Intermittent hypoxia-induced TLR4 gene expression in microglia differs by time and CNS region.

<p>TLR4 gene expression was analyzed by qRT-PCR in immunomagnetically-separated microglia from the cortex, brainstem and spinal cord of healthy male rats exposed either to normoxia or IH for 1, 3 or 14 days. Means +/− 1 SEM are presented relative to expression in normoxic controls. **p<0.01; ***p<0.001; ^#p = 0.085.</p

Intermittent hypoxia-induced inflammatory gene expression is rapid and transient in spinal microglia but sustained in spinal tissue homogenates.

<p>iNOS, COX-2, TNFα, IL-1β and IL-6 gene expression was analyzed by qRT-PCR in immunomagnetically-separated microglia (A) or tissue homogenates (B) from the spinal cord of healthy male rats exposed either to normoxia or IH for 1, 3 or 14 days. Means +/− 1 SEM are presented relative to expression in normoxic controls. *p<0.05; **p<0.01; ***p<0.001; ^#p = 0.074.</p

Intermittent hypoxia-induced inflammatory gene expression differs temporally in brainstem microglia and brainstem tissue homogenates.

<p>iNOS, COX-2, TNFα, IL-1α and IL-6 gene expression was analyzed by qRT-PCR in immunomagnetically-separated microglia (A) or tissue homogenates (B) from the brainstem of healthy male rats exposed either to normoxia or IH for 1, 3 or 14 days. Means +/− 1 SEM are presented relative to expression in normoxic controls. *p<0.05; **p<0.01; ^#p = 0.06.</p

Intermittent hypoxia-induced inflammatory gene expression peaks in cortical microglia at 14 days of exposure.

<p>iNOS, COX-2, TNFα, IL-1β and IL-6 gene expression was analyzed by qRT-PCR in immunomagnetically-separated microglia (A) or tissue homogenates (B) from the cortex of healthy male rats exposed either to normoxia or IH for 1, 3 or 14 days. Means +/− 1 SEM are presented relative to expression in normoxic controls. *p<0.05; **p<0.01; ***p<0.001; ^#p = 0.09.</p

Location of slices for electrophysiological recordings and immunohistochemical analyses.

<p>Spontaneous neuronal activity was recorded in cortical slices <i>(</i><b><i>A</i></b><i>)</i>, and medullary slices <i>(</i><b><i>B</i></b><i>)</i> containing the rostral ventral respiratory group (rVRG), Pre Bötzinger (preBötC) and Bötzinger Complex (BötC) <i>(</i><b><i>C</i></b><i>)</i>. Electrode arrays placed in cortical slices were centered on primary motor/somatosensory cortex (<b><i>A</i></b>; hatched area). Electrode arrays in medullary slices were centered on the region containing the ventral respiratory column (VRC) <i>(</i><b><i>B</i></b><i>)</i>. The gray bar indicates the rostrocaudal extent of coronal sections used for <i>in vitro</i> electrophysiology and well as immunohistochemistry. During the initial equilibration, medullary neurons exhibited rhythmic bursts <i>(</i><b><i>D top, bottom</i></b><i>)</i>, slow tonic activity <i>(</i><b><i>E top</i></b><i>)</i> or fast tonic activity <i>(</i><b><i>E bottom</i></b><i>)</i>. Raw traces are shown above overlaid action potential waveforms extracted from raw data. (<b><i>D, E</i></b>). NA, nucleus ambiguus; IV, fourth ventricle. NTS, nucleus of the solitary tract; XII, hypoglossal nucleus; cVRG, caudal ventral respiratory group; rVRG, rostral ventral respiratory group; Pre-BötC, pre-Bötzinger complex; BötC, Bötzinger complex.</p

Increased GABA_AR ε subunit mRNA levels in medulla and cortex in 30d isoflurane-treated rats.

<p>Medullary GABA_AR ε mRNA levels were more than 3-fold greater in 30d isoflurane-treated rats compared to untreated control rats. Likewise, cortical GABA_AR ε mRNA levels were more than 4-fold increased in 30d isoflurane-treated rats. Statistics were conducted on delta CT values. Asterisk denotes p<0.05. Error bars indicate SEM.</p

Phrenic nerve activity is insensitive to pentobarbital application during pregnancy.

<p><b><i>A,</i></b> Representative, compressed phrenic neurograms from virgin female, G17 pregnant and 30 d post partum rats. After establishing baseline conditions, 10 mg/kg of pentobarbital was administered every 5 min up to a maximum total dose of 100 mg/kg. The protocol ended with 5 min of hypercapnic hypoxia to assess phrenic response to maximal chemosensory input under 100 mg/kg of pentobarbital. <b><i>B,</i></b> Survival plot of phrenic nerve activity by condition. 0% of post partum rats generated measurable phrenic nerve output above 50 mg/kg pentobarbital. No virgin female rats generated measureable phrenic nerve output above 60 mg/kg pentobarbital. 100% of pregnant animals generated phrenic nerve output at 60 mg/kg, and 17% generated activity at 90 mg/kg. <b><i>C,</i></b> Pentobarbital IC50 curve of phrenic nerve burst frequency demonstrates a significant rightward shift during pregnancy compared to virgin female and post partum animals. The hill slope of the IC50 curve during pregnancy is significantly increased as well. <b><i>D,</i></b> Pentobarbital IC50 curve of phrenic nerve amplitude demonstrates a significant rightward shift during pregnancy compared to virgin female and post partum animals. The hill slope of the amplitude IC50 during pregnancy is not significantly altered. <b><i>E,</i></b> Hypercapnic hypoxia produced a measurable response in the phrenic nerve in a greater number of pregnant animals (4/5) than virgin female (2/6), post partum (1/6), or male (0/6; not shown).</p

mRNA for the GABA_AR ε subunit is increased in the medulla during pregnancy.

<p>Quantitative analysis of mRNA for GABA_AR ε subunit in the medulla of virgin female, male, G17 pregnant, and 20–30 d post-partum rats. Medullary GABA_A ε mRNA was nearly 4-fold greater in pregnant than virgin female or male rats (p<0.05). Medullary GABA_AR ε mRNA appeared elevated in post-partum rats, but the difference was not statistically significant compared to virgin females. For graphical representation, delta delta CT values were normalized to virgin females. Statistics were conducted on delta CT values. * denotes different than virgin female, p<0.05. Error bars indicate S.E.M.</p

Neuronal responses to pentobarbital are altered in the medulla but not cortex of pregnant animals.

<p>Spontaneous activity of neurons in the VRC including rVRG, PreBötC, and BötC are insensitive to pentobarbital (300 µM) during pregnancy. <b><i>A,</i></b> Mean normalized firing rate of VRC (n = 37 closed circles) and CTX (n = 47, open squares) neurons from virgin female rats (n = 5 animals) in response to 300 µM pentobarbital (applied at vertical bar, t = 30 min). <b><i>B,</i></b> Pentobarbital had equivalent effects on VRC (n = 62) and CTX (n = 37) neurons from male rats (n = 6 animals). <b><i>C,</i></b> Pentobarbital (solid vertical line) failed to depress spontaneous activity of VRC neurons (n = 31) from pregnant animals (n = 4), yet inhibited CTX neurons (n = 26) in the same animals. Neuronal activity was modulated by barbiturate insensitive GABA_ARs, as 20 µM muscimol (dashed vertical line) rapidly silenced VRC neurons. <b><i>D,</i></b> This effect is reversible as both VRC (n = 51) post partum animals (n = 4) were inhibited by pentobarbital. <b><i>E,</i></b> Quantification of the normalized spontaneous activity of CTX (filled bars) and VRC (open bars) after 60 min of pentobarbital (300 µM) application. Single unit activity was normalized to mean baseline activity for that neuron (time 0–30 min). * denotes significantly different from virgin female. p<0.05.</p

Spontaneous neuronal activity resistance to bath-applied pentobarbital in medullary and cortical slices from 7d isoflurane-treated rats.

<p>Spontaneous activity of neurons in the region anatomically consistent with the VRC and PreBötC (all labeled as VRC neurons) are insensitive to bath-applied pentobarbital (300 μM) following 7d isoflurane treatment. <b><i>A</i></b>, Mean normalized firing rate of VRC (n = 62, open circles) and CTX (n = 37, filled circles) neurons from male rats in response to 300 μM pentobarbital (applied at vertical bar, t = 30 min). <b><i>B</i></b>, Pentobarbital had equivalent depressive effects on VRC (n = 37) and CTX (n = 47) neurons from virgin female rats. <b><i>C</i></b>, Following pentobarbital treatment, spontaneous activity of VRC neurons (n = 62) from male rats treated with intermittent isoflurane for 7d were significantly more active than VRC neurons from untreated control rats. The response of cortical neurons (n = 49) to pentobarbital was non-significantly shifted in the same direction. <b><i>D</i></b>, Likewise, in female rats treated with isoflurane for 7d, pentobarbital failed to inhibit spontaneous neuronal activity in the VRC neurons (n = 40). CTX neurons (n = 51) trended non-significantly towards an increase in activity compared to untreated control female rats. <b><i>E</i></b>, This effect was reversible, as 7d of recovery following 7d of intermittent isoflurane shifted spontaneous activity of VRC (n = 27) and CTX neurons (n = 38) in rats toward baseline values. <b><i>F</i></b>., 30d of intermittent isoflurane significantly attenuated the pentobarbital response in VRC (n = 40) and CTX neurons (n = 32) in male rats. To confirm the presence of pentobarbital-insensitive GABA_ARs, muscimol (20 μM) was applied at 90 min (dashed vertical line). Nearly all neurons that were resistant to pentobarbital were inhibited by muscimol. <b><i>G</i></b>, In female rats exposed to 30d isoflurane treatment, the response of VRC (n = 123) and CTX (n = 83) neurons to pentobarbital was significantly attenuated. These neurons were also silenced by muscimol (dashed vertical line). (<b><i>H</i>,<i>I</i>)</b> The average steady-state responses to bath-applied pentobarbital are quantified for the different isoflurane treatments and recovery for male rats (<b><i>C</i></b>) and female rats (<b><i>D</i></b>). The asterisk indicates p<0.05 for comparison to control while the pound sign indicates p<0.05 for comparison to 7d treatment.</p