Data Files

Focal brain cooling experiments with anaesthetized male Sprague-Dawley rats induced with epilepsy using Penicillin G potassium. Cooling was performed in five different rats for each cooling temperature (15, 20, and 25 degrees Celcius)

Apoptosis (TUNEL staining) in the cortex 24 h after hypoxic ischemia injury.

<p><i>A</i>: Tissue sections are shown from brain hemispheres subjected to mild ischemic injury (<i>top</i>) or severe ischemic injury in mice from the normothermia (<i>middle</i>) and hypothermia (<i>bottom</i>) groups. <i>Arrow</i> indicates TUNEL-positive cells. Scale, 500 µm. <i>B</i>: <i>(left</i>) Average number of TUNEL-positive cells (mean±SE) in the superficial and deep cortical layers in the normothermia (n = 9) and hypothermia (n = 8) groups. *<i>P</i><0.05 (Wilcoxon test). <i>(Right</i>) Distribution of the number of TUNEL-positive cells in the superficial layers and deep cortical layers.</p

Disruptions in laminar structure of mouse brain cortex after hypoxic ischemia injury.

<p><i>A</i>: (<i>left</i>) Histological tissue sections show the laminar structure of the cortex in the normothermia group. Scale bar, 500 µm. The designated areas are enlarged in the bottom panels for clarity. Scale bar, 200 µm; <i>(right</i>) average areas of superficial and deep cortical layers measured on mouse brain sections (n = 6, mean±SE). *<i>P</i><0.05 (Wilcoxon test). <i>B:</i> Laminar structure in the hypothermia group (n = 6, mean±SE). Other notations defined in (<i>A</i>).</p

Examination of hypoxic ischemia injury in predevelopmental mouse brain.

<p><i>A</i>: Cerebral blood flow immediately after carotid artery ligation in a P2–P3 mouse. <i>B</i>: Experimental paradigm. Hypoxic injury was followed by 24 h of normothermia or hypothermia, and mice were sacrificed at the indicated time points (P4, 5 weeks). Note that behavioral tests were conducted at 4 weeks of age. <i>C, D</i>: Representative examples of hematoxylin staining for normothermia (C) or hypothermia (D) treated mice. In the normothermia group, the boundary was obscure between the superficial layers and the deep layers (white asterisk). Scale, 500 µm. High magnification images of the contralateral (<i>left</i>) and ipsilateral (<i>right</i>) hemisphere. Scale, 200 µm.</p

Neuronal cell density reduced in adult mouse brains after hypoxic ischemia injury.

<p><i>A</i>: Immunohistochemistry shows NeuN-positive cells in the cortex (<i>Top</i>) and striatum (<i>Bottom</i>) of mice subjected to hypoxic ischemia injury, followed by normothermia. Scale, 500 µm. Average density of NeuN cells (n = 11 mouse brains, mean±SE) was assessed in the contralateral (black) and ischemic ipsilateral (white) hemispheres. <i>B</i>: High magnification images of the cortices of mice in normothermia and hypothermia groups. Scale, 200 µm. *<i>P<</i>0.05 (Wilcoxon test). Other notations are defined in (<i>A</i>).</p

Histological Characterization of the Tumorigenic “Peri-Necrotic Niche” Harboring Quiescent Stem-Like Tumor Cells in Glioblastoma

<div><p>Background</p><p>Characterization of the niches for stem-like tumor cells is important to understand and control the behavior of glioblastomas. Cell-cycle quiescence might be a common mechanism underlying the long-term maintenance of stem-cell function in normal and neoplastic stem cells, and our previous study demonstrated that quiescence induced by hypoxia-inducible factor (HIF)-1α is associated with a high long-term repopulation capacity of hematopoietic stem cells. Based on this, we examined human astrocytoma tissues for HIF-1α-regulated quiescent stem-like tumor cells as a candidate for long-term tumorigenic cells and characterized their niche histologically.</p><p>Methods</p><p>Multi-color immunohistochemistry was used to visualize HIF-1α-expressing (HIF-1α⁺) quiescent stem-like tumor cells and their niche in astrocytoma (WHO grade II–IV) tissues. This niche was modeled using spheroids of cultured glioblastoma cells and its contribution to tumorigenicity was evaluated by sphere formation assay.</p><p>Results</p><p>A small subpopulation of HIF-1α⁺ quiescent stem-like tumor cells was found in glioblastomas but not in lower-grade astrocytomas. These cells were concentrated in the zone between large ischemic necroses and blood vessels and were closer to the necrotic tissues than to the blood vessels, which suggested that a moderately hypoxic microenvironment is their niche. We successfully modeled this niche containing cells of HIF-1α⁺ quiescent stem-like phenotype by incubating glioblastoma cell spheroids under an appropriately hypoxic condition, and the emergence of HIF-1α⁺ quiescent stem-like cells was shown to be associated with an enhanced sphere-forming activity.</p><p>Conclusions</p><p>These data suggest that the “peri-necrotic niche” harboring HIF-1α⁺ quiescent stem-like cells confers a higher tumorigenic potential on glioblastoma cells and therefore may be a therapeutic target to control the behavior of glioblastomas.</p></div

Effect of <i>Q</i>_{10,<i>syn</i>}.

<p>As <i>Q</i>_{10,<i>syn</i>} is increased from unity, frequency of epileptic discharges during cooling (60 s–120 s) becomes less until complete termination. (From top to bottom: <i>Q</i>_{10,<i>syn</i>} = 1.0, 1.007, 1.013, 1.085.)</p

SOX2⁺ (or NANOG⁺) HIF-1α⁺ RNApII-S2P^-/low tumor cells are not found in zones around small pseudopalisading necroses or in areas showing no necrotic change.

<p><b>These cells are found in glioblastomas (WHO grade IV), but not in diffuse astrocytomas (grade II) and anaplastic astrocytomas (grade III). a, b:</b> Serial sections of glioblastoma tissue containing small pseudopalisading necroses. <b>c, d:</b> Serial sections of glioblastoma tissue showing no necrotic changes. H-E staining (a, c) and triple immunostaining for SOX2/HIF-1α/RNApII-S2P (b, d) are shown. Although SOX2⁺ and/or HIF-1α⁺ tumor cells were found, they were RNApII-S2P⁺. Therefore, SOX2⁺ HIF-1α⁺ RNApII-S2P^-/low cells were not observed. N_S, small pseudopalisading necrosis; V, blood vessel. Scale bars, 50 μm. <b>e:</b> Triple immunostaining for SOX2/HIF-1α/RNApII-S2P in astrocytomas of WHO grade II and III. No SOX2⁺ HIF-1α⁺ RNApII-S2P^-/low cells were observed. Scale bars, 25 μm. <b>f, g:</b> Frequency of SOX2⁺ HIF-1α⁺ RNApII-S2P^-/low cells (f) and NANOG⁺ HIF-1α⁺ RNApII-S2P^-/low cells (g) in cases of astrocytic tumors of WHO grade II–IV. *, <i>P</i> < 0.05; **, <i>P</i> < 0.01 (grade IV <i>vs</i>. combined group of grades II and III).</p

Simulated activity from rat 4.

<p>Suppression of epileptic discharges is replicated by the different models. (From top to bottom: Experimental data, SYN_INT, EXC_INH, EXC_SIN_FIN)</p

Neural mass model by Wendling et al.

<p>Block diagram of the model showing the interconnections among the neural populations (left) and the processes involved for each population (right).</p