSP27, and dephosphorylated hHSP27 groups. Data are means±SEM of 3 mice in each group. **<i>P</i><0.001 vs. controls. <b><i>B– D,</i></b> Dephosphorylated and phosphorylated hHSP27 proteins were separated by SDS-PAGE (<b><i>B</i></b>) and native-PAGE (<b><i>C</i></b>), stained with Coomassie brilliant blue (<b><i>B,C</i></b>), and immunoblotted with anti-phosphorylated S15 HSP27, S78 HSP27, and S82 HSP27 antibodies (<b><i>D</i></b>). <b><i>E</i></b>, Photomicrographs of infarct areas stained with cresyl violet in hHSP27 and dephosphorylated hHSP27 groups prepared 24 h after reperfusion. Scale bar = 1 mm. hHSP27, human heat shock protein.</p

Effects of hHSP27 on oxidative stress and inflammatory response.

<p><b><i>A,</i></b> Photomicrographs of 8-OHdG-, HHE-, Iba-1-, and GFAP-immunostaining in the infarct boundary zones in the control and hHSP27 groups 24 h after reperfusion. Bars = 50 µm. <b><i>B,</i></b> Numbers of 8-OHdG-, HHE–, Iba-1-, and GFAP-positive cells in control and hHSP27-treated mice. Data are means±SEM (<b><i>B</i></b>). **<i>P</i><0.001 vs. controls. 8-OHdG, 8-hydroxydeoxyguanosine; HHE, 4-hydroxy-2-hexenal; Iba-1, ionized calcium-binding adapter molecule-1; GFAP, glial fibrillary acidic protein; hHSP27, human heat shock protein.</p

Localization of injected FITC-hHSP27 on the ischemic and non-ischemic sides of mouse brain.

<p><b><b><i>A–H,</i></b></b> FITC-hHSP27 (<i>green</i>, <b><i>A,B</i></b>); NeuN, a neuronal marker protein (<i>red</i>, <b><i>C,D</i></b>); merge (<b><i>E–H</i></b>), FITC, fluorescein isothiocyanate. Scale bar = 100 um.</p

Effects of hHSP27 on cell death.

<p><b><i>A,</i></b> Photomicrographs of anti-cytochrome c, anti-cleaved caspase-9, anti-cleaved caspase-3, and TUNEL staining in the infarct boundary zones in controls and the hHSP27-treated group prepared 24 h after reperfusion. Scale bars = 50 µm. <b><i>B,</i></b> Number of cytochrome c-, cleaved caspase-9-, cleaved caspase-3-, and TUNEL-positive cells. <b><i>C,</i></b> Immunoblots of cytochrome c, Tom20 (mitochondrial marker), and actin, 24 h after reperfusion. <b><i>D,</i></b> Densitometric analysis of cytochrome c protein in cytosolic fractions of isolated hHSP27. Data are means±SEM (<b><i>B,D</i></b>). **<i>P</i><0.001 vs. controls. TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling; hHSP27, human heat shock protein.</p