Experimental design.

<p>(A) Cranial window in a C57/BL6 mouse. Experimental design of the closed cranial window. (B) Experimental design (upper panel). Representative two-dimensional images of cerebral blood flow measured by laser speckle perfusion imaging before MCAO (a), during MCAO (b), and after reperfusion (c) are shown in the lower panels. MCAO: middle cerebral artery occlusion.</p

Imaging of HIF-1-active regions in the focal brain ischemia model.

<p>(A) Representative <i>in vivo</i> fluorescence images visualized through a cranial window before and at 5 min, 1 h, and 6 h after POH-N administration are shown. Magnified head images are shown in the lower left panels. Arrowheads indicate accumulation of the probe in the right ischemic hemisphere. (B) The relative fluorescence intensity of the ischemic hemisphere to the non-ischemic hemisphere. Fluorescence intensities were measured at the indicated times after POH-N administration. *<i>P</i><0.05, n = 3. (C) <i>Ex vivo</i> imaging of the coronal brain sections after POH-N injection. (D) Relative fluorescence of the ischemic hemisphere compared with the non-ischemic hemisphere at 6 h after probe administration (n = 3/group: *<i>P</i><0.05). Relative fluorescence values were calculated using ROIs mirrored along the midline of the cerebral hemispheres. (E) <i>In vivo</i> fluorescence images visualized without preparation of a cranial window before and at 5 min, 1 h, and 6 h after POH-N administration. Anesthetized C57BL/6J mice were shaved and depilated top of the head 24 h before experimentation. Arrowheads indicate accumulation of the probe in the right ischemic hemisphere.</p

HIF-1α accumulation after focal brain ischemia.

<p>(A) Western blot analysis of HIF-1α in the ischemic and non-ischemic hemispheres of mice subjected to MCAO followed by reperfusion. (B) Densitometric analysis of HIF-1α protein levels in the ischemic hemispheres. Data were normalized relative to β-actin levels, and the values obtained from sham-operated controls (S) were arbitrarily defined as 1. *<i>P</i><0.05 (vs. sham, n = 4).</p

Stabilization of POH-N under hypoxic conditions.

<p>SH-SY5Y neuroblastoma cells cultured under normoxic (N) or hypoxic (H) conditions were treated with POH probe. (A) HIF-1α protein levels were analyzed by western blotting (a representative blot is shown). (B) The fluorescence intensity of POH probe in cells was measured. (C) Representative fluorescence images are shown. *<i>P</i><0.02 (vs. normoxic condition).</p

No clear visualization of HIF-1-active regions in the permanent brain ischemia model or with delayed injection of POH-N in the focal brain ischemia model.

<p>(A) Representative <i>in vivo</i> fluorescence images visualized through a cranial window before and at 5 min, 1 h, and 6 h after POH-N administration are shown. POH-N was injected intravenously at 60 min after permanent MCA occlusion. (B) Representative <i>in vivo</i> fluorescence images visualized through a cranial window before and at 5 min, 1 h, and 6 h following POH-N administration at 24 h after reperfusion. Magnified head images are shown in the lower left panels.</p

Immunohistochemical detection of HIF-1-active cells and POH-N probe.

<p>(A) Immunohistochemical analysis of HIF-1α, POH-N (ODD) and HaloTag (green), with or without DAPI nuclear staining (blue), at 1 day after probe administration. Panels at the bottom show magnified images. (B) Similar distributions of HIF-1α, HaloTag, and HSP70 in pyramidal neurons of the cortical layer bordering the infarct. Scale bars, 50 μm.</p

Chronic cerebral hypoperfusion reduced the Aβ1-42 stained area in the APP<i>_Sw/Ind</i>-Tg mice.

<p>Immunohistochemistry for Aβ1-42 showed intense staining in the hippocampus (A, B, E, F) and in the cerebral cortex (C, D) of the APP<i>_Sw/Ind</i>-Tg/sham mice (n = 4; A, C, E, G) and APP<i>_Sw/Ind</i>-Tg/BCAS mice (n = 6; B, D, F, H). (I) The area positive for Aβ1-42 was analysed in the cortex and the hippocampus. Scale bars: 400 µm (A, B), 300 µm (C–F), 80 µm (G, H).</p

Interaction between APP overexpression and chronic cerebral hypoperfusion.

<p>Barnes maze test showed that APP<i>_Sw/Ind</i>-Tg/BCAS mice exhibited prolonged latency to reach the desired target (A), increased number of errors (B), and increased distance before reaching the desired target (C) compared to the other groups.</p

Neuronal density in the cerebral cortex and the hippocampal CA1 and CA3 areas.

<p>(A) The schematic illustration of the regions of interest depicted in the cerebral cortex (size, 0.05×0.5 mm) and the hippocampal CA1 and CA3 areas (0.05×0.25 mm each) of the coronal section of the brain. (B) The density of Nissl-stained neurons was lowest in APP<i>_Sw/Ind</i>-Tg/BCAS mice.</p

Chronic cerebral hypoperfusion affected the number of silver-stained cored plaques in the APP<i>_Sw/Ind</i>-Tg mice.

<p>Modified Bielschowsky staining showed cored plaques in the hippocampus (A, B, G, H) and the cerebral cortex (C, D), and diffuse plaques in the hippocampus (E, F) of the APP<i>_Sw/Ind</i>-Tg/sham mice (n = 6; A, C, E, G) and APP<i>_Sw/Ind</i>-Tg/BCAS mice (n = 4; B, D, F, H). Number of cored plaques was counted in the cortex and the hippocampus (I). Scale bars: 400 µm (A, B), 300 µm (C-F), 80 µm (G, H).</p