Acute Intermittent Hypoxia Research Articles

Acute intermittent hypoxia evokes ventilatory long-term facilitation and active expiration in unanesthetized rats

Acute intermittent hypoxia (AIH) modifies the functioning of the respiratory network, causing respiratory motor facilitation in anesthetized animals and a compensatory increase in pulmonary ventilation in freely behaving animals. However, it is still unclear whether the ventilatory facilitation induced by AIH in unanesthetized animals is associated with changes in the respiratory pattern. We found that Holtzman male rats (80–150 g) exposed to AIH (10 × 6% O2 for 30–40 s every 5 min, n = 9) exhibited a prolonged (30 min) increase in baseline minute ventilation (P < 0.05) compared to control animals (n = 13), combined with the occurrence of late expiratory peak flow events, suggesting the presence of active expiration. The increase in ventilation after AIH was also accompanied by reductions in arterial CO2 and body temperature (n = 5–6, P < 0.05). The systemic treatment with ketanserin (a 5-HT2 receptor antagonist) before AIH prevented the changes in ventilation and active expiration (n = 11) but potentiated the hypothermic response (n = 5, P < 0.05) when compared to appropriate control rats (n = 13). Our findings indicate that the ventilatory long-term facilitation elicited by AIH exposure in unanesthetized rats is linked to the generation of active expiration by mechanisms that may depend on the activation of serotonin receptors. In contrast, the decrease in body temperature induced by AIH may not require 5-HT2 receptor activation.

The long-term impact of ovariectomy on ventilation and expression of phrenic long-term facilitation in female rats.

New Findings What is the central question of this study? Would ovariectomy cause prolonged changes in ventilation and sustained loss of acute, intermittent hypoxia‐induced neuroplasticity or would these outcomes be restored with time? What is the main finding and its importance? Our main findings demonstrate that ovariectomy elicits minimal alteration in overall breathing function but impairs acute, intermittent hypoxia‐induced plasticity for ≤ 12 weeks. Sex hormones are necessary to enable respiratory neuroplasticity, including phrenic long‐term facilitation (pLTF), a form of respiratory motor plasticity elicited by acute, intermittent hypoxia (AIH). Female rats exhibit a progressive increase in phrenic nerve amplitude after AIH characteristic of pLTF only during pro‐oestrus, the stage of the oestrous cycle notable for elevated circulating oestradiol levels. Removal of the ovaries [ovariectomy (OVX)], the primary source of circulating oestradiol, also eliminates AIH‐induced pLTF after 1 week. Ovariectomy is used routinely as a model to examine the impact of sex hormones on CNS structure and function, but the long‐term impact of OVX is rarely examined. Extra‐ovarian sites of oestradiol synthesis, including multiple CNS sites, have been identified and might possess the capacity to restore oestradiol levels, in part, over time, impacting respiratory function and the expression of respiratory neuroplasticity. We examined both ventilation in awake, freely behaving female rats, using barometric plethysmography, and the expression of AIH‐induced pLTF in anaesthetized, ventilated female rats 2 and 12 weeks after OVX and compared them with age‐matched ovarian‐intact female rats. Our findings indicate that chronic OVX had little impact on baseline breathing or in the response to respiratory challenge (10% O2, 5% CO2, balance N2) during plethysmography. However, OVX rats at both 2 and 12 weeks demonstrated a persistent loss of AIH‐induced pLTF relative to control animals (P < 0.01), suggesting that other sources of oestradiol synthesis were insufficient to restore pLTF. These data are consistent with our previous work indicating that oestradiol plays a key role in expression of AIH‐induced respiratory neuroplasticity.

Daily acute intermittent hypoxia enhances phrenic motor output and stimulus-evoked phrenic responses in rats.

Plasticity is a hallmark of the respiratory neural control system. Phrenic long-term facilitation (pLTF) is one form of respiratory plasticity characterized by persistent increases in phrenic nerve activity following acute intermittent hypoxia (AIH). Although there is evidence that key steps in the cellular pathway giving rise to pLTF are localized within phrenic motor neurons (PMNs), the impact of AIH on the strength of breathing-related synaptic inputs to PMNs remains unclear. Furthermore, the functional impact of AIH is enhanced by repeated/daily exposure to AIH (dAIH). Here, we explored the effects of AIH versus 2 wk of dAIH preconditioning on spontaneous and evoked phrenic responses in anesthetized, paralyzed, and mechanically ventilated rats. Evoked phrenic potentials were elicited by respiratory cycle-triggered lateral funiculus stimulation at the C2 spinal level delivered before and 60 min post-AIH (or the equivalent in time controls). Charge-balanced biphasic pulses (100 μs/phase) of progressively increasing intensity (100-700 μA) were delivered during the inspiratory and expiratory phases of the respiratory cycle. Although robust pLTF (∼60% from baseline) was observed after a single exposure to moderate AIH (3 × 5 min; 5-min intervals), there was no effect on evoked phrenic responses, contrary to our initial hypothesis. However, in rats preconditioned with dAIH, baseline phrenic nerve activity and evoked responses were increased, suggesting that repeated exposure to AIH enhances functional synaptic strength when assessed using this technique. The impact of daily AIH preconditioning on synaptic inputs to PMNs raises interesting questions that require further exploration.NEW & NOTEWORTHY Two weeks of daily acute intermittent hypoxia (dAIH) preconditioning enhanced stimulus-evoked phrenic responses to lateral funiculus stimulation (targeting respiratory bulbospinal projection to phrenic motor neurons). Furthermore, dAIH preconditioning enhanced baseline phrenic motor output responses to maximal chemoreflex activation in intact rats.

Oxidative stress in the heart of rats exposed to acute intermittent hypobaric hypoxia

Oxidative stress in the heart of rats exposed to acute intermittent hypobaric hypoxia

Acute morphine blocks spinal respiratory motor plasticity via long-latency mechanisms that require toll-like receptor 4 signalling.

While respiratory complications following opioid use are mainly mediated via activation of mu opioid receptors, long-latency off-target signalling via innate immune toll-like receptor 4 (TLR4) may impair other essential elements of breathing control such as respiratory motor plasticity. In adult rats, pre-treatment with a single dose of morphine blocked long-term facilitation (LTF) of phrenic motor output via a long-latency TLR4-dependent mechanism. In the phrenic motor nucleus, morphine triggered TLR4-dependent activation of microglial p38 MAPK - a key enzyme that orchestrates inflammatory signalling and is known to undermine phrenic LTF. Morphine-induced LTF loss may destabilize breathing, potentially contributing to respiratory side effects. Therefore, we suggest minimizing TLR-4 signalling may improve breathing stability during opioid therapy. Opioid-induced respiratory dysfunction is a significant public health burden. While respiratory effects are mediated via mu opioid receptors, long-latency off-target opioid signalling through innate immune toll-like receptor 4 (TLR4) may modulate essential elements of breathing control, particularly respiratory motor plasticity. Plasticity in respiratory motor circuits contributes to the preservation of breathing in the face of destabilizing influences. For example, respiratory long-term facilitation (LTF), a well-studied model of respiratory motor plasticity triggered by acute intermittent hypoxia, promotes breathing stability by increasing respiratory motor drive to breathing muscles. Some forms of respiratory LTF are exquisitely sensitive to inflammation and are abolished by even a mild inflammation triggered by TLR4 activation (e.g. via systemic lipopolysaccharides). Since opioids induce inflammation and TLR4 activation, we hypothesized that opioids would abolish LTF through a TLR4-dependent mechanism. In adult Sprague Dawley rats, pre-treatment with a single systemic injection of the prototypical opioid agonist morphine blocks LTF expression several hours later in the phrenic motor system - the motor pool driving diaphragm muscle contractions. Morphine blocked phrenic LTF via TLR4-dependent mechanisms because pre-treatment with (+)-naloxone - the opioid inactive stereoisomer and novel small molecule TLR4 inhibitor - prevented impairment of phrenic LTF in morphine-treated rats. Morphine triggered TLR4-dependent activation of microglial p38 MAPK within the phrenic motor system - a key enzyme that orchestrates inflammatory signalling and undermines phrenic LTF. Morphine-induced LTF loss may destabilize breathing, potentially contributing to respiratory side effects. We suggest minimizing TLR-4 signalling may improve breathing stability during opioid therapy by restoring endogenous mechanisms of plasticity within respiratory motor circuits.

The effect of acute intermittent hypoxia on postprandial triglyceride levels in humans: a randomized crossover trial

BackgroundObstructive sleep apnea (OSA), a sleep disorder frequently observed in individuals living with obesity, consists of repeated involuntary breathing obstructions during sleep, leading to intermittent hypoxia (IH). In humans, acute continuous hypoxia slightly increases plasma triglycerides (TG). However, no study yet compared the postprandial TG response of individuals with or without OSA under intermittent hypoxia.MethodsUsing a randomized crossover design, seven individuals diagnosed with moderate OSA and eight healthy individuals without OSA were given a meal after which they were exposed for 6 h to normoxia or intermittent hypoxia (e.g., 15 hypoxic events per hour). Blood lipid levels were measured hourly during each session.ResultsPeak postprandial TG concentrations tended to be 22% higher under IH irrespective of group (IH × time interaction, p = 0.068). This trend toward higher total plasma TG was attributable to increased levels of denser TG-rich lipoproteins such as very low-density lipoproteins (VLDL) and chylomicrons (CM) remnants. Irrespective of group, the postprandial TG concentrations in denser TG-rich lipoproteins was 20% higher under IH (IH × time interaction, p = 0.036), although IH had virtually no impact on denser TG-rich lipoprotein concentrations in the OSA group.ConclusionAcute intermittent hypoxia tends to negatively affect postprandial TG levels in healthy individuals, which is attributable to an increase in denser TG-carrying lipoprotein levels such as VLDL and CM remnants. This altered postprandial TG response to acute intermittent hypoxia was not observed in individuals with OSA.

Cardiorespiratory plasticity in humans following two patterns of acute intermittent hypoxia.

What is the central question of this study? Do cardiorespiratory experience-dependent effects (EDEs) differ between two different stimulus durations of acute isocapnic intermittent hypoxia (IHx; 5-min vs. 90-s cycles between hypoxia and normoxia)? What is the main finding and its importance? There was long-term facilitation in ventilation and blood pressure in both IHx protocols, but there was no evidence of progressive augmentation or post-hypoxia frequency decline. Not all EDEs described in animal models translate to acute isocapnic IHx responses in humans, and cardiorespiratory responses to 5-min versus 90-s on/off IHx protocols are largely similar. Peripheral respiratory chemoreceptors monitor breath-by-breath changes in arterial CO2 and O2 , and mediate ventilatory changes to maintain homeostasis. Intermittent hypoxia (IHx) elicits hypoxic ventilatory responses, with well-described experience-dependent effects (EDEs), derived mostly from animal work involving intermittent 5-min cycles of hypoxia and normoxia. These EDEs include post-hypoxia frequency decline (PHxFD), progressive augmentation (PA) and long-term facilitation (LTF). Comparisons of these EDEs between animal models and humans using similar IHx protocols are lacking. In addition, it is unknown whether shorter bouts of hypoxia, which may be more relevant to clinical conditions, elicit EDEs of similar magnitudes in humans. Respiratory (frequency, tidal volume and minute ventilation ( ) and cardiovascular (heart rate and mean arterial pressure (MAP)) variables were measured during and following two patterns of acute isocapnic IHx in 14 healthy human participants (four female): (1) 5×5min and (2) 5×90s on/off hypoxia. Participants' end-tidal was clamped at 45Torr during hypoxia and 100Torr during normoxia. We found that (1) PHxFD and PA were not present in either IHx pattern (P>0.14), (2) LTF was present in following both 5-min (P<0.001) and 90-s isocapnic IHx trials (P<0.001), and (3) LTF was present in MAP following 5-min isocapnic IHx (P<0.001), and trended towards significance following 90-s IHx (P=0.058). We demonstrate that acute isocapnic IHx alone may not elicit all of the EDEs that have been described in animal models. Additionally, ventilatory LTF occurred regardless of the length of hypoxia-normoxia cycles.

Muscle Metaboreflex Control of Sympathetic Activity Is Preserved after Acute Intermittent Hypercapnic Hypoxia.

In normotensive patients with obstructive sleep apnea (OSA), the muscle sympathetic nerve activity (MSNA) response to exercise is increased while metaboreflex control of MSNA is decreased. We tested the hypotheses that acute intermittent hypercapnic hypoxia (IHH) in males free from OSA and associated comorbidities would augment the MSNA response to exercise but attenuate the change in MSNA during metaboreflex activation. Thirteen healthy males (age = 24 ± 4 yr) were exposed to 40 min of IHH. Before and after IHH, the pressor response to exercise was studied during 2 min of isometric handgrip exercise (at 30% maximal voluntary contraction), whereas the metaboreflex was studied during 4 min of postexercise circulatory occlusion (PECO). Mean arterial pressure (MAP), heart rate (HR), and fibular MSNA were recorded continuously. MSNA was quantified as burst frequency (BF) and total activity (TA). Mixed effects linear models were used to compare the exercise pressor and metaboreflex before and after IHH. As expected, IHH led to significant increases in MSNA BF, TA, and MAP at baseline and throughout exercise and PECO. However, during handgrip exercise, the change from baseline in MAP, HR, MSNA BF, and TA was similar before and after IHH (All P > 0.31). During PECO, the change from baseline in MSNA BF and TA was similar after IHH, whereas the change from baseline in MAP (Δ14 mm Hg, 95% CI = 7-19, vs Δ16 mm Hg, 95% CI = 10-21; P < 0.01) was modestly increased. After acute IHH, MSNA response to handgrip exercise and metaboreflex activation were preserved in healthy young males despite overall increases in resting MSNA and MAP. Chronic IHH and comorbidities often associated with OSA may be required to modulate the exercise pressor reflex and metaboreflex.

Blood pressure and high altitude: physiological response and clinical management

High altitude is an extreme environment that challenges human beings exposed because of work, recreational activities, or habitat. Exposure to hypobaric hypoxia results in physiological adaptations in response to the geography and the associated extreme environmental conditions. These acclimatization responses can be diverse and result from evolutionary changes and comorbidities. In this context, this review aims to identify the available evidence on the effects of high altitude on blood pressurefrom the physiological to clinical aspects at rest and during exerciseand the underlying mechanisms and possible clinical implications of acute and chronic intermittent hypoxia.

The combination of intermittent electrical stimulation with acute intermittent hypoxia strengthens genioglossus muscle discharge in chronic intermittent hypoxia-pretreated rats

ObjectiveExploring whether the genioglossus discharge in chronic intermittent hypoxia(CIH) – pretreated rats could be enhanced by intermittent electrical stimulation combined with acute intermittent hypoxia(AIH). MethodsRats were pretreated with CIH for 4 weeks and then were randomly divided into 6 groups: time control, intermittent electric stimulation, AIH, intermittent electric stimulation + AIH, continuous electric stimulation and continuous hypoxia exposure. The genioglossus discharges were recorded and compared before and after stimulation. Normoxic-treated rats were grouped and treated with the same stimulation protocols. ResultsIntermittent electrical stimulation or AIH temporarily increased the activity of the genioglossus discharge, in which the degree of the increase was significantly higher in CIH-pretreated rats than in normoxic rats.After intermittent electrical stimulation, AIH evoked a sustained elevation of genioglossus discharge activities in CIH-pretreated rats, in which the degree of the increase was significantly higher than in rats induced by a single intermittent electric stimulation. ConclusionIntermittent electrical stimulation combined with AIH strengthens the genioglossus plasticity in CIH-pretreated rats.

Single-session effects of acute intermittent hypoxia on breathing function after human spinal cord injury

After spinal cord injury (SCI) respiratory complications are a leading cause of morbidity and mortality. Acute intermittent hypoxia (AIH) triggers spinal respiratory motor plasticity in rodent models, and repetitive AIH may have the potential to restore breathing capacity in those with SCI. As an initial approach to provide proof of principle for such effects, we tested single-session AIH effects on breathing function in adults with chronic SCI. 17 adults (13 males; 34.1 ± 14.5 years old; 13 motor complete SCI; >6 months post injury) completed two randomly ordered sessions, AIH versus sham. AIH consisted of 15, 1-min episodes (hypoxia: 10.3% O2; sham: 21% O2) interspersed with room air breathing (1.5 min, 21% oxygen); no attempt was made to regulate arterial CO2 levels. Blood oxygen saturation (SpO2), maximal inspiratory and expiratory pressures (MIP; MEP), forced vital capacity (FVC), and mouth occlusion pressure within 0.1 s (P0.1) were assessed. Outcomes were compared using nonparametric Wilcoxon's tests, or a 2 × 2 ANOVA. Baseline SpO2 was 97.2 ± 1.3% and was unchanged during sham experiments. During hypoxic episodes, SpO2 decreased to 84.7 ± 0.9%, and returned to baseline levels during normoxic intervals. Outcomes were unchanged from baseline post-sham. Greater increases in MIP were evident post AIH vs. sham (median values; +10.8 cmH2O vs. -2.6 cmH2O respectively, 95% confidence interval (−18.7) – (−4.3), p = .006) with a moderate Cohen's effect size (0.68). P0.1, MEP and FVC did not change post-AIH. A single AIH session increased maximal inspiratory pressure generation, but not other breathing functions in adults with SCI. Reasons may include greater spared innervation to inspiratory versus expiratory muscles or differences in the capacity for AIH-induced plasticity in inspiratory motor neuron pools. Based on our findings, the therapeutic potential of AIH on breathing capacity in people with SCI warrants further investigation.

The Vascular Response to Sympathetic Activation is Attenuated in Young Women Following Acute Intermittent Hypoxia

INTRODUCTION : Acute intermittent hypoxia (AIH) elicits an increase in sympathetic nervous activity (SNA) that persists following return to room air. This AIH-mediated sympathetic activation is associated with an increase in blood pressure in men, but not women. These data suggest the vascular responsiveness to SNA is attenuated in young women, although mechanisms are unclear. We sought to examine the vascular responsiveness to sympathetic activation before and after AIH in heathy young men and women. We hypothesized the vascular response to sympathetic activation would be preserved following AIH in men. We further hypothesized the vascular response to sympathetic activation would be attenuated in women following AIH, with the greatest effect observed when endogenous estradiol was high. METHODS : Twelve men (25±1 yrs) and nine women (24±1 yrs) participated. Women were studied twice, during the early follicular (EF, Estradiol 28±4 pg/mL) and late follicular (LF, Estradiol 185±51 pg/mL) phases of the menstrual cycle. Before and after 30-min AIH, a 2-min cold pressor test (CPT) was conducted to increase SNA. Forearm blood flow (FBF, venous occlusion plethysmography) and blood pressure (finger photoplethysmography) were measured continuously. FBF was normalized for mean blood pressure (forearm vascular conductance, FVC) and a change in response to CPT was calculated (ΔFVC = Baseline - CPT). RESULTS : Mean blood pressure increased following AIH in men (p=0.03) with no effect in women (p>0.05). In men, the vascular response to sympathetic activation was preserved following AIH in men (ΔFVC -0.5±0.4 vs -0.4±0.6 mL/min/dL/100 mmHg, p=0.85). In contrast, the vascular response to sympathetic activation was attenuated in women following AIH (ΔFVC: EF -1.0±0.3 vs -0.4±0.2 mL/min/dL/100 mmHg; LF -0.8±0.4 vs -0.1±0.2 mL/min/dL/100 mmHg, p<0.01) with no effect of menstrual cycle phase (p=0.99). CONCLUSION : The vascular response to sympathetic activation is preserved in men following AIH, but is attenuated in women and is independent of menstrual cycle phase. Present data highlight sex-related differences in the neurovascular response to AIH and indicate sex differences in the blood pressure response to AIH may be due, in part, to an attenuated vascular response to SNA in women.

Fractalkine signaling in the cervical spinal cord orchestrates microglia‐neuron interactions regulating intermittent hypoxia‐induced phrenic motor plasticity

Microglia are innate immune cells with many non-immune functions in the healthy nervous system. For example, microglia engage in bidirectional communication with central nervous system neurons, regulating diverse processes including neuronal architecture, excitability, and synaptic plasticity. Although neuron-microglia interactions are known to regulate several forms of neuroplasticity, their role in regulating respiratory motor plasticity is unknown. Acute intermittent hypoxia (AIH) elicits a form of plasticity in the phrenic motor system known as phrenic long-term facilitation (pLTF) — a progressive, long-lasting increase in phrenic motor output following AIH. Whereas AIH consisting of moderate hypoxic episodes elicits pLTF by a serotonin-dependent, adenosine-constrained mechanism, severe AIH elicits pLTF by an adenosine-dominant mechanism. Here, we test the hypothesis that communication between cervical spinal neurons and microglia differentially regulates moderate versus severe AIH-induced pLTF. Specifically, we hypothesized that neuron-microglia signaling modulates pLTF expression by: 1) neuronal fractalkine (CX3CL1) release and activation of its receptor, CX3CR1, which is uniquely expressed in CNS microglia; and 2) fractalkine-induced ATP release, and breakdown to adenosine, which differentially regulates moderate and severe AIH-induced pLTF. In anesthetized, adult male Sprague Dawley rats, cervical spinal CX3CR1 inhibition enhanced serotonin-dependent moderate AIH-induced pLTF, but blocked adenosine-dependent, severe AIH-induced pLTF. Further, spinal CX3CR1 activation with recombinant fractalkine induced phrenic motor facilitation in a dose-dependent manner— an effect that required both cervical spinal ATP breakdown and adenosine 2A receptor activation. We thus propose a hypothetical model whereby hypoxia triggers dose-dependent phrenic motor neuron fractalkine release that activates CX3CR1 on nearby microglia. Subsequent microglial ATP release and eventual breakdown increases extracellular adenosine and undermines serotonin-dependent pLTF, but drives severe AIH-induced (adenosine-dependent) pLTF by activating phrenic motor neuron adenosine 2A receptors. These findings enhance our understanding of how spinal, intercellular interactions regulate AIH-induced plasticity in the phrenic motor circuit.

Daily acute intermittent hypoxia therapy 1‐week after moderate contusion spinal cord injury improves lower urinary tract function in rats

There is an increasing interest in the use of non-invasive acute intermittent hypoxia (AIH) as a therapeutic intervention to improve respiratory and locomotor function in spinal cord injury (SCI). SCI, however, also significantly disrupts normal bladder function, leading to detrusor overactivity, decreased voiding efficiency, and increased residual volume. Ongoing studies in our laboratory investigating the effects of AIH exposure in rats as a strategy for improving lower urinary tract (LUT) function following mid-thoracic SCI indicate that a single bout of AIH decreases the incidence of non-voiding bladder contractions (NVCs) and increases voiding efficiency. In the current study, we begin to evaluate the potential for AIH to be used as a therapy for inducing long-lasting improvements in SCI-induced LUT dysfunction. To accomplish this, we examined the effects of daily AIH (dAIH) administered for 7 consecutive days on both spontaneous awake micturition patterns and reflex micturition event (rME) behaviors in adult female Sprague-Dawley rats with mid-thoracic moderate contusion (200 kdyn) SCI. For these experiments, dAIH composed of five alternating 3-min exposures of hypoxia (12% O2) and normoxia (21% O2) was initiated 1-week after SCI, with awake micturition patterns being evaluated by overnight metabolic chamber measurements weekly for 2-3-weeks before and 4 weeks after SCI and rME behaviors elicited by continuous infusion of saline (0.04-0.09 ml/min) into the bladder being evaluated by cystometry at 4-weeks after SCI; daily normoxia (dNx; 21% O2 episodes) served as a control. The impact of SCI on hindlimb motor and LUT function prior to initiating dAIH or dNx treatment was evaluated using BBB scores and manually expressed urine volumes during post-operative care, and these measurements revealed that deficits and initial stages of recovery were similar (as expected) in both treatment groups. Serial metabolic chamber measurements in both dAIH- and dNx-treated rats that exhibited functional voids at 1-week post-SCI demonstrated that most rats exhibited some degree of functional recovery of micturition pattern behaviors. Only dAIH-treated rats, however, exhibited a recovery of void frequency pattern (i.e., number of voids and inter-void interval)to pre-SCI levels albeit with slightly increased void volumes; dNx-treated rats also had increased void volumes. Intravesical bladder pressure (BP) measurements during terminal cystometry experiments in urethane-anesthetized (1.4 g/kg) rats revealed that dAIH-treated rats exhibited a decreased incidence of NVCs and that voiding contractions had increased delta BP (peak BP-minimum BP) and dynamic voiding efficiency (void volume/fill volume) compared to dNx-treated rats. Taken together, these findings suggest that dAIH treatment produces beneficial improvements in LUT function lasting at least two weeks that include more efficient voiding and decreased NVCs, and further support the use of dAIH therapy to treat SCI-induced LUT dysfunction.

Sex Differences in the Effect of Acute Intermittent Hypoxia on Respiratory‐Sympathetic Coupling in Humans

Background Sex-related differences in respiratory-sympathetic coupling have been described in preclinical models of intermittent hypoxia (IH) and have been implicated in the development of neurogenic hypertension. Based on sex disparities in the respiratory and blood pressure response to acute IH in humans as well as changes in respiratory-sympathetic coupling in clinical sleep apnea, we examined sex-related differences in respiratory modulation of muscle sympathetic nerve activity (MSNA) following acute IH in healthy humans. We hypothesized the magnitude of respiratory-sympathetic coupling would increase in both men and women following IH; however, the respiratory patterning of MSNA following IH would be sex-specific. Methods Heart rate, MSNA, and respiration were assessed in 21 healthy young men (30±1 yrs) and 10 healthy young women (28±1 yrs) during normoxic rest prior to and following IH. IH was achieved by alternating between a hypercapnic hypoxic (3% carbon dioxide, 5% oxygen) gas cylinder and room air (21% oxygen) to target 15 hypoxic events over 30-min. Respiratory modulation of MSNA was assessed by fitting polynomials to cross-correlation histograms constructed between sympathetic spikes and respiration. Results MSNA was elevated following IH in both men (34±2 to 41±3 bursts/100 heart beats) and women (30±4 to 35±5 bursts/100 heart beats) (p<0.01). When examining the normalized temporal profile of MSNA, both men and women exhibited changes in respiratory patterning of MSNA following IH; however, the pattern of MSNA differed by sex. Following IH, women exhibited more sympathetic activity during late expiration and less during post-inspiration (interaction of IH and time, p<0.01). In contrast, men exhibited greater sympathetic activity during post-inspiration following IH (interaction of IH and time, p<0.01). Conclusions Both men and women exhibit changes in respiratory patterning of MSNA following acute IH; however, this pattern differs by sex. These data support sex disparities in respiratory-sympathetic coupling and advance our understanding of sex-specific changes in sympathetic activation following IH which may have important implications for the development of neurogenic hypertension.

The Long‐Term Impact of Ovariectomy on Ventilation and Expression of Phrenic Long‐Term Facilitation in Female Rats

Estrogen is necessary for the expression of phrenic long-term facilitation (pLTF) in female rats. pLTF, a well-studied model of respiratory neuroplasticity, is characterized by a progressive increase in phrenic nerve amplitude following acute intermittent hypoxia (AIH). Gonadally-intact, adult female rats only develop pLTF in stages of the estrous cycle with high levels of circulating estrogen. Removal of the ovaries (ovariectomy, OVX), the primary source of circulating estrogen in females, also prevents AIH-induced pLTF and estrogen supplementation is sufficient to restore pLTF in OVX rats, supporting a key role for estrogen in the development of pLTF. Respiratory neuroplasticity studies using OVX female rats are generally completed ~7-10 days post-OVX; a timeframe sufficient for surgery recovery and for circulating estrogen levels to significantly decline. However, few studies have explored the long-term impact of OVX on respiratory function or the development of respiratory neuroplasticity. Here, we examined ventilation in awake, freely behaving rats and the development of AIH-induced pLTF in adult female rats 12 weeks-post OVX (or sham control surgeries) and compared them to female rats 2 weeks-post OVX. Our hypothesis was that chronic OVX would have minimal long-term impact on ventilation but would continue to impair development of AIH-induced pLTF. Whole body plethysmography was used to measure baseline breathing (20.9% O2, N2 balance) and ventilatory responses to strong chemoreceptor stimulation (10% O2, 5% CO2, N2 balance). Respiratory measures including frequency, tidal volume, minute ventilation, and respiratory neural drive were measured and compared between groups. Following plethysmography, phrenic nerve recordings were used to quantify pLTF. Results indicate that chronic OVX had little impact on baseline breathing function and rats in all experimental groups responded appropriately to respiratory challenge during plethysmography. However, 12 week OVX rats showed a significant increase in tidal volume relative to baseline during respiratory challenge (p≤0.001), different from other groups. This increase in tidal volume production was also reflected in the calculation of tidal volume/inspiratory time, a measure of relative respiratory neural drive (p≤0.01) but not in minute ventilation. OVX had sustained effects on development of respiratory plasticity, as only rats in the 12 week sham control group displayed pLTF, indicated by significantly increased phrenic nerve amplitude 60-min following AIH (p≤0.001). This increase in amplitude was higher than both 12 week and 2 week OVX groups (p≤0.01). ELISA assays also showed a significant correlation between circulating estrogen levels and magnitude of pLTF (p= 0.039) In summary, OVX appears to limit the expression of respiratory neuroplasticity in female rats for up to 12 weeks. Since no significant changes in circulating estrogen are observed in OVX rats over that time, these data are consistent with our prior work indicating that estrogen plays a key role in development of respiratory neuroplasticity.

Daily administration of ketoprofen restores AIH‐induced phrenic long term facilitation with prolonged chronic intermittent hypoxia

Acute intermittent hypoxia (AIH) elicits a form of spinal, respiratory motor plasticity known as phrenic long-term facilitation (pLTF). Preconditioning with a single day of chronic intermittent hypoxia (8 hours, 2 min hypoxic episodes with 2 min intervals) abolishes AIH-induced pLTF through a mechanism that requires systemic inflammation and spinal p38 MAP kinase activation (Huxtable et al., 2015). pLTF is restored by high-dose, systemic administration of the non-steroidal anti-inflammatory drug, ketoprofen, or spinal p38 MAP kinase inhibition. Similarly, preconditioning with 7 days of CIH (8 hours per day, 2 min episodes, 2 min intervals) abolishes pLTF, but we observed that phospho-p38 within phrenic motor neurons is unchanged from control rats (Gonzalez-Rothi and colleagues, unpublished). Thus, mechanisms whereby 7 days of CIH undermines AIH-induced pLTF are unclear. Since CIH induces time-dependent spinal inflammatory gene expression profiles over time (Smith et al., 2013), we hypothesize that prolonged CIH (versus a single day) blunts LTF through an inflammation-dependent mechanism that is independent of p-38MAP kinase. Thus, in ongoing experiments, the impact of anti-inflammatory drugs on AIH-induced pLTF in rats preconditioned with prolonged CIH (7 days). pLTF was restored when ketoprofen was administered each day just prior to the IH7 exposure (70% increase, p=.0013 vs. vehicle), but not when a single dose of ketoprofen was delivered just prior to assessment of pLTF (19% increase, p=0.9975 vs. vehicle). Upcoming experiments will explore the role of p38 MAP kinase with prolonged CIH exposure. These experiments are the first to explore shifting mechanisms whereby prolonged CIH and associated inflammation undermine phrenic motor plasticity. Such experiments are essential since chronic intermittent hypoxia is a hallmark of sleep apnea, a condition highly prevalent in individuals living with neurological disorders (spinal cord injury, ALS, etc.). It is therefore important to identify and mitigate factors which may undermine the potential for therapeutic interventions (i.e., repetitive AIH) to harness endogenous plasticity and ultimately improve breathing.

Acute Intermittent Optogenetic Stimulation of Median Preoptic Nucleus (MnPO) Induces Sympathetic Long‐term Facilitation (sLTF)

Acute intermittent hypoxia in the rodent model can produce a ramp increase in sympathetic nerve activity (SNA) that progressively increases and is maintained for up to an hour after stimulation. The induction of this increase, termed sympathetic long-term facilitation (sLTF), involves circulating Angiotensin II acting on the subfornical organ (SFO) and the paraventricular nucleus of the hypothalamus (PVN). The median preoptic nucleus (MnPO) receives inputs from the SFO and projects to the PVN and might mediate the induction of sLTF. This study used an intersectional viral approach to test the ability of acute intermittent optogenetic stimulation of PVN-projecting MnPO neurons to induce sLTF. Male Sprague-Dawley rats (250 g) anesthetized with isoflurane were stereotaxically microinjected with CRE-containing retrograde AAV (AAV9.hSyn.HI.eGFP-cre.WPRE.SV40) in PVN and with either Channel rhodopsin 2 (ChR2)-containing AAV (rAAV2/Ef1a-DIO-hchR2(H134R)-mCherry) or control virus (rAAV2/Ef1a-DIO-mCherry) in MnPO. This resulted in CRE-dependent expression of either ChR2 or mCherry in PVN-projecting MnPO neurons. Following a 4-wk recovery, rats underwent acute intermittent optogenetic (AIO) experiments where splanchnic and or renal SNA, blood pressure (BP), and heart rate (HR) were recorded. Each rat was anesthetized with urethane (800mg/kg) and α-chloralose (80 mg/kg) cocktail delivered by intraperitoneal injection, paralyzed with gallamine triethiodide (20 mg/ml, 0.25 ml/h, iv), bilaterally vagotomized, and mechanically ventilated. Noise levels was determined after a bolus injection of hexamethonium (30 mg/kg iv). MnPO terminals in the PVN were stimulated to induce AIO with a train of 10 stimuli, once per 6 minutes. Each stimuli consisted of a duty cycle of 20-ms light pulse delivered at 5 Hz for 1 min. A single optogenetic duty cycle did not alter splanchnic SNA (P=0.216, ChR2 n = 5 vs control n =2), renal SNA (P=0.234, ChR2 n =3 vs control n =2), blood pressure (P=0.928, ChR2 n = 6 vs control n =4), nor heart rate (P=0.174, ChR2 n = 6 vs control n =4). AIO significantly increased splanchnic SNA (P=0.0163) in rats with ChR2 as compared to rats injected with the control vector 1h post-AIO (ChR2 percent baseline: 160.51±20.08%, n= 5 vs control percent baseline: 77.03±11.76%, n = 2). There were no differences in renal SNA (P=0.302, ChR2 n =3 vs control n =2), BP (P=0.339, ChR2 n = 6 vs control n =4), and HR (P=0.731, ChR2 n =3 vs control n =2) between rats injected with ChR2 as compared to those in injected with the control vector 1h post-AIO. These results suggest that intermittent activation of PVN-projecting MnPO neurons may induce sLTF in the splanchnic SNA but not in renal SNA. Further study is needed to clarify the mechanisms responsible for the differential effects of MnPO-PVN AIO.

Conversion of Testosterone to Estradiol is Necessary for the Development of Long‐term Facilitation in Adult Male Rats

Phrenic long-term facilitation (pLTF) and ventilatory long-term facilitation (vLTF) are well-defined models of respiratory neuroplasticity. In each model, acute intermittent hypoxia (AIH) stimulates persistent increases in respiratory neural drive or minute ventilation respectively. Recent findings from our laboratory indicate that sex hormones are necessary for the induction of respiratory neuroplasticity in both male and female rats. For example, AIH induced pLTF and vLTF only develop during the proestrus phase of the estrous cycle in female rats, notable for high levels of circulating 17β-estradiol, the most neuroactive form of estrogen. Removal of the ovaries (ovariectomy, OVX), the primary source of circulating sex hormones in females, also eliminates the development of pLTF and vLTF. Plasticity can be restored in OVX females with estradiol supplementation supporting a key role for estradiol signaling in development of respiratory plasticity. Testosterone, the principle sex hormone in males, is also important for development of respiratory plasticity. Male rats demonstrate reduced pLTF magnitude with aging, in correlation with reduced circulating testosterone levels, and removal of the gonads (gonadectomy, GDX) eliminates AIH-induced pLTF. Testosterone replacement is sufficient to restore pLTF in GDX male rats, but requires the activity of aromatase, the enzyme responsible for converting testosterone to estradiol, suggesting that estradiol may also play a key role in development of respiratory neuroplasticity in male rats. It is unknown whether conversion of testosterone to estradiol is necessary for development of AIH-induced plasticity in gonadally-intact male rats. To address this question, we used systemic injections of Letrozole, an aromatase inhibitor, as a first step towards determining the necessity of testosterone conversion to estradiol for development of AIH-induced respiratory neuroplasticity in gonadally-intact adult male rats. We hypothesized that aromatization of testosterone to estradiol would be necessary for induction of both AIH-induced vLTF and pLTF. Letrozole (1mg/kg), or a vehicle control, was administered subcutaneously over four days. On day three, vLTF was measured via whole-body plethysmography. On the fourth day, pLTF was quantified via phrenic nerve recordings. Researchers were blinded to treatment for all experiments and data analysis. Preliminary findings suggest that daily Letrozole injections impaired development of AIH-induced vLTF and pLTF, supporting our hypothesis. These data provide further evidence that estradiol plays a critical role in the development of respiratory neuroplasticity in both males and females.

CO 2 Effects on Intermittent Hypoxia‐Induced Respiratory Motor Plasticity in Humans: In Progress Study

Intermittent hypoxia (IH) elicits respiratory motor plasticity (known as ventilatory long-term facilitation [LTF]) in humans and other mammals. However, awake humans exhibit ventilatory LTF only when CO2 levels are raised slightly above eupneic levels in a sustained or intermittent fashion during the IH protocol. In this ongoing study, we seek to determine the effect of CO2 on IH-induced respiratory motor plasticity by examining multiple, distinct mechanisms contributing to the ventilatory response during and following acute intermittent hypercapnic hypoxia (IHH). We hypothesized that IHH elicits a prolonged increase in cortico-diaphragmatic conduction, resting neural drive to breathe, and respiratory motor output versus poikilocapnic IH and a normoxic, normocapnic control (CTRL). Five healthy young adults (age = 32 ± 5 years; 3 female) completed four study visits. Day 1 consisted of general subject characterization. On Days 2, 3 and 4 (separated by ≥72 h), subjects were randomly assigned to receive either: IHH (PETO2 = 55 ± 4 Torr, PETCO2 = 44 ± 3 Torr), IH (PETO2 = 55 ± 3 Torr, PETCO2 = 37 ± 2 Torr), or CTRL (PETO2 = 107 ± 3 Torr, PETCO2 = 36 ± 2 Torr). Protocols consisted of 15 episodes of 60 s exposure with 90 s breathing ambient room air. Cardiovascular responses (heart rate, blood pressure and oxygen saturation) were monitored throughout. Cortico-diaphragmatic conduction was quantified by recording motor evoked potentials (MEPs) using transcranial magnetic stimulation and surface EMG electrodes. Resting neural drive to breathe was assessed using the mouth occlusion pressure technique (i.e. P0.1). Respiratory motor output was measured as minute ventilation () during resting isocapnic conditions (PETCO2 maintained within 1 mmHg of baseline) and adjusted based on the rate of metabolic CO2 production (/). Dependent variables were assessed at baseline, and 30-60 min post. Following IHH, IH and CTRL, the mean change in MEP amplitude was +34 ± 48%, +9 ± 21%, and +2 ± 22%; the mean change in P0.1 was +26 ± 17%, +15 ± 16%, and +7 ± 16%; and the mean change in was +0.3 ± 1.0 L·min-1, +0.3 ± 1.1 L·min-1, and +0.3 ± 0.8 L·min-1. When adjusted for , the mean change in / was -0.5 ± 1.0, +0.4 ± 1.8, and -0.2 ± 1.1. Based on these preliminary data in 5 subjects, we suggest that IHH elicits greater increases in cortico-diaphragmatic conduction and resting neural drive to breathe vs. poikilocapnic IH and CTRL. Conversely, our IHH protocol does not elicit ventilatory LTF. We reason that measures such as MEP amplitude and P0.1 are less sensitive to behavioral influences vs. , and may provide unique insights into mechanisms of IH-induced respiratory motor plasticity in awake healthy humans.