Impact of Serotonergic 5HT1A and 5HT2A Receptor Activation on the Respiratory Response to Hypercapnia in a Rat Model of Parkinson's Disease.

In Parkinson's disease (PD), along with typical motor dysfunction, abnormal breathing is present; the cause of which is not well understood. The study aimed to analyze the effects of stimulation of the serotonergic system with 5-HT1A and 5-HT2A agonists in a model of PD induced by injection of 6-hydroxydopamine (6-OHDA). To model PD, bilateral injection of 6-OHDA into both striata was performed in male Wistar rats. Respiratory disturbances in response to 7% hypercapnia (CO2 in O2) in the plethysmographic chamber before and after stimulation of the serotonergic system and the incidence of apnea were studied in awake rats 5 weeks after 6-OHDA or vehicle injection. Administration of 6-OHDA reduced the concentration of serotonin (5-HT), dopamine (DA) and norepinephrine (NA) in the striatum and the level of 5-HT in the brainstem of treated rats, which have been associated with decreased basal ventilation, impaired respiratory response to 7% CO2 and increased incidence of apnea compared to Sham-operated rats. Intraperitoneal (i.p.) injection of the 5-HT1AR agonist 8-OH-DPAT and 5-HT2AR agonist NBOH-2C-CN increased breathing during normocapnia and hypercapnia in both groups of rats. However, it restored reactivity to hypercapnia in 6-OHDA group to the level present in Sham rats. Another 5-HT2AR agonist TCB-2 was only effective in increasing normocapnic ventilation in 6-OHDA rats. Both the serotonergic agonists 8-OH-DPAT and NBOH-2C-CN had stronger stimulatory effects on respiration in PD rats, compensating for deficits in basal ventilation and hypercapnic respiration. We conclude that serotonergic stimulation may have a positive effect on respiratory impairments that occur in PD.

Evaluation of calibrated and uncalibrated optical imaging approaches for relative cerebral oxygen metabolism measurements in awake mice.

Objective. The continuous delivery of oxygen is critical to sustain brain function, and therefore, measuring brain oxygen consumption can provide vital physiological insight. In this work, we examine the impact of calibration and cerebral blood flow (CBF) measurements on the computation of the relative changes in the cerebral metabolic rate of oxygen consumption (rCMRO2) from hemoglobin-sensitive intrinsic optical imaging data. Using these data, we calculate rCMRO2, and calibrate the model using an isometabolic stimulus.Approach. We used awake head-fixed rodents to obtain hemoglobin-sensitive optical imaging data to test different calibrated and uncalibrated rCMRO2models. Hypercapnia was used for calibration and whisker stimulation was used to test the impact of calibration.Main results. We found that typical uncalibrated models can provide reasonable estimates of rCMRO2with differences as small as 7%-9% compared to their calibrated models. However, calibrated models showed lower variability and less dependence on baseline hemoglobin concentrations. Lastly, we found that supplying the model with measurements of CBF significantly reduced error and variability in rCMRO2change calculations.Significance. The effect of calibration on rCMRO2calculations remains understudied, and we systematically evaluated different rCMRO2calculation scenarios that consider including different measurement combinations. This study provides a quantitative comparison of these scenarios to evaluate trade-offs that can be vital to the design of blood oxygenation sensitive imaging experiments for rCMRO2calculation.

Prospective Randomized Pilot Trial on the Effects of Mild Hypercapnia on Cerebral Oxygen Saturation in Patients Undergoing Off-Pump Coronary Artery Bypass Grafting.

OBJECTIVE AND DESIGN: A single-center prospective randomized controlled study was conducted to assess the effect of targeted mild hypercapnia (TMH) on cerebral oxygen saturation (rSO2) in patients undergoing off-pump coronary artery bypass grafting (CABG). SETTING AND PARTICIPANTS: A prospective randomized controlled study involving 100 patients undergoing off-pump CABG at U. N. Mehta Hospital, Ahmedabad, Gujarat, India. INTERVENTION: Patients were randomized to either the TMH (PaCO2 45-55 mmHg) or the targeted normocapnia (TN; PaCO2 35-45 mmHg) group, containing 50 patients in each group. MEASUREMENTS: Monitoring of rSO2, heart rate, mean arterial pressure (MAP), PaCO2, and peripheral oxygen saturation was done at baseline, after induction, after left internal mammary artery harvesting, at each grafting (distal and proximal), after protamine, and after shifting to the intensive care unit. The standardized minimental-state examination (SMMSE) was performed preoperatively and at 8, 12, and 24 hours postextubation. Data were analyzed using an independent sample t test. RESULTS: The TMH group had higher MAP during grafting (p < 0.001) and higher rSO2 on both sides during distal and proximal grafting (p < 0.001) and after protamine (p < 0.05), as compared to the TN group. Compared to preoperative values, SMMSE scores in the TN group were significantly lower at 12 and 24 hours postextubation (p < 0.001). CONCLUSION: TMH during grafting increased the cerebral blood flow and rSO2 when hemodynamic instability was very common. It has a protective role on the brain and helps maintain cognition postoperatively.

Effects of therapeutic hypercapnia on the expression and function of γδT cells in transplanted lungs in rats.

OBJECTIVE: To investigate the effect of therapeutic hypercapnia on the expression and function of gamma delta T (γδ T) cells during ischemia-reperfusion injury (IRI) after lung transplantation. METHODS: We randomly divided male Wistar rats into three groups (n = 6 in each group), the control group (group N), the IRI group (group I), and the therapeutic hypercapnia group (group H). We then assessed pulmonary edema, neutrophil infiltration, wet-to-dry (W/D) weight ratio, and microscopic histopathology and separately measured the levels of γδT cell surface antigen (TCR) and Interleukin-17 (IL-17) using flow cytometry and enzyme-linked immunosorbent assays (ELISAs). RESULTS: The infiltration of neutrophils and the expression of TCR and IL-17 were significantly increased in the I group compared to the control, and the biopsy edema in group I was more severe. Arterial partial pressure of oxygen (PaO2) was decreased after reperfusion in group I compared with the control group. W/D weight ratio, neutrophil infiltration, and the expression of TCR and IL-17 decreased drastically in the H group compared to the I group. CONCLUSION: Our findings suggest that γδ T lymphocytes were directly involved in lung injury. In addition, therapeutic hypercapnia effectively reduced the expression of γδ T cells and IL-17, and this has the potential to become a treatment strategy for IRI and an intervention to improve lung function.

Oxygen imaging of hypoxic pockets in the mouse cerebral cortex.

Consciousness is lost within seconds upon cessation of cerebral blood flow. The brain cannot store oxygen, and interruption of oxidative phosphorylation is fatal within minutes. Yet only rudimentary knowledge exists regarding cortical partial oxygen tension (Po2) dynamics under physiological conditions. Here we introduce Green enhanced Nano-lantern (GeNL), a genetically encoded bioluminescent oxygen indicator for Po2 imaging. In awake behaving mice, we uncover the existence of spontaneous, spatially defined "hypoxic pockets" and demonstrate their linkage to the abrogation of local capillary flow. Exercise reduced the burden of hypoxic pockets by 52% compared with rest. The study provides insight into cortical oxygen dynamics in awake behaving animals and concurrently establishes a tool to delineate the importance of oxygen tension in physiological processes and neurological diseases.

Short-term exposure to high pCO2 leads to decreased branchial cytochrome C oxidase activity in the presence of octopamine in a decapod.

In a recent mechanistic study, octopamine was shown to promote proton transport over the branchial epithelium in green crabs, Carcinus maenas. Here, we follow up on this finding by investigating the involvement of octopamine in an environmental and physiological context that challenges acid-base homeostasis, the response to short-term high pCO2 exposure (400 Pa) in a brackish water environment. We show that hyperregulating green crabs experienced a respiratory acidosis as early as 6 h of exposure to hypercapnia, with a rise in hemolymph pCO2 accompanied by a simultaneous drop of hemolymph pH. The slightly delayed increase in hemolymph HCO3- observed after 24 h helped to restore hemolymph pH to initial values by 48 h. Circulating levels of the biogenic amine octopamine were significantly higher in short-term high pCO2 exposed crabs compared to control crabs after 48 h. Whole animal metabolic rates, intracellular levels of octopamine and cAMP, as well as branchial mitochondrial enzyme activities for complex I + III and citrate synthase were unchanged in posterior gill #7 after 48 h of hypercapnia. However, application of octopamine in gill respirometry experiments suppressed branchial metabolic rate in posterior gills of short-term high pCO2 exposed animals. Furthermore, branchial enzyme activity of cytochrome C oxidase decreased in high pCO2 exposed crabs after 48 h. Our results indicate that hyperregulating green crabs are capable of quickly counteracting a hypercapnia-induced respiratory acidosis. The role of octopamine in the acclimation of green crabs to short-term hypercapnia seems to entail the alteration of branchial metabolic pathways, possibly targeting mitochondrial cytochrome C in the gill. Our findings help advancing our current limited understanding of endocrine components in hypercapnia acclimation. SUMMARY STATEMENT: Acid-base compensation upon short-term high pCO2 exposure in hyperregulating green crabs started after 6 h and was accomplished by 48 h with the involvement of the biogenic amine octopamine, accumulation of hemolymph HCO3-, and regulation of mitochondrial complex IV (cytochrome C oxidase).

Superoxide dismutase 2 deficiency is associated with enhanced central chemoreception in mice: Implications for breathing regulation.

AIMS: In mammals, central chemoreception plays a crucial role in the regulation of breathing function in both health and disease conditions. Recently, a correlation between high levels of superoxide anion (O2.-) in the Retrotrapezoid nucleus (RTN), a main brain chemoreceptor area, and enhanced central chemoreception has been found in rodents. Interestingly, deficiency in superoxide dismutase 2 (SOD2) expression, a pivotal antioxidant enzyme, has been linked to the development/progression of several diseases. Despite, the contribution of SOD2 on O2.- regulation on central chemoreceptor function is unknown. Accordingly, we sought to determine the impact of partial deletion of SOD2 expression on i) O2.-accumulation in the RTN, ii) central ventilatory chemoreflex function, and iii) disordered-breathing. Finally, we study cellular localization of SOD2 in the RTN of healthy mice. METHODS: Central chemoreflex drive and breathing function were assessed in freely moving heterozygous SOD2 knockout mice (SOD2+/- mice) and age-matched control wild type (WT) mice by whole-body plethysmography. O2.- levels were determined in RTN brainstem sections and brain isolated mitochondria, while SOD2 protein expression and tissue localization were determined by immunoblot, RNAseq and immunofluorescent staining, respectively. RESULTS: Our results showed that SOD2+/- mice displayed reductions in SOD2 levels and high O2.- formation and mitochondrial dysfunction within the RTN compared to WT. Additionally, SOD2+/- mice displayed a heightened ventilatory response to hypercapnia and exhibited overt signs of altered breathing patterns. Both, RNAseq analysis and immunofluorescence co-localization studies showed that SOD2 expression was confined to RTN astrocytes but not to RTN chemoreceptor neurons. Finally, we found that SOD2+/- mice displayed alterations in RTN astrocyte morphology compared to RTN astrocytes from WT mice. INNOVATION & CONCLUSION: These findings provide first evidence of the role of SOD2 in the regulation of O2.- levels in the RTN and its potential contribution on the regulation of central chemoreflex function. Our results suggest that reductions in the expression of SOD2 in the brain may contribute to increase O2.- levels in the RTN being the outcome a chronic surge in central chemoreflex drive and the development/maintenance of altered breathing patterns. Overall, dysregulation of SOD2 and the resulting increase in O2.- levels in brainstem respiratory areas can disrupt normal respiratory control mechanisms and contribute to breathing dysfunction seen in certain disease conditions characterized by high oxidative stress.

Hypercapnia increases ACE2 expression and pseudo-SARS-CoV-2 entry in bronchial epithelial cells by augmenting cellular cholesterol.

Patients with chronic lung disease, obesity, and other co-morbid conditions are at increased risk of severe illness and death when infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Hypercapnia, the elevation of CO2 in blood and tissue, commonly occurs in patients with severe acute and chronic lung disease, including those with pulmonary infections, and is also associated with high mortality risk. We previously reported that hypercapnia increases viral replication and mortality of influenza A virus infection in mice. We have also shown that culture in elevated CO2 upregulates expression of cholesterol synthesis genes in primary human bronchial epithelial cells. Interestingly, factors that increase the cholesterol content of lipid rafts and lipid droplets, platforms for viral entry and assembly, enhance SARS-CoV-2 infection. In the current study, we investigated the effects of hypercapnia on ACE2 expression and entry of SARS-CoV-2 pseudovirus (p-SARS-CoV-2) into airway epithelial cells. We found that hypercapnia increased ACE2 expression and p-SARS-CoV-2 uptake by airway epithelium in mice, and in cultured VERO and human bronchial epithelial cells. Hypercapnia also increased total cellular and lipid raft-associated cholesterol in epithelial cells. Moreover, reducing cholesterol synthesis with inhibitors of sterol regulatory element binding protein 2 (SREBP2) or statins, and depletion of cellular cholesterol, each blocked the hypercapnia-induced increases in ACE2 expression and p-SARS-CoV-2 entry into epithelial cells. Cigarette smoke extract (CSE) also increased ACE2 expression, p-SARS-CoV-2 entry and cholesterol accumulation in epithelial cells, an effect not additive to that of hypercapnia, but also inhibited by statins. These findings reveal a mechanism that may account, in part, for poor clinical outcomes of SARS-CoV-2 infection in patients with advanced lung disease and hypercapnia, and in those who smoke cigarettes. Further, our results suggest the possibility that cholesterol-lowering therapies may be of particular benefit in patients with hypercapnia when exposed to or infected with SARS-CoV-2.

Ultrastructural Changes in Hippocampal Region CA1 Neurons After Exposure to Permissive Hypercapnia and/or Normobaric Hypoxia.

Isolated exposure to intermittent hypoxia and permissive hypercapnia activates signaling mechanisms that induce ultrastructural changes in mitochondria and endoplasmic reticulum, accompanied by the development of maximal ischemic tolerance in neurons under the combined influence of these factors. However, there are a lack of data on the combined impact of these factors on the ultrastructure of neuronal organelles. The present study aims to comparatively assess the ultrastructural changes in neurons following isolated and combined exposure to hypoxia and hypercapnia, as well as to correlate these changes with the neuroprotective potential previously observed for these factors. Following a 15-session course of 30-min exposures to permissive hypercapnia (PCO2 ≈ 50 mmHg) and/or normobaric hypoxia (PO2 ≈ 150 mmHg), morphometric assessment was conducted to evaluate the extent of ultrastructural changes in hippocampal neurons (mitochondria, perinuclear space, and granular endoplasmic reticulum). It was found that in hippocampal neurons from the CA1 region, permissive hypercapnia resulted in increased mitochondrial size, expansion of membranous compartments of the granular endoplasmic reticulum, and perinuclear space. Normobaric hypoxia affected only mitochondrial size, while hypercapnic hypoxia specifically widened the perinuclear space. These ultrastructural changes objectively reflect varying degrees of the influence of hypoxia and hypercapnia on organelles responsible for energy metabolism, anti-apoptotic, and synthetic functions of neurons. This confirms the effect of potentiation of their neuroprotective effects under combined exposure and highlights the dominant role of the hypercapnic component in this mechanism.

Mild and moderate chronic hypercapnia elicit distinct transcriptomic responses of immune function in cardiorespiratory nuclei.

Chronic hypercapnia (CH) is a hallmark of respiratory-related diseases, and the level of hypercapnia can acutely or progressively become more severe. Previously, we have shown time-dependent adaptations in steady-state physiology during mild (arterial Pco2 ∼55 mmHg) and moderate (∼60 mmHg) CH in adult goats, including transient (mild CH) or sustained (moderate CH) suppression of acute chemosensitivity suggesting limitations in adaptive respiratory control mechanisms as the level of CH increases. Changes in specific markers of glutamate receptor plasticity, interleukin-1ß, and serotonergic modulation within key nodes of cardiorespiratory control do not fully account for the physiological adaptations to CH. Here, we used an unbiased approach (bulk tissue RNA sequencing) to test the hypothesis that mild or moderate CH elicits distinct gene expression profiles in important brain stem regions of cardiorespiratory control, which may explain the contrasting responses to CH. Gene expression profiles from the brain regions validated the accuracy of tissue biopsy methodology. Differential gene expression analyses revealed greater effects of CH on brain stem sites compared with the medial prefrontal cortex. Mild CH elicited an upregulation of predominantly immune-related genes and predicted activation of immune-related pathways and functions. In contrast, moderate CH broadly led to downregulation of genes and predicted inactivation of cellular pathways related to the immune response and vascular function. These data suggest that mild CH leads to a steady-state activation of neuroinflammatory pathways within the brain stem, whereas moderate CH drives the opposite response. Transcriptional shifts in immune-related functions may underlie the cardiorespiratory network's capability to respond to acute, more severe hypercapnia when in a state of progressively increased CH.NEW & NOTEWORTHY Mild chronic hypercapnia (CH) broadly upregulated immune-related genes and a predicted activation of biological pathways related to immune cell activity and the overall immune response. In contrast, moderate CH primarily downregulated genes related to major histocompatibility complex signaling and vasculature function that led to a predicted inactivation of pathways involving the immune response and vascular endothelial function. The severity-dependent effect on immune responses suggests that neuroinflammation has an important role in CH and may be important in the maintenance of proper ventilatory responses to acute and chronic hypercapnia.

Umbilical cord pH, blood gases, and lactate at birth: normal values, interpretation, and clinical utility.

Normal birth is a eustress reaction, a beneficial hedonic stress with extremely high catecholamines that protects us from intrauterine hypoxia and assists in the rapid shift to extrauterine life. Occasionally the cellular O2 requirement becomes critical and an O2 deficit in blood (hypoxemia) may evolve to a tissue deficit (hypoxia) and finally a risk of organ damage (asphyxia). An increase in H+ concentration is reflected in a decrease in pH, which together with increased base deficit is a proxy for the level of fetal O2 deficit. Base deficit (or its negative value, base excess) was introduced to reflect the metabolic component of a low pH and to distinguish from the respiratory cause of a low pH, which is a high CO2 concentration. Base deficit is a theoretical estimate and not a measured parameter, calculated by the blood gas analyzer from values of pH, the partial pressure of CO2, and hemoglobin. Different brands of analyzers use different calculation equations, and base deficit values can thus differ by multiples. This could influence the diagnosis of metabolic acidosis, which is commonly defined as a pH <7.00 combined with a base deficit ≥12.0 mmol/L in umbilical cord arterial blood. Base deficit can be calculated as base deficit in blood (or actual base deficit) or base deficit in extracellular fluid (or standard base deficit). The extracellular fluid compartment represents the blood volume diluted with the interstitial fluid. Base deficit in extracellular fluid is advocated for fetal blood because a high partial pressure of CO2 (hypercapnia) is common in newborns without concomitant hypoxia, and hypercapnia has a strong influence on the pH value, then termed respiratory acidosis. An increase in partial pressure of CO2 causes less increase in base deficit in extracellular fluid than in base deficit in blood, thus base deficit in extracellular fluid better represents the metabolic component of acidosis. The different types of base deficit for defining metabolic acidosis in cord blood have unfortunately not been noticed by many obstetrical experts and organizations. In addition to an increase in H+ concentration, the lactate production is accelerated during hypoxia and anaerobic metabolism. There is no global consensus on definitions of normal cord blood gases and lactate, and different cutoff values for abnormality are used. At a pH <7.20, 7% to 9% of newborns are deemed academic; at <7.10, 1% to 3%; and at <7.00, 0.26% to 1.3%. From numerous studies of different eras and sizes, it can firmly be concluded that in the cord artery, the statistically defined lower pH limit (mean -2 standard deviations) is 7.10. Given that the pH for optimal enzyme activity differs between different cell types and organs, it seems difficult to establish a general biologically critical pH limit. The blood gases and lactate in cord blood change with the progression of pregnancy toward a mixed metabolic and respiratory acidemia because of increased metabolism and CO2 production in the growing fetus. Gestational age-adjusted normal reference values have accordingly been published for pH and lactate, and they associate with Apgar score slightly better than stationary cutoffs, but they are not widely used in clinical practice. On the basis of good-quality data, it is reasonable to set a cord artery lactate cutoff (mean +2 standard deviations) at 10 mmol/L at 39 to 40 weeks' gestation. For base deficit, it is not possible to establish statistically defined reference values because base deficit is calculated with different equations, and there is no consensus on which to use. Arterial cord blood represents the fetus better than venous blood, and samples from both vessels are needed to validate the arterial origin. A venoarterial pH gradient of <0.02 is commonly used to differentiate arterial from venous samples. Reference values for pH in cord venous blood have been determined, but venous blood comes from the placenta after clearance of a surplus of arterial CO2, and base deficit in venous blood then overestimates the metabolic component of fetal acidosis. The ambition to increase neonatal hemoglobin and iron depots by delaying cord clamping after birth results in falsely acidic blood gas and lactate values if the blood sampling is also delayed. Within seconds after birth, sour metabolites accumulated in peripheral tissues and organs will flood into the central circulation and further to the cord arteries when the newborn starts to breathe, move, and cry. This influence of "hidden acidosis" can be avoided by needle puncture of unclamped cord vessels and blood collection immediately after birth. Because of a continuing anaerobic glycolysis in the collected blood, it should be analyzed within 5 minutes to not result in a falsely high lactate value. If the syringe is placed in ice slurry, the time limit is 20 minutes. For pH, it is reasonable to wait no longer than 15 minutes if not in ice. Routine analyses of cord blood gases enable perinatal audits to gain the wisdom of hindsight, to maintain quality assurance at a maternity unit over years by following the rate of neonatal acidosis, to compare results between hospitals on regional or national bases, and to obtain an objective outcome measure in clinical research. Given that the intrapartum cardiotocogram is an uncertain proxy for fetal hypoxia, and there is no strong correlation between pathologic cardiotocograms and fetal acidosis, a cord artery pH may help rather than hurt a staff person subjected to a malpractice suit based on undesirable cardiotocogram patterns. Contrary to common beliefs and assumptions, up to 90% of cases of cerebral palsy do not originate from intrapartum events. Future research will elucidate whether cell injury markers with point-of-care analysis will become valuable in improving the dating of perinatal injuries and differentiating hypoxic from nonhypoxic injuries.

Venous Blood Oxygenation Measurements Using TRUST and T2-TRIR MRI During Hypoxic and Hypercapnic Gas Challenges.

BACKGROUND: Oxygen extraction fraction (OEF) and cerebral metabolic rate of oxygen (CMRO2 ) may serve as biomarkers in several diseases. OEF and CMRO2 can be estimated from venous blood oxygenation (Yv ) levels, which in turn can be calculated from venous blood T2 values (T2b ). T2b can be measured using different MRI sequences, including T2-relaxation-under-spin-tagging (TRUST) and T2-prepared-blood-relaxation-imaging-with-inversion-recovery (T2-TRIR). The latter measures both T2b and T1 (T1b ) but was found previously to overestimate T2b compared to TRUST. It remained unclear, however, if this bias is constant across higher and lower oxygen saturations. PURPOSE: To compare TRUST and T2-TRIR across a range of O2 saturations using hypoxic and hypercapnic gas challenges. STUDY TYPE: Prospective. POPULATION: Twelve healthy volunteers (four female, age 36 ± 10 years). FIELD STRENGTH/SEQUENCE: A 3T; turbo-field echo-planar-imaging (TFEPI), echo-planar-imaging (EPI), and fast-field-echo (FFE). ASSESSMENT: TRUST- and T2-TRIR-derived T2b , Yv , OEF, and CMRO2 were compared across different respiratory challenges. T1b from T2-TRIR was used to estimate Hct (HctTRIR ) and compared with venipuncture (HctVP ). STATISTICAL TESTS: Shapiro-Wilk, one-sample and paired-sample t-test, repeated measures ANOVA, Friedman test, Bland-Altman, and correlation analysis. Bonferroni multiple-comparison correction was performed. Significance level was 0.05. RESULTS: A significant bias was observed between TRUST- and T2-TRIR-derived T2b , Yv , and OEF values (-13 ± 11 msec, -5.3% ± 3.5% and 5.9 ± 4.1%, respectively). For Yv and OEF, this bias was constant across the range of measured values. T1b was significantly lower during severe hypoxia and hypercapnia compared to baseline (1712 ± 86 msec and 1634 ± 79 msec compared to 1757 ± 90 msec). While no significant bias was found between HctVP and HctTRIR (0.02% ± 0.06%, P = 0.20), the correlation between these Hct values was significant but weak (r = 0.19). DATA CONCLUSION: Given the constant bias, TRUST- and T2-TRIR-derived venous T2b values can be used interchangeably to estimate Yv , OEF, and CMRO2 across a broad range of oxygen saturations. Hct from T2-TRIR-derived T1-values only weakly correlated with Hct from venipuncture. EVIDENCE LEVEL: 2 TECHNICAL EFFICACY: Stage 2.

Hypercapnia alters mitochondrial gene expression and acylcarnitine production in monocytes.

CO2 is produced during aerobic respiration. Normally, levels of CO2 in the blood are tightly regulated but pCO2 can rise (hypercapnia, pCO2 > 45 mmHg) in patients with lung diseases, for example, chronic obstructive pulmonary disease (COPD). Hypercapnia is a risk factor in COPD but may be of benefit in the context of destructive inflammation. The effects of CO2 per se, on transcription, independent of pH change are poorly understood and warrant further investigation. Here we elucidate the influence of hypercapnia on monocytes and macrophages through integration of state-of-the-art RNA-sequencing, metabolic and metabolomic approaches. THP-1 monocytes and interleukin 4-polarized primary murine macrophages were exposed to 5% CO2 versus 10% CO2 for up to 24 h in pH-buffered conditions. In hypercapnia, we identified around 370 differentially expressed genes (DEGs) under basal and about 1889 DEGs under lipopolysaccharide-stimulated conditions in monocytes. Transcripts relating to both mitochondrial and nuclear-encoded gene expression were enhanced in hypercapnia in basal and lipopolysaccharide-stimulated cells. Mitochondrial DNA content was not enhanced, but acylcarnitine species and genes associated with fatty acid metabolism were increased in hypercapnia. Primary macrophages exposed to hypercapnia also increased activation of genes associated with fatty acid metabolism and reduced activation of genes associated with glycolysis. Thus, hypercapnia elicits metabolic shifts in lipid metabolism in monocytes and macrophages under pH-buffered conditions. These data indicate that CO2 is an important modulator of monocyte transcription that can influence immunometabolic signaling in immune cells in hypercapnia. These immunometabolic insights may be of benefit in the treatment of patients experiencing hypercapnia.

The Pedunculopontine Tegmental Nucleus is not Important for Breathing Impairments Observed in a Parkinson's Disease Model.

Parkinson's disease (PD) is a motor disorder resulting from degeneration of dopaminergic neurons of substantia nigra pars compacta (SNpc), with classical and non-classical symptoms such as respiratory instability. An important region for breathing control, the Pedunculopontine Tegmental Nucleus (PPTg), is composed of cholinergic, glutamatergic, and GABAergic neurons. We hypothesize that degenerated PPTg neurons in a PD model contribute to the blunted respiratory activity. Adult mice (40 males and 29 females) that express the fluorescent green protein in cholinergic, glutamatergic or GABAergic cells were used (Chat-cre Ai6, Vglut2-cre Ai6 and Vgat-cre Ai6) and received bilateral intrastriatal injections of vehicle or 6-hydroxydopamine (6-OHDA). Ten days later, the animals were exposed to hypercapnia or hypoxia to activate PPTg neurons. Vglut2-cre Ai6 animals also received retrograde tracer injections (cholera toxin b) into the retrotrapezoid nucleus (RTN) or preBötzinger Complex (preBötC) and anterograde tracer injections (AAV-mCherry) into the SNpc. In 6-OHDA-injected mice, there is a 77% reduction in the number of dopaminergic neurons in SNpc without changing the number of neurons in the PPTg. Hypercapnia activated fewer Vglut2 neurons in PD, and hypoxia did not activate PPTg neurons. PPTg neurons do not input RTN or preBötC regions but receive projections from SNpc. Although our results did not show a reduction in the number of glutamatergic neurons in PPTg, we observed a reduction in the number of neurons activated by hypercapnia in the PD animal model, suggesting that PPTg may participate in the hypercapnia ventilatory response.

Hypercapnia alters stroma-derived Wnt production to limit ß-catenin signaling and proliferation in AT2 cells.

Persistent symptoms and radiographic abnormalities suggestive of failed lung repair are among the most common symptoms in patients with COVID-19 after hospital discharge. In mechanically ventilated patients with acute respiratory distress syndrome (ARDS) secondary to SARS-CoV-2 pneumonia, low tidal volumes to reduce ventilator-induced lung injury necessarily elevate blood CO2 levels, often leading to hypercapnia. The role of hypercapnia on lung repair after injury is not completely understood. Here - using a mouse model of hypercapnia exposure, cell lineage tracing, spatial transcriptomics, and 3D cultures - we show that hypercapnia limits ß-catenin signaling in alveolar type II (AT2) cells, leading to their reduced proliferative capacity. Hypercapnia alters expression of major Wnts in PDGFRα+ fibroblasts from those maintaining AT2 progenitor activity toward those that antagonize ß-catenin signaling, thereby limiting progenitor function. Constitutive activation of ß-catenin signaling in AT2 cells or treatment of organoid cultures with recombinant WNT3A protein bypasses the inhibitory effects of hypercapnia. Inhibition of AT2 proliferation in patients with hypercapnia may contribute to impaired lung repair after injury, preventing sealing of the epithelial barrier and increasing lung flooding, ventilator dependency, and mortality.

Acid-base and hematological regulation in chicken embryos during internal progressive hypercapnic hypoxia.

Development of the capacity to mitigate potential disturbances to blood physiology in bird embryos is incompletely understood. We investigated regulation of acid-base and hematology in day 15 chicken embryos exposed to graded intrinsic hypercapnic hypoxia created by varying degrees of water submersion. Metabolic acidosis with additional respiratory or metabolic acidosis occurred at 2 h according to magnitude of submersion. Acid-base disturbance was partially compensated by metabolic alkalosis at 6 h, but compensatory metabolic alkalosis was absent at 24 h. Following submersion with only air cell exposed to air, both hypercapnic respiratory acidosis and metabolic acidosis occurred within 10 min. Subsequently, both forms of acidosis created lethal levels of [HCO3-] at ∼120 min. Blood hematology showed small but significant effects associated with induced acid-base disturbance. Increased Hct occurring during partial egg submersion lasting 24 h was attributed to an increase in MCV. By day 15 of development chicken embryos are able to partially compensate for and withstand all but severe induced internal hypoxic hypercapnia.

The many layers of BOLD. The effect of hypercapnic and hyperoxic stimuli on macro- and micro-vascular compartments quantified by CVR, M, and CBV across cortical depth.

Ultra-high field functional magnetic resonance imaging (fMRI) offers the spatial resolution to measure neuronal activity at the scale of cortical layers. However, cortical depth dependent vascularization differences, such as a higher prevalence of macro-vascular compartments near the pial surface, have a confounding effect on depth-resolved blood-oxygen-level dependent (BOLD) fMRI signals. In the current study, we use hypercapnic and hyperoxic breathing conditions to quantify the influence of all venous vascular and micro-vascular compartments on laminar BOLD fMRI, as measured with gradient-echo (GE) and spin-echo (SE) scan sequences, respectively. We find that all venous vascular and micro-vascular compartments are capable of comparable theoretical maximum signal intensities, as represented by the M-value parameter. However, the capacity for vessel dilation, as reflected by the cerebrovascular reactivity (CVR), is approximately two and a half times larger for all venous vascular compartments combined compared to the micro-vasculature at superficial layers. Finally, there is roughly a 35% difference in estimates of CBV changes between all venous vascular and micro-vascular compartments, although this relative difference was approximately uniform across cortical depth. Thus, our results suggest that fMRI BOLD signal differences across cortical depth are likely caused by differences in dilation properties between macro- and micro-vascular compartments.

Metabolic and respiratory chemosensitivity and brain monoaminergic responses to cold exposure in chicks subjected to thermal manipulation during incubation.

We evaluated ventilation (V˙E), body temperature (TB), oxygen consumption (V˙ O2), respiratory equivalent (V˙E/ V˙ O2), and monoamine concentrations of 14-day-old (14d) male and female chicks from eggs incubated at low (LT, 36 °C), control (CT, 37.5 °C) and high (HT, 39 °C) temperature during the early embryonic phase, to normoxia, hypercapnia and hypoxia under exposure to cold environment (20 °C). At normoxia, acute cold exposure did not affect the ventilatory variables, with the exception of HT males, in which cold prevented the reduced V˙E observed under thermoneutral conditions. Exposure to 20 °C caused a decrease in TB in both sexes, and LT and HT females presented a greater hypothermic response. Hypercapnia combined with cold did not alter the ventilatory variables, but LT females and CT males and females showed a blunted CO2-induced hyperventilation due to a higher V˙ O2, compared to the same groups in thermoneutral conditions. Unlike with thermoneutral conditions, the blunted hypercapnic hyperventilation observed in the HT groups was not observed during cold challenge. CO2 exposure promoted a similar decrease in TB in the thermoneutral and acutely cold exposed groups, while LT females under cold condition presented a blunted hypothermic response. During hypoxia, cold challenge attenuated the increase in V˙E in LT females and HT males, due to changes in VT. Hypoxic metabolic depression was greater in LT females and males and HT males during cold exposure, while no change in V˙E/ V˙ O2 was observed. The only alteration in monoaminergic concentration under cold challenge was an increase in brainstem 5-HIAA and 5-HIAA/5-HT ratio in HT females, and an enhanced 5-HT concentration in HT males. In summary, thermal manipulation during embryogenesis induces 14d old chicks to respond differently to cold stress with LT females and HT males being more sensitive.

Analyzing the brainstem circuits for respiratory chemosensitivity in freely moving mice.

Regulation of systemic PCO2 is a life-preserving homeostatic mechanism. In the medulla oblongata, the retrotrapezoid nucleus (RTN) and rostral medullary Raphe are proposed as CO2 chemosensory nuclei mediating adaptive respiratory changes. Hypercapnia also induces active expiration, an adaptive change thought to be controlled by the lateral parafacial region (pFL). Here, we use GCaMP6 expression and head-mounted mini-microscopes to image Ca2+ activity in these nuclei in awake adult mice during hypercapnia. Activity in the pFL supports its role as a homogenous neuronal population that drives active expiration. Our data show that chemosensory responses in the RTN and Raphe differ in their temporal characteristics and sensitivity to CO2, raising the possibility these nuclei act in a coordinated way to generate adaptive ventilatory responses to hypercapnia. Our analysis revises the understanding of chemosensory control in awake adult mouse and paves the way to understanding how breathing is coordinated with complex non-ventilatory behaviours.

CO2 exposure enhances Fos expression in hypothalamic neurons in rats during the light and dark phases of the diurnal cycle.

Orexinergic (OX) neurons in the lateral hypothalamus (LH), perifornical area (PFA) and dorsomedial hypothalamus (DMH) play a role in the hypercapnic ventilatory response, presumably through direct inputs to central pattern generator sites and/or through interactions with other chemosensitive regions. OX neurons can produce and release orexins, excitatory neuropeptides involved in many functions, including physiological responses to changes in CO2/pH. Thus, in the present study, we tested the hypothesis that different nuclei (LH, PFA and DMH) where the orexinergic neurons are located, show distinct activation by CO2 during the light-dark cycle phases. For this purpose, we evaluated the Fos and OXA expression by immunohistochemistry to identify neurons that co-localize Fos + OXA in the LH, LPeF, MPeF and DMH in the light-inactive and dark-active phase in Wistar rats subjected to 3 h of normocapnia or hypercapnia (7% CO2). Quantitative analyses of immunoreactive neurons show that hypercapnia caused an increase in the number of neurons expressing Fos in the LH, LPeF, MPeF and DMH in the light and dark phases. In addition, the number of Fos + OXA neurons increased in the LPeF and DMH independently of the phases of the diurnal cycle; whereas in the MPeF, this increase was observed exclusively in the light phase. Thus, we suggest that OX neurons are selectively activated by hypercapnia throughout the diurnal cycle, reinforcing the differential role of nuclei in the hypothalamus during central chemosensitivity.