Ventilatory Acclimatization To Hypoxia Research Articles

Consumption of probiotic SLAB51 enhances ventilatory acclimatization to high altitude

Ventilatory acclimatization to hypoxia (VAH) is crucial for adjusting to physiologically stressful high-altitude environments. Increased ventilatory and cardiac responses to low-oxygen (O2) may partially mitigate deleterious effects at high altitude. Previous studies show that administration of the probiotic SLAB51 (O2booster; Hecto, Seoul, South Korea) improves O2 saturation in patients suffering from COVID-19 and O2-treated prematurely born babies. Since changes in ventilation may contribute to increase O2 saturation, we hypothesize that acute administration of SLAB51 to sojourners traveling to high altitude will increase respiratory responses to hypoxia. We measured minute ventilation (VI) and heart rate (HR) during normoxia and acute isocapnic hypoxia in 17 healthy participants (9 males, 8 females) of Latino ancestry with residence at sea level in San Diego and surrounding areas. We performed control experiments at sea level and then the participants were taken to White Mountain Barcroft Station (12,470ft / 3,800m) to repeat the measurements. At high altitude, the participants underwent a double-blind, placebo-controlled study with administration of placebo (n=8) or SLAB51 probiotic (n=9) up to three times per day for up to four days. Recordings of physiological parameters were obtained during steady-state at different gas mixtures with continuous monitoring of O2 saturation to target 10% desaturation during the acute hypoxia stimulus. We measured hypoxic ventilatory and heart rate responses (HVR and HHRR, respectively) and compared results between placebo and probiotic groups (mean ± SEM). As expected, high altitude increased VI and HR values compared to sea-level measurements in all participants. However, at high altitude, we found a significant increase of VI (p<0.02) in participants consuming SLAB51 (0.275+0.015 L/min per Kg) when compared to those administered with placebo (0.207±0.025 L/min per Kg) during exposure to acute hypoxia. Administration of SLAB51 did not produce differences in HR responses at high altitude, and HVR and HHRR were not significantly different with SLAB51 treatment. Our results show that consumption of the probiotic SLAB51 increases ventilatory responses to hypoxia at high altitude without affecting hypoxia-induced changes in HR, suggesting that SLAB51 probiotic intervention may contribute to ventilatory acclimatization to hypoxia, although future studies are required to determine the mechanisms underlying this potential response. Supported by LEA Altitude Performance Fund. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Influence of chronic hypoxia on the hypoxic ventilatory response of juvenile and adult rats

Chronic hypoxia (CH) from birth attenuates the acute hypoxic ventilatory response (HVR) in rats and other mammals, but CH is often reported to augment the HVR in adult mammals. To test the hypothesis that this transition – from blunting to augmenting the HVR – occurs in the third or fourth postnatal week in rats, juvenile and adult rats were exposed to normobaric CH (12% O2) for 7 days and the HVR was assessed by whole-body plethysmography. No transition was observed, however, and the acute HVR was reduced by 61 – 85% across all ages studied. The failure to observe an augmented HVR in adult rats could not be explained by the substrain of Sprague Dawley rats used, the duration of the CH exposure, the order in which test gases were presented, the level of hypoxia used for CH and to assess the HVR, or the effects of CH on the metabolic response to hypoxia and the hypercapnic ventilatory response. A literature survey revealed several distinct patterns of ventilatory acclimatization to hypoxia (VAH) in adult rats, with most studies (77%) revealing a decrease or no change in the acute HVR after CH. In conclusion, the effects of CH on respiratory control are qualitatively similar across age groups, at least within the populations of Sprague Dawley rats used in the present study, and there does not appear to be one “typical” pattern for VAH in adult rats.

Absence of Erythropoietin and Cardio‐respiratory Responses to Hypobaric Hypoxia in Olfactory Receptor 78 Null mice

Hypobaric hypoxia (HH) evokes a series of physiological adaptations, including carotid body (CB)–dependent ventilatory acclimatization to hypoxia (VAH), elevated blood pressure (BP), and stimulation of erythropoietin (Epo) gene in the kidney. Olfactory receptor 78 (Olfr78) is a G‐protein coupled receptor that has been implicated in CB response to acute hypoxia. Given that both CB and kidney express Olfr78, we tested the hypothesis that Olfr78 plays a role in the cardio‐respiratory and renal Epo gene responses to HH. Studies were performed on wild type (WT) and Olfr78 null mice reared in room air (control) or exposed to 18h of HH (0.4 atmospheres). HH treated WT exhibited: 1) VAH manifested as increased baseline breathing, and enhanced hypoxic ventilatory response (HVR; 12% O2); 2) elevated BP and plasma norepinephrine levels; and 3) increased baseline CB sensory nerve activity and augmented CB sensory nerve response to subsequent acute hypoxia‐ all these responses to HH are either markedly attenuated or absent in Olfr78 null mice. WT mice responded to HH with activation of the renal Epo gene expression and elevated plasma Epo levels, and these effects were attenuated or absent in Olfr78 null mice. The attenuated Epo activation by HH was accompanied with markedly reduced hypoxia‐inducible factor (HIF)‐2α protein and reduced activation of HIF‐2 target gene Sod‐1 in Olfr78 null mice, suggesting impaired transcriptional activation of HIF‐2 contributes to attenuated Epo responses to HH. These results suggest a role for Olfr78 in physiological adaptations to HH experienced at high altitude

Investigation of vascular endothelial growth factor receptor-dependent neuroplasticity on rat nucleus tractus solitarius and phrenic nerve after chronic sustained hypoxia.

The neuronal system that controls respiration creates plasticity in response to physiological changes. Chronic sustained hypoxia causes neuroplasticity that contributes to ventilatory acclimatization to hypoxia (VAH). The purpose of this study is to explain the potential roles of the VAH mechanism developing because of chronic sustained hypoxia on respiratory neuroplasticity of vascular endothelial growth factor (VEGF) receptor activationon the nucleus tractus solitarius (NTS) and phrenic nerve. In this study 24 adult male Sprague-Dawley rats were used. Subjects were separated into four groups, amoderate-sham (mSHAM), severed-sham (sSHAM), moderate chronic sustained hypoxia (mCSH), and severed chronic sustained hypoxia (sCSH). Normoxic group (mSHAM and sSHAM) rats were exposed to 21% O₂ level (7 days) in the normobaric room while hypoxia group (mCSH and sCSH) rats were exposed to 13% and 10% O₂ level (7 days). Different protocols were applied for normoxic and hypoxia groups and ventilation, respiratory frequency, and tidal volume measurements were made with whole-body plethysmography. After the test HIF-1α, erythropoietin (EPO), and VEGFR-2 expressions on the NTS region in the medulla oblongata and phrenic nerve motor neurons in spinal cord tissue were analyzed using the immunohistochemical stain method. Examinations on the medulla oblongata and spinal cord tissues revealed that HIF-1α, EPO, and VEGFR-2 expressions increased inhypoxia groups compared to normoxic groups while a similar increase was also seen when respiratory parameters were assessed. Consequently, learning about VAH-related neuroplasticity mechanisms developed as a result of chronic continuous hypoxia will contribute to developing new therapeutical approaches to various diseases causing respiratory failure using brain plasticity without recourse to medicines.

Neuronal HIF-1α in the nucleus tractus solitarius contributes to ventilatory acclimatization to hypoxia.

We hypothesized that hypoxia inducible factor 1α (HIF-1α) in CNS respiratory centres is necessary for ventilatory acclimatization to hypoxia (VAH); VAH is a time-dependent increase in baseline ventilation and the hypoxic ventilatory response (HVR) occurring over days to weeks of chronic sustained hypoxia (CH). Constitutive deletion of HIF-1α in CNS neurons in transgenic mice tended to blunt the increase in HVR that occurs in wild-type mice with CH. Conditional deletion of HIF-1α in glutamatergic neurons of the nucleus tractus solitarius during CH significantly decreased ventilation in acute hypoxia but not normoxia in CH mice. These effects are not explained by changes in metabolic rate, nor CO2 , and there were no changes in the HVR in normoxic mice. HIF-1α mediated changes in gene expression in CNS respiratory centres are necessary in addition to plasticity of arterial chemoreceptors for normal VAH. Chronic hypoxia (CH) produces a time-dependent increase of resting ventilation and the hypoxic ventilatory response (HVR) that is called ventilatory acclimatization to hypoxia (VAH). VAH involves plasticity in arterial chemoreceptors and the CNS [e.g. nucleus tractus solitarius (NTS)], although the signals for this plasticity are not known. We hypothesized that hypoxia inducible factor 1α (HIF-1α), an O2 -sensitive transcription factor, is necessary in the NTS for normal VAH. We tested this in two mouse models using loxP-Cre gene deletion. First, HIF-1α was constitutively deleted in CNS neurons (CNS-HIF-1α-/- ) by breeding HIF-1α floxed mice with mice expressing Cre-recombinase driven by the calcium/calmodulin-dependent protein kinase IIα promoter. Second, HIF-1α was deleted in NTS neurons in adult mice (NTS-HIF-1α-/- ) by microinjecting adeno-associated virus that expressed Cre-recombinase in HIF-1α floxed mice. In normoxic control mice, HIF-1α deletion in the CNS or NTS did not affect ventilation, nor the acute HVR (10-15min hypoxic exposure). In mice acclimatized to CH for 1week, ventilation in hypoxia was blunted in CNS-HIF-1α-/- and significantly decreased in NTS-HIF-1α-/- compared to control mice (P<0.0001). These changes were not explained by differences in metabolic rate or CO2 . Immunofluorescence showed that HIF-1α deletion in NTS-HIF-1α-/- was restricted to glutamatergic neurons. The results indicate that HIF-1α is a necessary signal for VAH and the previously described plasticity in glutamatergic neurotransmission in the NTS with CH. HIF-1α deletion had no effect on the increase in normoxic ventilation with acclimatization to CH, indicating this is a distinct mechanism from the increased HVR with VAH.

Time Course of Astrocyte Activation in Mouse Nucleus Tractus Solitarius During Exposure to Hypoxia

Chronic sustained hypoxia (CH) occurs in populations living at high altitude and in patients with chronic pulmonary disease. Exposure to CH produces ventilatory acclimatization to hypoxia (VAH) and increases the hypoxic ventilatory response (HVR) by mechanisms that involve areas of the brainstem that control breathing. The nucleus tractus solitarius (NTS) is a sensory integrative center in the medulla receiving carotid body afferents and known to be crucial for VAH. Our previous results demonstrated that glia cells in the rat NTS contribute to VAH but these mechanisms have not been studied in mice exposed to CH beyond 24 hours. We hypothesized that CH produces an early transient activation of astrocytes and microglia in the mouse brainstem as observed in rats. To study the activation of glial cells with CH, we exposed mice to normobaric hypoxia (10% FiO2) for 0.5, 1 and 4 hours and 1 and 7 days. Mice were perfused with 4% paraformaldehyde and brainstem sections were obtained. We quantified astrocyte activation by measuring glial fibrillary acidic protein (GFAP) intensity with immunofluorescence, and microglial activation by measuring branch morphology using Iba‐1 marker. Exposure to CH significantly increased GFAP intensity after 30 minutes and 1day while microglia morphology did not change significantly. The results differ from those in rats (J. Neurophysiol.117: 1625–1635, 2017) and suggest that time‐dependent astrocyte activation in the NTS may contribute to VAH.Support or Funding InformationSupported by NIH RO1 HL‐081823

Microglia Activation and IL‐1β Expression in Select Ventilatory Control Regions Following Exposure to Chronic Sustained Hypoxia

Ventilatory acclimatization to hypoxia (VAH) is defined as the time‐dependent increase in ventilation which occurs with chronic sustained hypoxia (CSH) of several hours to months. Previous research has shown that astrocytes and microglia undergo a morphology shift, indicating a possible change in activity, upon exposure to hypoxic conditions and may contribute to VAH. Understanding when and how the different cell types in respiratory control regions are activated is pertinent to understanding ventilatory control during hypoxic conditions. The first part of this study aimed to optimize the Sholl analysis method to detect morphological changes in microglia. Two different microglia antibodies, Iba‐1 and CD11b[Ox42], were compared using Sholl analysis to determine which antibody best represents the branching pattern of the individual microglia. Analysis indicated that neither antibody was statistically different from the other in terms of microglia branching (p&gt;0.05), so CD11b[Ox42] was chosen to simplify the immunohistochemistry protocol. The second part of this study assessed the morphology shift of microglia in the nucleus tractus solitarius (NTS) and Pre‐Bötzinger Complex (PBC) following CSH exposure. Based on previous research, we hypothesized that microglia in the NTS and PBC would be activated following CSH exposure, as assessed via a morphology shift to a more amoeboid state indicated by less microglia branch crossings of the Sholl brackets. To address this hypothesis, rats were exposed to either normoxic, 60‐minutes of CSH, or 12‐hours of CSH. Microglia morphology was assessed in perfused brainstem tissue via immunohistochemistry, confocal imaging, and image analysis. In the NTS, microglia branching analysis revealed a trend towards a more amoeboid morphology at the 60‐minute CSH time point, but only one of the Sholl analysis brackets, 21–30μM, was statistically significant from normoxic conditions (p&lt;0.05). In the PBC, microglia branching analysis also revealed a trend towards a more amoeboid morphology at the 60‐minute CSH time point with statistically significant (p&lt;0.05) branch patterns at the 11–20μM, 21–30μM, and 31–40 μM Sholl brackets. A morphology shift of microglia to a more amoeboid state could indicate their localized response to neurotransmitters or cytokines. In a follow‐up study we investigated the expression of IL‐1β in the NTS region using immunofluorescent labeling. Rat brainstem tissue from normoxic conditions, 15‐minute CSH, and 60‐minute CSH was labeled with antibodies against IL‐1β, GFAP, and Cd11b[Ox42]. IL‐1β positive cells were counted in the NTS region. This preliminary set of cell counts (n=2) suggests that there is an increase in IL‐1β positive cells with CHS exposure (normoxic 17 ± 2 IL‐1β positive cells, 15‐minute CSH 31.5 ± 0.5 IL‐1β positive cells, 60‐minute CSH 29.5 ± 9.5 IL‐1β positive cells). Taken together these data provide a great starting point in assessing the activation profiles of glial cells and cytokine activity in select respiratory control regions in the brainstem.Support or Funding InformationCentenary College Student‐Faculty Summer Research Award 2018 and 2019 (JRF and JAS); NIH R01HL081823 (FLP)

Ventilatory acclimatization to hypoxia: Time to express a critical central message.

Ventilatory acclimatization to hypoxia: Time to express a critical central message.

Evolved changes in breathing and CO2 sensitivity in deer mice native to high altitudes.

We examined the control of breathing by O2 and CO2 in deer mice native to high altitude to help uncover the physiological specializations used to cope with hypoxia in high-altitude environments. Highland deer mice ( Peromyscus maniculatus) and lowland white-footed mice ( P. leucopus) were bred in captivity at sea level. The first and second generation progeny of each population was raised to adulthood and then acclimated to normoxia or hypobaric hypoxia (12 kPa O2, simulating hypoxia at ~4,300 m) for 6-8 wk. Ventilatory responses to poikilocapnic hypoxia (stepwise reductions in inspired O2) and hypercapnia (stepwise increases in inspired CO2) were then compared between groups. Both generations of lowlanders appeared to exhibit ventilatory acclimatization to hypoxia (VAH), in which hypoxia acclimation enhanced the hypoxic ventilatory response and/or made the breathing pattern more effective (higher tidal volumes and lower breathing frequencies at a given total ventilation). In contrast, hypoxia acclimation had no effect on breathing in either generation of highlanders, and breathing was generally similar to hypoxia-acclimated lowlanders. Therefore, attenuation of VAH may be an evolved feature of highlanders that persists for multiple generations in captivity. Hypoxia acclimation increased CO2 sensitivity of breathing, but in this case, the effect of hypoxia acclimation was similar in highlanders and lowlanders. Our results suggest that highland deer mice have evolved high rates of alveolar ventilation that are unaltered by exposure to chronic hypoxia, but they have preserved ventilatory sensitivity to CO2.

Serotonin and Adenosine G-protein Coupled Receptor Signaling for Ventilatory Acclimatization to Sustained Hypoxia.

Different patterns of hypoxia evoke different forms of plasticity in the neural control of ventilation. For example, acute intermittent hypoxia produces long term facilitation (LTF) of ventilation, while chronic sustained hypoxia (CH) causes ventilatory acclimatization to hypoxia (VAH). In both LTF and VAH, ventilation in normoxia is greater than normal after the hypoxic stimulus is removed and the acute hypoxic ventilatory response can increase. However, the mechanisms of LTF and VAH are thought to be different based on previous results showing serotonin 5HT2 receptors, which are G protein coupled receptors (GPCR) that activate GQ signaling, contribute to LTF but not VAH. Newer results show that a different GPCR, namely adenosine A2A receptors and the GS signaling pathway, cause LTF with more severe intermittent hypoxia, i.e., PaO2 = 25–30 Torr for GS versus 35–45 Torr for LTF with the GQ signaling pathway. We hypothesized adenosine A2A receptors and GS signaling are involved in establishing VAH with longer term moderate CH and tested this in adult male rats by measuring ventilatory responses to O2 and CO2 with barometric pressure plethysmography after administering MSX-3 or ketanserin (A2A and 5HT2 antagonists, respectively, both 1 mg/Kg i.p.) during CH for 7 days. Blocking GS or GQ signals throughout CH exposure, significantly decreased VAH. After VAH was established, GQ blockade did not affect ventilation while GS blockade increased VAH. Similar to LTF, data support roles for both GQ and GS pathways in the development of VAH but after VAH has been established, the GS pathway inhibits VAH.

Astrocyte‐specific deletion of Kir4.1 increases normoxic ventilation after acclimatization to chronic sustained hypoxia.

Chronic sustained hypoxia (CH) produces ventilatory acclimatization to hypoxia (VAH), which involves time‐dependent increases of ventilation in normoxia and the hypoxic ventilatory response (HVR). Previous results show that glia are activated by CH and contribute to VAH but the contribution of astrocytes involved in central CO2‐sensitivity to VAH is not known. We tested the hypothesis that inwardly rectifying K+ 4.1 channels in astrocytes contribute to VAH using conditional gene deletion in transgenic mice. Conditional knockout (cKO) Kir4.1 mice had loxP sites at both ends of the entire Kir4.1 open reading frame and WT mice did not. WT and cKO Kir4.1 mice expressed tdTomato reporter and Cre‐recombinase/ERT2 receptors in astrocytes under the control of hGFAP promoter. Immunofluorescence against markers of astrocytes and Kir4.1 confirmed that tamoxifen treatment decreased Kir4.1 expression in astrocytes in CO2‐senstivie areas of the medulla of cKO Kir4.1 but not WT mice. After CH (PiO2 = 70 mmHg for 1 week), ventilation measured with barometric pressure plethysmography increased in normoxia, acute hypoxia and acute hypercapnia relative to chronically normoxic control mice. Ventilation was greater in normoxia after CH in Kir4.1cKO than in WT because of greater respiratory frequency and tidal volume. Ventilation in acute hypoxia or hypercapnia after CH was not significantly different between genotypes and metabolic rate was similar between genotypes at all O2 levels. The results support a role in astrocytes in the persistent hyperventilation in normoxia with VAH.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Short-Term Sustained Hypoxia Elevates Basal and Hypoxia-Induced Ventilation but Not the Carotid Body Chemoreceptor Activity in Rats.

Exposure to chronic sustained hypoxia (SH), as experienced in high altitudes, elicits an increase in ventilation, named ventilatory acclimatization to hypoxia (VAH). We previously showed that rats exposed to short-term (24 h) SH exhibit enhanced abdominal expiratory motor activity at rest, accompanied by augmented baseline sympathetic vasoconstrictor activity. In the present study, we investigated whether the respiratory and sympathetic changes elicited by short-term SH are accompanied by carotid body chemoreceptor sensitization. Juvenile male Holtzman rats (60–80 g) were exposed to SH (10% O2 for 24 h) or normoxia (control) to examine basal and hypoxic-induced ventilatory parameters in unanesthetized conditions, as well as the sensory response of carotid body chemoreceptors in artificially perfused in situ preparations. Under resting conditions (normoxia/normocapnia), SH rats (n = 12) exhibited higher baseline respiratory frequency, tidal volume, and minute ventilation compared to controls (n = 11, P < 0.05). SH group also showed greater hypoxia ventilatory response than control group (P < 0.05). The in situ preparations of SH rats (n = 8) exhibited augmented baseline expiratory and sympathetic activities under normocapnia, with additional bursts in abdominal and thoracic sympathetic nerves during late expiratory phase that were not seen in controls (n = 8, P < 0.05). Interestingly, basal and potassium cyanide-induced afferent activity of carotid sinus nerve (CSN) was similar between SH and control rats. Our findings indicate that the maintenance of elevated resting ventilation, baseline sympathetic overactivity, and enhanced ventilatory responses to hypoxia in rats exposed to 24 h of SH are not dependent on increased basal and sensorial activity of carotid body chemoreceptors.

Effects of 5‐HT2 and A2A Antagonists on pERK and pAKT Levels in the NTS and Spinal Cord of Rats Exposed to Chronic Sustained Hypoxia

Different patterns of hypoxic exposure can result in long‐term changes of ventilation and the hypoxic ventilatory response. For example, acute intermittent hypoxia (AIH) causes long term facilitation (LTF) of ventilation and phrenic nerve activity, with an increased acute HVR and persistent increase in ventilation in normoxia after AIH. Others have shown that LTF involves plasticity in serotonin 5HT2 neurotransmission (Gq pathway) and A2A adenosine neurotransmission (Gs pathway) in the spinal cord. Chronic sustained hypoxia (CSH) also increases the acute HVR and ventilation remains greater than control when normoxia is restored, which is called ventilatory acclimatization to hypoxia (VAH). We previously showed VAH involves plasticity in the nucleus tractus solitarius (NTS). Here we tested the hypothesis that Gq and Gs signaling play similar roles in LTF and VAH. Systemic inhibition of the Gq pathway (5‐HT2 antagonist Ketanserin, 1 mg/Kg per day) and the Gs pathway (A2A receptor antagonist MSX‐3, 0.5 mg/Kg per day) administered with subcutaneous osmotic pumps during 7 days of CSH (inspired PO2 = 70 mm Hg) decreased but did not eliminate VAH in male adult rats. This is different than previous reports from other laboratories showing LTF is abolished by blocking the Gq pathway with ketanserin but increased by blocking the Gs pathway with MSX‐3 administered before exposure to moderate AIH. To understand the site of action of Gq and Gs effects on VAH, we collected tissue from (1) the NTS, and (2) the spinal cord (between C3 and C5). We used Western blots of pERK and pAKT to measure Gq and Gs activation, respectively, in 4 groups of rats (n≥6 each) administered drugs or vehicle (DMSO) for 7 days as previously described above for ventilation studies: CON = DMSO in normoxia, CSH = DMSO in hypoxia, KET = ketanserin in CSH, MSX = MSX‐3 in CSH. CSH decreased pERK and pAKT in the spinal cord but had no effect on them in the NTS, indicating Gq and Gs pathways respond differently to CSH in the NTS versus spinal cord. In the NTS, pERK decreased with KET but not MSX during CSH. In the spinal cord, MSX during CSH restored pERK and pAKT to normoxic CON levels. Effects of KET on pERK and pAKT in the spinal cord during CSH are not available yet. The data indicate the Gq and Gs signaling with CSH is different than previously reported for AIH in the spinal cord. Also, Gq signaling in the NTS may contribute to VAH. Further study is necessary to determine how such differences in Gq and Gs signaling in the NTS and spinal cord depend on specific neurons in these locations.Support or Funding InformationNIH RO1 HL‐081823

Ventilatory acclimatization to hypoxia in deer mice native to high altitudes

Hypoxia at high altitudes constrains O2 supply to support metabolism, thermoregulation in the cold, and the aerobic scope needed for exercise. Many highland taxa appear to have physiological specializations for overcoming these challenges, but relatively little is known about the importance of hypoxia acclimation responses in high‐altitude natives, such as ventilatory acclimatization to hypoxia (VAH) and erythropoiesis. Highland taxa often possess a high haemoglobin‐O2 binding affinity, which safeguards arterial O2 saturation (SaO2) and tissue O2 delivery in hypoxia, but could foreseeably affect hypoxia acclimation responses. We examined this issue by comparing captive populations of deer mice (Peromyscus maniculatus) from high and low altitudes. Mice from each population were acclimated to normoxia and moderate hypoxia (12 kPa O2, simulating the PO2 at the native altitude of highlanders at 4300m) for 6–8 weeks. Highland mice were also acclimated to more severe hypoxia (9 kPa, the PO2 at ~7000m), which induces the same SaO2 in highlanders that lowlanders experience at 12 kPa. In lowlanders, hypoxia acclimation augmented the hypoxic ventilatory response (HVR), increased SaO2 in hypoxia, blunted the hypoxic depression of body temperature, increased blood [haemoglobin], and induced right ventricle hypertrophy (a sign of maladaptive pulmonary hypertension). Hypoxia acclimation had little effect on the HVR in highlanders, such that the hypoxic control of breathing appeared to be fixed like that in hypoxia‐acclimated lowlanders. Right ventricle hypertrophy did not occur in either moderate or severe hypoxia in highlanders, suggesting that they have blunted hypoxic pulmonary vasoconstriction and hypertension. The increase in blood [haemoglobin] was lower in highlanders than in lowlanders at 12 kPa, but highlanders mounted a robust increase in severe hypoxia (9 kPa), suggesting that differences in SaO2 and not the erythropoietic process contribute to differences in blood [haemoglobin] between populations. Therefore, high‐altitude adaptation appears to have blunted several hypoxia acclimation responses in deer mice.Support or Funding InformationSupported by NSERC of Canada.

Minocycline blocks glial cell activation and ventilatory acclimatization to hypoxia.

Ventilatory acclimatization to hypoxia (VAH) is the time-dependent increase in ventilation, which persists upon return to normoxia and involves plasticity in both central nervous system respiratory centers and peripheral chemoreceptors. We investigated the role of glial cells in VAH in male Sprague-Dawley rats using minocycline, an antibiotic that inhibits microglia activation and has anti-inflammatory properties, and barometric pressure plethysmography to measure ventilation. Rats received either minocycline (45mg/kg ip daily) or saline beginning 1 day before and during 7 days of chronic hypoxia (CH, PiO2 = 70 Torr). Minocycline had no effect on normoxic control rats or the hypercapnic ventilatory response in CH rats, but minocycline significantly (P < 0.001) decreased ventilation during acute hypoxia in CH rats. However, minocycline administration during only the last 3 days of CH did not reverse VAH. Microglia and astrocyte activation in the nucleus tractus solitarius was quantified from 30 min to 7 days of CH. Microglia showed an active morphology (shorter and fewer branches) after 1 h of hypoxia and returned to the control state (longer filaments and extensive branching) after 4 h of CH. Astrocytes increased glial fibrillary acidic protein antibody immunofluorescent intensity, indicating activation, at both 4 and 24 h of CH. Minocycline had no effect on glia in normoxia but significantly decreased microglia activation at 1 h of CH and astrocyte activation at 24 h of CH. These results support a role for glial cells, providing an early signal for the induction but not maintenance of neural plasticity underlying ventilatory acclimatization to hypoxia.NEW & NOTEWORTHY The signals for neural plasticity in medullary respiratory centers underlying ventilatory acclimatization to chronic hypoxia are unknown. We show that chronic hypoxia activates microglia and subsequently astrocytes. Minocycline, an antibiotic that blocks microglial activation and has anti-inflammatory properties, also blocks astrocyte activation in respiratory centers during chronic hypoxia and ventilatory acclimatization. However, minocycline cannot reverse ventilatory acclimatization after it is established. Hence, glial cells may provide signals that initiate but do not sustain ventilatory acclimatization.

Ventilatory acclimatization to hypoxia in mice: Methodological considerations

We examined ventilatory acclimatization to hypoxia (VAH) in CD1 mice, and contrasted results obtained using the barometric method on unrestrained mice with pneumotachography and pulse oximetry on restrained mice. Responses to progressive step reductions in O2 fraction (21%–8%) were assessed in mice acclimated to normoxia and hypobaric hypoxia (barometric pressure of 60kPa for 6–8 weeks). Hypoxia acclimation increased the hypoxic ventilatory response (primarily by increasing breathing frequency rather than tidal volume), arterial O2 saturation (SaO2) and heart rate in deep hypoxia, hypoxic chemosensitivity (ventilatory O2/CO2 equivalents versus SaO2), and respiratory water loss, and it blunted the hypoxic depression of metabolism and body temperature. Although some effects of hypoxia acclimation were qualitatively similar between methods, the effects were often greater in magnitude when assessed using pneumotachography. Furthermore, whereas hypoxia acclimation reduced ventilatory O2 equivalent and increased pulmonary O2 extraction in barometric experiments, it had the opposite effects in pneumotachography experiments. Our findings highlight the importance of considering the impact of how breathing is measured on the apparent responses to hypoxia.

Mouth Occlusion Pressure (P0.1) Provides Valuable Information in the Clinical Assessment and Follow‐up of Ventilatory Drive in Obstructive and Restrictive Lung Disease

Ventilatory acclimatization to hypoxia (VAH) includes a time‐dependent increase in minute ventilation with re‐setting of the arterial partial pressure of CO2 “set point” that results in persistent hyperventilation with concomitant changes in arterial oxygen levels. One of the methods used to evaluate the neuromuscular drive to breathe is the airway occlusion pressure (P0.1), which is the pressure generated at the airway opening 100 ms after the onset of occluded inspiratory effort. We hypothesized that P0.1 is a marker of disease that should be follow as a part of the treatment in patients with lung disease, we studied the correlation between P0.1 with FEV1, FVC, FEV1/FVC in patients with lung disease. We retrospectively studied 100 subjects that had PFTs and P0.1 done between September and October 2014. Mean age was 59.3 (range, 19‐89 years). In restrictive lung disease (39/100, 39%) P0.1 was correlated with the FVC and FEV1/FVC, in obstructive lung disease (61/100, 61%) P0.1 was correlated with FEV1 and FEV1/FVC. In subjects with diagnosis of obstructive lung disease P0.1 had a significant correlation with FEV1 and FEV1/FVC (p=0.001 and 0.003), in restrictive lung disease, P0.1 had a significant correlation with FVC (p=0.001) but no significant correlation with FEV1/FVC (p=0.2). Our results showed that P0.1 has a significant correlation with disease severity in lung disease. Previous studies showed that ventilatory drive decreases as a consequence of treatment, and hypoxia correction, is likely that P0.1 is a good marker of disease progression and can help diagnose and guide treatment in both obstructive and restrictive pulmonary disease, more studies are necessary to further evaluate the limitations of the P0.1 in different lung mechanics.

No evidence of a role for neuronal nitric oxide synthase in the nucleus tractus solitarius in ventilatory responses to acute or chronic hypoxia in awake rats.

When exposed to a hypoxic environment, the body's first response is a reflex increase in ventilation, termed the hypoxic ventilatory response (HVR). With chronic sustained hypoxia (CSH), such as during acclimatization to high altitude, an additional time-dependent increase in ventilation occurs, which increases the HVR and is termed ventilatory acclimatization to hypoxia (VAH). This secondary increase persists after exposure to CSH and involves plasticity within the circuits in the central nervous system that control breathing. The mechanisms of HVR plasticity are currently poorly understood. We hypothesized that changes in neuronal nitric oxide synthase (nNOS) activity or expression in the nucleus tractus solitarius contribute to this plasticity and underlie VAH in rats. To test this, we treated rats held in normoxia or 10% O2 (CSH, PIO2 = 70 Torr) for 7-9 days and measured ventilation in conscious, unrestrained animals before and after microinjecting the general NOS antagonist L-NG-Nitroarginine methyl ester into the nucleus tractus solitarius (NTS) or systemically injecting the nNOS-specific antagonist S-methyl-l-thiocitrulline. Localization of injection sites in the NTS was confirmed by histology following the experiment. We found that 1) neither NTS-specific nor systemic nNOS antagonism had any effect on hypoxia-mediated changes in breathing or metabolism (P > 0.05), but 2) nNOS protein expression was increased in the middle and caudal NTS by CSH. A persistent HVR after nNOS blockade in the NTS contrasts with results in awake mice, and our findings do not support the hypotheses that nNOS in the NTS contribute to the HVR or VAH in awake rats.

The effect of combined glutamate receptor blockade in the NTS on the hypoxic ventilatory response in awake rats differs from the effect of individual glutamate receptor blockade.

Ventilatory acclimatization to hypoxia (VAH) increases the hypoxic ventilatory response (HVR) and causes persistent hyperventilation when normoxia is restored, which is consistent with the occurrence of synaptic plasticity in acclimatized animals. Recently, we demonstrated that antagonism of individual glutamate receptor types (GluRs) within the nucleus tractus solitarii (NTS) modifies this plasticity and VAH (J. Physiol. 592(8):1839–1856); however, the effects of combined GluR antagonism remain unknown in awake rats. To evaluate this, we exposed rats to room air or chronic sustained hypobaric hypoxia (CSH, PiO2 = 70 Torr) for 7–9 days. On the experimental day, we microinjected artificial cerebrospinal fluid (ACSF: sham) and then a “cocktail” of the GluR antagonists MK‐801 and DNQX into the NTS. The location of injection sites in the NTS was confirmed by glutamate injections on a day before the experiment and with histology following the experiment. Ventilation was measured in awake, unrestrained rats breathing normoxia or acute hypoxia (10% O2) in 15‐min intervals using barometric pressure plethysmography. In control (CON) rats, acute hypoxia increased ventilation; NTS microinjections of GluR antagonists, but not ACSF, significantly decreased ventilation and breathing frequency in acute hypoxia but not normoxia (P <0.05). CSH increased ventilation in hypoxia and acute normoxia. In CSH‐conditioned rats, GluR antagonists in the NTS significantly decreased ventilation in normoxia and breathing frequency in hypoxia. A persistent HVR after combined GluR blockade in the NTS contrasts with the effect of individual GluR blockade and also with results in anesthetized rats. Our findings support the hypotheses that GluRs in the NTS contribute to, but cannot completely explain, VAH in awake rats.

Activation of microglia and astrocytes in the nucleus tractus solitarius during ventilatory acclimatization to 10% hypoxia in unanesthetized mice

Nucleus tractus solitarius (NTS) is the integrative sensory relay of autonomic functions in the brainstem. To explore the nonneuronal cellular basis of central chemosensitivity during the first 24 hr of ventilatory acclimatization to hypoxia (VHA), we have investigated glial activation markers in the NTS. Conscious mice (C57/BL6) were placed in a hermetic hypoxia chamber containing a plethysmograph to record ventilation. After 4 days of habituation to the normoxic environment, mice were subjected to physiological hypoxia (10% O2 ) for 1, 6, or 24 hr. To dissociate interactions between microglia and astrocytes, another group received daily minocycline, a microglia activation blocker. By immunochemical localization of astrocytes (GFAP), activated microglia (Cd11b), and total microglia (Iba-1), we identified an oxygen-sensing glial layer in the NTS, in which astrocytes are first activated after 1-6 hr of hypoxia, followed by microglia after 6-24 hr of hypoxia. Minocycline administration suppressed microglial activation and decreased astrocyte activation at 6 hr and VHA at 24 hr of hypoxia. These results suggest that astrocytes contribute to the neuronal response during the first hour of hypoxia, whereas microglial cells, via cross-talk with astrocytes, are involved in the VHA during the first 24 hr of acclimatization.