Additional file 1: Figure S1. of The glucagon-like peptide-1 receptor agonist exendin-4 ameliorates warfarin-associated hemorrhagic transformation after cerebral ischemia

The PT-INR values after warfarin withdrawal. After warfarin withdrawal, INR values remained stable for the next 6 h and dropped to normal values after 24 h. Data are shown as mean ± SD

Additional file 3: Figure S3. of The glucagon-like peptide-1 receptor agonist exendin-4 ameliorates warfarin-associated hemorrhagic transformation after cerebral ischemia

Representative immunofluorescence images showed co-localization of Iba1 (green) and TNF-α (red) in microglia. Immunostaining of Iba1(green), TNF-α(red), and DAPI (blue) was performed in the cortical and subcortical areas supplied by the middle cerebral artery. (A) Representative immunofluorescence images showed the percentage of Iba1+/TNF-α + cells to total Iba1+ cells was increased after warfarin treatment. EX-4 treatment reduced the Iba1+/TNF-α + cells percentage, whereas wortmannin blocked this effect of EX-4. Scale bar 50 μm. (B) Quantitative analysis of Iba1 and TNF-α double positive cells/Iba1-positive cells

E3d HPC reversed the upregulation of CD39 and CD73 and induced ENT-1 mRNA expression.

<p>A single episode of HPC and E6d HPC significantly increased the mRNA expression levels of CD39 (A), CD73 (B) and had no effect on ENT-1 (C) expression. E3d HPC had the opposite effect, having no effect on CD39 (A) and CD73 (B) expression, but significantly increasing ENT-1 (C) expression. Data are shown as mean ± SD; <i>n</i> = 6 mice in each group; data analyzed by one-way ANOVA; * <i>P</i><0.05 compared to the control.</p

CD39 and CD73 were highly upregulated after HPC and were mainly distributed in neurons.

<p>Immunofluorescence assay was used to show the expression of CD39 (A, B, C) and CD73 (D, E, F) in neurons and astrocytes in different groups. Neuron and astrocyte cytoskeletal proteins, MAP-2 and GFAP, were labeled with green fluorescence, while CD39 or CD73 were labeled with red fluorescence. CD39 and CD73 were highly upregulated after HPC but not after E3d HPC (A, B, D, E). Quantitative analysis of receptor expression showed that both CD39 and CD73 were mainly localized in neurons (C, F). Data are shown as mean ± SD; <i>n</i> = 4 mice in each group.</p

E3d HPC-induced reduction of extracellular adenosine levels were totally reversed by ENT1 inhibitor NBTI.

<p>After the last hypoxic exposure, animals received either CD73 inhibitor (AOPCP) or ENT1 inhibitor (NBTI) through the microdialysis infusate. AOPCP blocked the elevation of extracellular adenosine levels induced by HPC or E6d HPC (A), while NBTI totally reversed the E3d HPC-induced extracellular adenosine reduction (B). Data are shown as mean ± SD; <i>n</i> = 6 mice in each group; data analyzed by one-way ANOVA; * <i>P</i><0.05 compared to the control.</p

E3d HPC failed to provide protection against cerebral ischemia.

<p>Stroke volume was evaluated 72 hours after MCAO. E3d HPC failed to protect from ischemia 3 days after the last hypoxic exposure (A). Neurological deficits were also determined 72 hours after MCAO. Prominent improvements were observed 3 days after the last hypoxic exposure in the HPC and E6d HPC group (C, D). However, E3d HPC failed to ameliorate the neurological deficits induced by MCAO (C). Data are shown as mean ± SD; <i>n</i> = 6–8 mice in each group; data analyzed by one-way ANOVA; * <i>P</i><0.01 compared to the control.</p

Hypoxia-inducible factor targets in the brain were still highly upregulated following E3d HPC.

<p>The mRNA expression levels of selected HIF targets in the region supplied by the MCA after the last hypoxic exposure are shown. A single episode of HPC, E3d HPC and E6d HPC significantly increased the expression of EPO (A), VEGF (B), MCT4 (C) and GLUT-1 (D). There was no significant difference between HPC groups. Data are shown as mean ± SD; <i>n</i> = 6 mice in each group; data analyzed by one-way ANOVA; * <i>P</i><0.01 compared to the control.</p

E3d HPC reversed the upregulation of CD39 and CD73 and induced ENT-1 protein expression.

<p>Consistent with our mRNA data, a single episode of HPC and E6d HPC significantly increased the protein expression levels of CD39 (A) and CD73 (B) and had no effect on ENT-1 (C) expression. E3d HPC had the opposite effect, having no effect on CD39 (A) and CD73 (B) expression, but significantly increasing ENT-1 (C) protein expression. Representative Western blots are shown in panel D. Data are shown as mean ± SD; <i>n</i> = 6 mice in each group; data analyzed by one-way ANOVA; * <i>P</i><0.05 compared to the control.</p

Physiological parameters of mice treated with HPC.

<p><b>BP,</b> blood pressure; <b>HGB</b>, hemoglobin; <b>WBC</b>, white blood cell count.</p

E3d HPC lead to the loss of HPC-induced neuroprotection.

<p>A schematic diagram shows the experimental protocols for HPC, E3dHPC and E6dHPC (A). Each black box represents an episode of hypoxia (8% O₂ for 5 hours). Bar charts shows the stroke volume in mice following a single episode of HPC (C), E3d HPC (C) and E6d HPC (D). Stroke volume was evaluated 24 hours after MCAO. A significantly reduced stroke volume was induced by a single episode of HPC and E6d HPC, 3 days after the last exposure, although no effects were observed following E3d HPC. Data are shown as mean ± SD; <i>n</i> = 6–8 mice in each group; data analyzed by one-way ANOVA; * <i>P</i><0.01 compared to the control.</p