Table 1.
Body weight and blood glucose levels among groups.
Fig 1.
(A) D-glucose was injected intraperitoneally at 15 min before 1-h transient middle cerebral artery occlusion (MCAO). Following MCAO, the mice were re-perfused for 2 h and then injected with recombinant tissue plasminogen activator (tPA, n = 7), phosphorylated recombinant heat shock protein 27 (prHSP27, n = 6), tPA plus prHSP27 (tPA+prHSP27, n = 7), or bovine serum albumin (BSA, n = 9). The mice were sacrificed 24 h later. (B) Representative photomicrographs of infarct areas stained with cresyl violet at 24 h after reperfusion. Infarct areas are circumscribed with dotted lines. prHSP27 decreased brain infarct size. Scale bar = 2 mm. (C) Brain swelling. (D) Infarct volume corrected for edema. *p < 0.05, §p < 0.001. (E) prHSP27 improved neurological severity scores at 1 h following MCAO and at 24 h following reperfusion in mice treated with the above substances. Data are mean ± standard error of the mean (SEM; n = same as above). *p < 0.05: tPA and prHSP27 vs. tPA, §p < 0.001: BSA vs. prHSP27.
Fig 2.
prHSP27 reduces mortality and hemorrhagic transformation at 24 h after reperfusion.
(A) At 24 h after reperfusion following MCAO, the mortality rate was 20% (6/30) in the BSA treatment group, 0% (0/15) in the prHSP27 group, 35% (11/31) in the tPA group, and 7% (2/28) in the tPA plus prHSP27 group. *p < 0.05: BSA vs. prHSP27, †p < 0.01: tPA plus prHSP27 vs. tPA. (B) Effect of administering tPA on macroscopic hemorrhages. (Upper) Representative coronal brain sections stained with 1% 2,3,5-triphenyl tetrazolium chloride at 24 h after reperfusion showing the five grades of hemorrhages: Grades 0–4. (Lower) whole, unstained brains with the same hemorrhages.
Fig 3.
prHSP27 attenuates blood-brain barrier permeability.
(A) Representative photomicrographs of endogenous IgG extravasation stained in BSA- (n = 7), prHSP27- (n = 6), tPA- (n = 7), and tPA plus prHSP27- (n = 7) treated mice at 24 h after reperfusion. Scale bar = 2 mm. (B) Quantitation of IgG staining in nine consecutive coronal brain slices from each mouse. Data are means ± SEM. *p < 0.05: tPA plus prHSP27 vs. tPA, ‡p < 0.005: BSA vs. prHSP27.
Fig 4.
prHSP27 maintains the cell wall structure of endothelial cells and tight junction proteins.
(A) Localization of endothelial cell-stained FITC-tomato lectin on the penumbra and core of the ischemic side of the brain from mice treated with tPA or tPA plus prHSP27. FITC-staining is higher in the mice treated with prHSP27. *p < 0.05: tPA vs tPA plus prHSP27. (B) Immunoblot analysis (upper) and quantitation (lower) of occludin. Protein loading was calculated relative to tubulin. ‡p < 0.005: tPA plus prHSP27 vs. tPA. (C) Photomicrographs (upper) and quantitation (lower) of type IV collagen immunostaining in the infarct boundary zones in BSA- (n = 5), tPA- (n = 5), and tPA plus prHSP27- (n = 5) treated mice at 24 h after reperfusion. prHSP27 maintained the type IV collagen structure of the basement membrane. Scale bars = 100 μm. Data are mean ± SEM. *p < 0.05: tPA plus prHSP27 vs. BSA, tPA plus prHSP27 vs. tPA.
Fig 5.
Effect of prHSP27 on the expression of matrix metalloproteinase-9 (MMP-9) on the ischemic side.
(A) Zymographic (upper) and densitometric (lower) analyses of MMP-9 protein in BSA- (n = 7), tPA- (n = 7), and tPA plus prHSP27- (n = 7) treated mice at 24 h after reperfusion. ‡p < 0.005: tPA plus prHSP27 vs. tPA. (B) Photomicrographs of ionized calcium binding adapter molecule-1 (Iba-1) immunostaining (upper) and number of Iba-1-positive cells (lower) in the infarct boundary zones in BSA- (n = 5), tPA- (n = 5), and tPA plus prHSP27- (n = 5) treated mice at 24 h after reperfusion. prHSP27 suppressed the inflammatory response. Scale bars = 100 μm. Data are mean ± SEM. *p < 0.05: tPA plus prHSP27 vs. BSA, §p < 0.001: tPA plus prHSP27 vs. tPA.
Fig 6.
Effect of cerebral ischemia on prHSP27 in the blood-brain barrier.
(A) Electron microscopic analysis of the blood-brain barrier on the non-ischemic side of tPA- (contralateral tPA, n = 3), ischemic side of tPA- (tPA, n = 3), and ischemic side of tPA plus prHSP27- (tPA and prHSP27, n = 3) treated mice. Magnification, ×5000 (left row) and ×15000 (right row). The left row shows magnifications of the boxed areas in the right row. Scale bars, 1 μm (left row) and 500 nm (right row). a: astrocyte endfeet; e: endothelial cell; L: lumen. Arrowheads: the space between the basement membrane and astrocyte endfeet. Asterisks indicate the space between the basement membrane and astrocyte endfeet (B).