Materials and Methods from Behavioural responses of naked mole rats to acute hypoxia and anoxia

Naked mole rats (NMRs) are among the most hypoxia-tolerant mammals. Other species respond to hypoxia by either escaping the hypoxic environment or drastically decreasing behavioural activity and body temperature (<i>T</i>_b) to conserve energy. However, NMRs rarely leave their underground burrows, which are putatively hypoxic and thermally stable near the NMRs' preferred <i>T</i>_b. Therefore, we asked whether NMRs are able to employ behavioural and thermoregulatory strategies in response to hypoxia despite their need to remain active and the minimal thermal scope in their burrows. We exposed NMRs to progressively deeper levels of hypoxia (from 21 to 0% O₂) while measuring their behaviour and <i>T</i>_b. Behavioural activity decreased 40–60% in hypoxia and <i>T</i>_b decreased slightly in moderate hypoxia (5–9%) and then further with deeper hypoxia (3% O₂). However, even at 3% O₂ NMRs remained somewhat active and warm, and continued to explore their environment. Remarkably, NMRs were active for greater than 90 s in acute anoxia and <i>T</i>_b and metabolic rate decreased rapidly. We conclude that NMRs are adapted to remain awake and functional even at the extremes of their hypoxia-tolerance. This adaptation likely reflects variable and challenging levels of environmental hypoxia in the natural habitat of this species

DIDS treated cells retain plasma membrane integrity despite ATP depletion and an apoptotic phenotype.

<p>DIDS treatment induced an apoptotic phenotype and also dose-dependant [ATP] depletion from neurons, but membrane integrity was preserved. (<b>A</b>) Sample TEM images of neurons treated as indicated for 24 hrs. TEM experiments were repeated 2 times and 10–20 cells were examined from each treatment group. (<b>B</b>) Summary of neuronal [ATP] versus [DIDS] (40 or 400 µM) following 24 hrs treatment, normalized to untreated controls at <i>t</i> = 0. ATP luciferase experiments were repeated 5 times. (<b>C</b>) Summary of neuronal propidium iodide (PI) uptake vs. [DIDS] following 24 hrs of treatment, normalized to untreated controls. PI exclusion experiments were repeated 4 times. [DMSO] = 0.4%. Data are mean ± SEM. Asterisks (*) indicate significant difference from normoxic controls; bars indicate significance between treatments (<i>p</i><0.05).</p

DIDS (400 µM) treated nuclei exhibit apoptotic chromatin condensation, nuclear envelope shrinkage and fragmentation of the nuclear membrane into apoptotic bodies.

<p>(<b>A</b>) Sample TEM images of nuclei from neurons treated as indicated for 24 hrs. Arrows indicate condensed chromatin beads. Inset: DAPI-stained chromatin distribution in healthy (ACSF) and apoptotic (DIDS-treated) nuclei. (<b>B</b>) Summary of nuclear volume density from samples in (A). (<b>C</b>) Summary of chromatin volume density from samples in (A). (<b>D</b>) Summary of cleaved lamin A expression normalized to DAPI-stained nuclei following 24 hrs treatment. (<b>E</b>) Sample immunohistochemical (IHC) fluorescent confocal microscopy images of cleaved lamin A (red) and DAPI (blue) staining in neurons from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060804#pone-0060804-g005" target="_blank">Fig. 5D</a>. Lamin A IHC experiments were repeated 4 times. Data are mean ± SEM. Asterisks (*) indicate significant difference from normoxic controls (<i>p</i><0.05).</p

DIDS induces Annexin V staining in a dose- and time-dependant manner.

<p>(<b>A</b>) Summary of Annexin V fluorescence from neurons treated for 2 hrs in 96-well microplates as indicated. (<b>B</b>) Sample confocal microscopy images of Annexin V (green) and DAPI (blue) fluorescence from neurons treated as indicated for 2 hrs. (<b>C</b>) Summary of Annexin V fluorescence from neurons treated for 24 hrs in 96-well microplates as indicated. (<b>D</b>) Sample confocal microscopy images of Annexin V (green, and arrows) and DAPI (blue) fluorescence from neurons treated as indicated for 24 hrs. Images are representative of 4 separate experiments. Data are mean ± SEM. Asterisks (*) indicated significant difference from normoxic controls; bars indicate significance between treatments (<i>p</i><0.05). AFU = artificial fluorescence units.</p

DIDS upregulates apoptotic pathways in neurons.

<p>(<b>A</b>) Sample Western blots of apoptosis-related protein expression from neurons treated as indicated for 6 hrs. (<b>B–D</b>) Summaries of fold-change in cytochrome C (<b>B</b>), caspase 3 (<b>C</b>), and JNK3 (<b>D</b>) protein expressions from (A) normalized to α–actin expression in the same sample. Data are mean ± SEM from 3 separate experiments for each protein and treatment. Asterisks (*) indicate significant difference from untreated controls (<i>p</i><0.05).</p

DIDS (400 µM) causes mitochondrial swelling and cristae reduction.

<p>DIDS treated mitochondria were longer, fewer in number, and had markedly reduced cristae density. (<b>A</b>) Sample TEM images of mitochondria from neurons treated as indicated for 24 hrs. Arrow indicates “ballooning” of outer mitochondrial membrane (OMM). (<b>B</b>) Summary of mitochondrial morphology-related parameters from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060804#pone-0060804-g004" target="_blank">Fig. 4A</a>. (<b>C</b>) Summary of cristae morphology-related parameters from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060804#pone-0060804-g004" target="_blank">Fig. 4A</a>. Data are mean ± SEM. Asterisks (*) indicate significant difference from normoxic controls (<i>p</i><0.05).</p

DIDS causes RNA and DNA degradation in a dose-dependant manner.

<p>(<b>A&B</b>) Summaries of the effect of ACSF±DMSO±DIDS (40 or 400 µm) on total RNA content (RNA_tot) (A) or RNA integrity (RIN) (B) from neurons treated for 1, 2, 4, 8 or 24 hrs as indicated. RNA_tot was normalized to untreated samples. RNA experiments were repeated 4 times at each time point. (<b>C</b>) Summary of the ratio of terminal transferase dUTP nick end-labeling (TUNEL)-positive neurons relative to DAPI-stained nuclei from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060804#pone-0060804-g006" target="_blank">Fig. 6D</a>. (<b>D</b>) Sample confocal microscopy images of TUNEL (green, arrows) and DAPI (blue) fluorescence from neurons treated as indicated for 24 hrs. TUNEL experiments were repeated 4 times. Data are mean ± SEM. Asterisks (*) indicate significant difference from normoxic controls; bars indicate significance between treatments (<i>p</i><0.05).</p

IS induces apoptotic Annexin V translocation in neurons and astrocytes.

<p>(<b>A</b>) Sample paired DIC (left panels) and Annexin V and DAPI (green and blue fluorescence, respectively; right panels) confocal microscopy images of neurons and astrocytes treated as indicated for 24 hrs. Images are representative of 4 separate experiments. (<b>B</b>) Summary of the ratio of Annexin V-positive stained cells to DAPI-stained nuclei. (<b>C</b>) Summary of Annexin V fluorescence from neurons or astrocytes treated in 96-well microplates as indicated for 24 hrs. Data are mean ± SEM. Asterisks (*) indicated significant difference from untreated controls (<i>p</i><0.05).</p

IS upregulates autophagy and apoptotic pathways in neurons and astrocytes.

<p>(<b>A</b>) Sample Western blots of autophagy- and apoptosis related protein expression from neurons (left panels) and astrocytes (right panels) treated as indicated for 6 hrs. (<b>B</b>) Summary of fold-change in protein expressions from (A) normalized to α–actin expression in the same sample. Data are mean ± SEM from 3 separate experiments for each protein, cell type, and treatment. Asterisks (*) indicate significant difference from untreated controls (<i>p</i><0.05).</p

IS causes mitochondrial fission and membrane rupture.

<p>(<b>A</b>) Sample TEM images of mitochondria from neurons (upper) and astrocytes (lower) treated as indicated for 24 hrs. (<b>B</b>) Sample western blots of apoptosis inducing factor (AIF) and cytochrome C (Cyto C) release from samples treated as indicated for 6 hrs. Blots are representative of 3 separate experiments.</p