Chrome c levels (15 kDa) in cytosolic fractions were also significantly lower (P = 0.00016) in the hHSP27 group vs. controls (Figure 6C,D). Because expansion of the damaged area following an ischemic insult has been attributed to immediate and direct cytotoxic effects of oxidative products [28,29], we examined the effects of hHSP27 on levels of 8hydroxydeoxyguanosine (8-OHdG), an oxidized nucleoside of DNA, and 4-hydroxy-2-hexenal (HHE), a major lipid Title Loaded From File peroxidation product. The numbers of cells immunopositive for these oxidative stress Title Loaded From File markers 24 h after reperfusion were significantly lower (P,0.001) in the hHSP27 group vs. controls (Figure 7A,B). The numbers of ionized calcium-binding adapter molecule-1 (Iba1)-positive activated microglia and astrocytes 24 h after reperfusion, were also significantly lower (P,0.001) in the hHSP27 group vs. controls (Figure 7A,B).DiscussionIn our experiments, delayed intravenous injections of phosphorylated, multimeric hHSP27 complexes following reperfusion after transient MCAO reduced infarct volume, neurological deficits, and apoptotic cell death, and at the same time decreased TUNEL reactions and the levels of cytochrome c, cleaved caspase9, and cleaved caspase-3. The hHSP27 complex also decreased oxidative DNA damage, lipid peroxidation, and glial activation. Thus, hHSP27 appears to protect brain by inhibiting apoptosis and oxidative stress following ischemia and reperfusion. We also confirmed that it was the HSP27 that protected the brain, as a specific anti-HSP27 antibody inhibited the protective effects.hHSP27 Suppressed Apoptotic Cell Death, Oxidative DNA Damage, Lipid Peroxidation and Glial ActivationThe numbers of cells immunopositive for cytochrome c, cleaved caspase-9, and cleaved caspase-3, and the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL)-positive cells 24 h after reperfusion were significantly lower (P = 0.00024) in the hHSP27 group than inFigure 3. Neuroprotective effects of hHSP27 against ischemic/reperfusion injury 72 h after reperfusion. A, 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, Infarct volumes in control and hHSP27 groups. C, Neurological deficit scores in control and hHSP27 groups. Data are presented as mean6SEM of 3 mice (B) and 5 mice (C) in each group. *P,0.05, **P,0.001 vs. controls. doi:10.1371/journal.pone.0066001.gHSP27 Protects against Ischemic Brain InjuryFigure 4. Anti-HSP27 antibody and dephosphorylation inhibit hHSP27 neuroprotective effects. A, Infarct volumes in control, hHSP27 (50 mg), hHSP27 plus HSP27 antibody cocktails, HSP27 elution peptide, recombinant HSP27, and dephosphorylated hHSP27 groups. Data are means6SEM of 3 mice in each group. **P,0.001 vs. controls. B?D, Dephosphorylated and phosphorylated hHSP27 proteins were separated by SDSPAGE (B) and native-PAGE (C), stained with Coomassie brilliant blue (B,C), and immunoblotted with anti-phosphorylated S15 HSP27, S78 HSP27, and S82 HSP27 antibodies (D). E, 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. doi:10.1371/journal.pone.0066001.gAdministered hHSP27 crossed the blood-brain barrier injured by ischemic insults and was localized in neurons on the ischemic side of brains, w.Chrome c levels (15 kDa) in cytosolic fractions were also significantly lower (P = 0.00016) in the hHSP27 group vs. controls (Figure 6C,D). Because expansion of the damaged area following an ischemic insult has been attributed to immediate and direct cytotoxic effects of oxidative products [28,29], we examined the effects of hHSP27 on levels of 8hydroxydeoxyguanosine (8-OHdG), an oxidized nucleoside of DNA, and 4-hydroxy-2-hexenal (HHE), a major lipid peroxidation product. The numbers of cells immunopositive for these oxidative stress markers 24 h after reperfusion were significantly lower (P,0.001) in the hHSP27 group vs. controls (Figure 7A,B). The numbers of ionized calcium-binding adapter molecule-1 (Iba1)-positive activated microglia and astrocytes 24 h after reperfusion, were also significantly lower (P,0.001) in the hHSP27 group vs. controls (Figure 7A,B).DiscussionIn our experiments, delayed intravenous injections of phosphorylated, multimeric hHSP27 complexes following reperfusion after transient MCAO reduced infarct volume, neurological deficits, and apoptotic cell death, and at the same time decreased TUNEL reactions and the levels of cytochrome c, cleaved caspase9, and cleaved caspase-3. The hHSP27 complex also decreased oxidative DNA damage, lipid peroxidation, and glial activation. Thus, hHSP27 appears to protect brain by inhibiting apoptosis and oxidative stress following ischemia and reperfusion. We also confirmed that it was the HSP27 that protected the brain, as a specific anti-HSP27 antibody inhibited the protective effects.hHSP27 Suppressed Apoptotic Cell Death, Oxidative DNA Damage, Lipid Peroxidation and Glial ActivationThe numbers of cells immunopositive for cytochrome c, cleaved caspase-9, and cleaved caspase-3, and the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL)-positive cells 24 h after reperfusion were significantly lower (P = 0.00024) in the hHSP27 group than inFigure 3. Neuroprotective effects of hHSP27 against ischemic/reperfusion injury 72 h after reperfusion. A, 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, Infarct volumes in control and hHSP27 groups. C, Neurological deficit scores in control and hHSP27 groups. Data are presented as mean6SEM of 3 mice (B) and 5 mice (C) in each group. *P,0.05, **P,0.001 vs. controls. doi:10.1371/journal.pone.0066001.gHSP27 Protects against Ischemic Brain InjuryFigure 4. Anti-HSP27 antibody and dephosphorylation inhibit hHSP27 neuroprotective effects. A, Infarct volumes in control, hHSP27 (50 mg), hHSP27 plus HSP27 antibody cocktails, HSP27 elution peptide, recombinant HSP27, and dephosphorylated hHSP27 groups. Data are means6SEM of 3 mice in each group. **P,0.001 vs. controls. B?D, Dephosphorylated and phosphorylated hHSP27 proteins were separated by SDSPAGE (B) and native-PAGE (C), stained with Coomassie brilliant blue (B,C), and immunoblotted with anti-phosphorylated S15 HSP27, S78 HSP27, and S82 HSP27 antibodies (D). E, 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. doi:10.1371/journal.pone.0066001.gAdministered hHSP27 crossed the blood-brain barrier injured by ischemic insults and was localized in neurons on the ischemic side of brains, w.