Chemical structure of the HIF-1 inhibitor YC-1.

<p>Chemical structure of the HIF-1 inhibitor YC-1.</p

Effect of YC-1 on the expression of HIF-1 in ischemic brains.

<p>The protein levels of HIF-1α and its down-stream proteins EPO, VEGF, and GLUT-3 were analyzed by western blotting in brains from rats subjected to 90 min ischemia and 24 hr reperfusion. Rats received YC-1 (2 mg/kg, i.v.) at 24 h and 30 min prior to the onset of ischemia. (<b>A</b>) Representative Western blots of HIF-1α and its down-stream proteins. (<b>B</b>) Quantification of the HIF-1α protein level. (<b>C</b>) Quantification of the VEGF protein level. (<b>D</b>) Quantification of the EPO protein level. (<b>E</b>) Quantification of the GLUT-3 protein level. Values were normalized to β-actin and contralateral hemispheres of control animals. Values are means ± SEM, n = 5. #<i>p</i><0.05 vs. the contralateral hemisphere of control animals, *<i>p</i><0.05 vs. the ipsilateral hemisphere of control animals.</p

YC-1-induced mortality of MCAO rats and group sizes for final analyses.

a<p>In this MCAO group, four animals were excluded due to a lack of obvious neurological deficits and one due to bleeding during the procedure. ^b In YC-1+MCAO group, one was excluded due to a lack of obvious neurological deficits. ^c Death rate after successfully completed MCAO.</p

Effect of YC-1 on brain tissue damage of MCAO rats.

<p>Brian damage was estimated by MRI and TTC staining after rats were subjected to 90 min ischemia and 24 hr reperfusion. Animals received YC-1 (2 mg/kg, i.v.) at 24 h and 30 min prior to the onset of ischemia. (<b>A</b>) Representative MRI images showing time-dependent progression of infarct volumes. T2 weighted MRI images were collected at 0, 3, 6, 12, and 24 hr after MCAO with white area representing infarct area. (<b>B</b>) Representative TTC staining (lower panel) and T2 (upper panel) images of brain sections of a MCAO rat. The brain was sectioned from the 4 mm position from the frontal pole and continued in 2-mm interval to 10 mm. (<b>C</b>) Quantification of infarct volume with T2-weighted MRI images of rat brain (n = 12 (control), 9 (YC-1)). (<b>D</b>) Quantification of brain damage estimated by TTC stained sections (n = 12 (control), 9 (YC-1)). (<b>E</b>) Quantification of brain edema volume estimated by TTC stained sections (n = 12 (control), 9 (YC-1)).Values are means ± SEM. *<i>p</i><0.05 vs. control. #p<0.05 vs. control striatum. ^□P<0.05 vs. control cortex.</p

Effect of YC-1 on HIF-1 expression in neurons in ischemic brains.

<p>The protein levels of HIF-1α and its down-stream genes were analyzed by double immunostaining with the neuronal marker NeuN after rats were subjected to 90 min ischemia and 24 hr reperfusion. Rats received YC-1 (2 mg/kg, i.v.) at 24 h and 30 min prior to the onset of ischemia. (<b>A</b>) TTC-stained rat brain coronal section. Labeled square areas represent locations of immuno images. (<b>B</b>) Double immunostaining of HIF-1α (green) and NeuN (red). (<b>C</b>) Double immunostaining of EPO (green) and NeuN (red). (<b>D</b>) Double immunostaining of VEGF (green) and NeuN (red). (<b>E</b>) Double immunostaining of GLUT-3 (green) and NeuN (red). Scale bar, 53 µm.</p

Effects of YC-1 on the BBB permeability of MCAO rats.

<p>BBB permeability was estimated by Evans blue leakage after rats were subjected to 90 min ischemia and 24 hr reperfusion. Animals received YC-1 (2 mg/kg, i.v.) at 24 h and 30 min prior to the onset of ischemia. (<b>A</b>) Representative images of Evans blue extravasation in a whole brain and coronal sections (bregma +0.70 mm). (<b>B</b>) Quantification of Evens blue leakage in ipsilateral and contralateral hemispheres of MCAO rats (n = 12 (control), 9 (YC-1)). Values are means ± SEM. *<i>p</i><0.05 vs. control.</p

Representative DWI (A and C) and corresponding permeability maps (B and D) of coronal sections of rat brains.

<p>Images shown are from two different groups: A and B from group 1 (scanned at 3 h, n = 8) and C and D from group 2 (studied at 48 h, n = 8). DWI images were used to calculate ADC maps and generate tissue signature maps shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006597#pone-0006597-g005" target="_blank">Fig. 5A and 5B</a>. Areas of the subcortex and cerebral cortex are demarcated in A and C.</p

Analysis of correlations between BBB permeability and the ADC values in cerebral cortex and subcortex.

<p>Color-coded K_i-ADC maps (A and B) show the three main populations of pixels with different K_i and ADC values for both time points. Quantitative changes of each area over time are presented in panels C and D. Based on the abnormality of ADC values and the BBB permeability; we have recognized three different areas in the ipsilateral side. These areas are labeled as tissue signatures corresponding to different pathophysiology in the evolution of lesion. Each tissue signature is represented by a different color at two different time points, 3 and 48 h of reperfusion. *p<0.05 and **p<0.01 with respect to 3 h.</p

Figure 3

<p>A. Changes in the area with hypointensive ADC at 3 and 48 h of reperfusion. Significant increases in areas with ADC abnormalities were found between 3 and 48 h in the cerebral cortex. Brain damage observed at 48 h in ADC maps (B) was confirmed histologically using TTC staining (C). Areas of the subcortex and cerebral cortex are demarcated in panels B and C.</p

Proposed model showing possible transitions between different states of tissue injury during the progression of ischemic stroke.

<p>Cytotoxic edema is characterized by low ADC values with preserved BBB function (normal K_i), while vasogenic edema is identified by high K_i values. The core of the infarct display high K_i and low ADC values (a mixture of cytotoxic and vasogenic edema). Based on data from the merged ADC+K_i maps (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006597#pone-0006597-g005" target="_blank">Fig. 5</a>), the main transition occurs from areas with cytotoxic edema to areas with both cytotoxic and vasogenic edema (core of the infarct), which is depicted with a thicker arrow in the schematic.</p