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.

Brainstem serotonergic, catecholaminergic, and inflammatory adaptations during chronic hypercapnia in goats.

Despite the prevalence of CO2 retention in human disease, little is known about the adaptive neurobiological effects of chronic hypercapnia. We have recently shown 30-d exposure to increased inspired CO2 (InCO2) leads to a steady-state ventilation that exceeds the level predicted by the sustained acidosis and the acute CO2/H+ chemoreflex, suggesting plasticity within respiratory control centers. Based on data showing brainstem changes in aminergic and inflammatory signaling during carotid body denervation-induced hypercapnia, we hypothesized chronic hypercapnia per se will lead to similar changes. We found that: 1) increased InCO2 increased IL-1ß in the medullary raphe (MR), ventral respiratory column, and cuneate nucleus after 24 h, but not after 30 d of hypercapnia; 2) the number of serotonergic and total neurons were reduced within the MR and ventrolateral medulla following 30 d of increased InCO2; 3) markers of tryptophan metabolism were altered following 24 h, but not 30 d of InCO2; and 4) there were few changes in brainstem amine levels following 24 h or 30 d of increased InCO2. We conclude that these changes may contribute to initiating or maintaining respiratory neuroplasticity during chronic hypercapnia but alone do not account for ventilatory acclimatization to chronic increased InCO2.-Burgraff, N. J., Neumueller, S. E., Buchholz, K. J., LeClaire, J., Hodges, M. R., Pan, L., Forster, H. V. Brainstem serotonergic, catecholaminergic, and inflammatory adaptations during chronic hypercapnia in goats.

Ventilatory and integrated physiological responses to chronic hypercapnia in goats.

KEY POINTS: Chronic hypercapnia per se has distinct effects on the mechanisms regulating steady-state ventilation and the CO2 /H+ chemoreflex. Chronic hypercapnia leads to sustained hyperpnoea that exceeds predicted ventilation based upon the CO2 /H+ chemoreflex. There is an integrative ventilatory, cardiovascular and metabolic physiological response to chronic hypercapnia. Chronic hypercapnia leads to deterioration of cognitive function. ABSTRACT: Respiratory diseases such as chronic obstructive pulmonary disease (COPD) often lead to chronic hypercapnia which may exacerbate progression of the disease, increase risk of mortality and contribute to comorbidities such as cognitive dysfunction. Determining the contribution of hypercapnia per se to adaptations in ventilation and cognitive dysfunction within this patient population is complicated by the presence of multiple comorbidities. Herein, we sought to determine the role of chronic hypercapnia per se on the temporal pattern of ventilation and the ventilatory CO2 /H+ chemoreflex by exposing healthy goats to either room air or an elevated inspired CO2 (InCO2 ) of 6% for 30 days. A second objective was to determine whether chronic hypercapnia per se contributes to cognitive dysfunction. During 30 days of exposure to 6% InCO2 , steady-state (SS) ventilation ( V̇I ) initially increased to 335% of control, and then within 1-5 days decreased and stabilized at ∼230% of control. There was an initial respiratory acidosis that was partially mitigated over time due to increased arterial [HCO3- ]. There was a transient decrease in the ventilatory CO2 /H+ chemoreflex, followed by return to pre-exposure levels. The SS V̇I during chronic hypercapnia was greater than predicted from the acute CO2 /H+ chemoreflex, suggesting separate mechanisms regulating SS V̇I and the chemoreflex. Finally, as assessed by a shape discrimination test, we found a sustained decrease in cognitive function during chronic hypercapnia. We conclude that chronic hypercapnia per se results in: (1) a disconnect between SS V̇I and the CO2 /H+ chemoreflex, and (2) deterioration of cognitive function.

Effects of sub-lethal doses of fentanyl on vital physiologic functions and withdrawal-like behaviors in adult goats.

Synthetic opioids like fentanyl have improved the standard of care for many patients in the clinical setting, but their abuse leads to tens of thousands of overdose deaths annually. The current opioid epidemic underscores a critical need for insights into the physiological effects of fentanyl on vital functions. High doses of opioids in small mammals cause opioid-induced respiratory depression (OIRD) leading to hypoventilation, hypoxemia, and hypercapnia. In addition, opioids can also increase the alveolar to arterial oxygen (A-a) gradient and airway dysfunction. However, little is known about the physiologic effects of sub-lethal doses of opioids in large mammals. Here we report the effects of a sub-lethal dose range of fentanyl (25-125 µg/kg; IV) on vital physiologic functions over 90 min (min) and withdrawal-like behaviors over the subsequent 4 h (h) in adult female goats (n = 13). Fentanyl induced decreases in breathing frequency in the first few min post-injection, but then led to a sustained increase in tidal volume, total ventilation, and blood pressure with a reduced heart rate for ≥90 min. These ventilatory changes resulted in time-dependent arterial hypocapnia and hypoxemia and an increased alveolar to arterial oxygen gradient ∼30 min post-injection indicative of impaired gas exchange in the lung. The predominant effects of fentanyl on breathing were stimulatory, underscored by an increased rate of rise of the diaphragm muscle activity and increased activation of upper airway, intercostal and abdominal muscles. Beginning 90 min post-injection we also quantified withdrawal-like behaviors over 4 h, demonstrating dose- and time-dependent increases in locomotor, biting, itching, and pawing behaviors. We conclude that fentanyl at sublethal doses induces multiple physiologic and behavior changes that emerge along different time courses suggesting multiple independent mechanisms underlying effects of opioids.

Chronic moderate hypercapnia suppresses ventilatory responses to acute CO₂ challenges.

Chronic hypercapnia (CH) is a hallmark of chronic lung disease, and CH increases the risk for acute-on-chronic exacerbations leading to greater hypoxemia/hypercapnia and poor health outcomes. However, the role of hypercapnia per se (duration and severity) in determining an individual's ability to tolerate further hypercapnic exacerbations is unknown. Our primary objective herein was to test the hypothesis that mild-to-moderate CH (arterial [Formula: see text] ∼50-70 mmHg) increases susceptibility to pathophysiological responses to severe acute CO₂ challenges. Three groups (GR) of adult female goats were studied during 14 days of exposure to room air (<i>GR 1</i>; control) or 6% inspired CO₂ (<i>GR 2</i>; mild CH), or 7 days of 6% inspired CO₂ followed by 7 days of 8% inspired CO₂ (<i>GR 3</i>; moderate CH). Consistent with previous reports, there were no changes in physiological parameters in <i>GR 1</i> (RA control), but mild CH (<i>GR 2</i>) increased steady-state ventilation and transiently suppressed CO₂/[H⁺] chemosensitivity. Further increasing InCO₂ from 6% to 8% (<i>GR 3</i>) transiently increased ventilation and arterial [H⁺]. Similar to mild CH, moderate CH increased ventilation to levels greater than predicted. However, in contrast to mild CH, acute ventilatory chemosensitivity was suppressed throughout the duration of moderate CH, and the arterial - mixed expired CO₂ gradient became negative. These data suggest that moderate CH limits physiological responses to acute severe exacerbations and provide evidence of recruitment of extrapulmonary systems (i.e., gastric CO₂ elimination) during times of moderate-severe hypercapnia.<b>NEW &amp; NOTEWORTHY</b> Moderate levels of chronic hypercapnia (CH; ∼70 mmHg) in healthy adult female goats elicited similar steady-state physiological adaptations compared with mild CH (∼55 mmHg). However, unlike mild CH, moderate CH chronically suppressed acute CO₂/[H⁺] chemosensitivity and reversed the arterial to mixed expired CO₂ gradient. These findings suggest that moderate CH suppresses vital mechanisms of ventilatory control and recruits additional physiological systems (i.e., gastric CO₂ release) to help buffer excess CO₂.

Physiological and neurochemical adaptations following abrupt termination of chronic hypercapnia in goats.

Chronic hypercapnia (CH) is a hallmark of respiratory diseases such as chronic obstructive pulmonary disease. In such patients, mechanical ventilation is often used to restore normal blood-gas homeostasis. However, little is known regarding physiological changes and neuroplasticity within physiological control networks after termination of CH. Utilizing our goat model of increased inspired CO2-induced CH, we determined whether termination of CH elicits time-dependent physiological and neurochemical changes within brain stem sites of physiological control. Thirty days of CH increased [Formula: see text] (+15 mmHg) and steady-state ventilation (SS V̇i; 283% of control). Within 24 h after terminating CH, SS V̇i, blood gases, arterial [H+], and most physiological measurements returned to control. However, the acute ventilatory chemoreflex (ΔV̇i/Δ[H+]) was greater than control, and measured SS V̇i exceeded ventilation predicted by arterial [H+] and ΔV̇i/Δ[H+]. Potentially contributing to these differences were increased excitatory neuromodulators serotonin and norepinephrine in the nucleus tractus solitarius, which contrasts with minimal changes observed at 24 h and 30 days of hypercapnia. Similarly, there were minimal changes found in markers of neuroinflammation and glutamate receptor-dependent neuroplasticity upon termination of CH, which were previously increased following 24 h of hypercapnia. Thus, following termination of CH: 1) ventilatory, renal, and other physiological functions rapidly return to control; 2) neuroplasticity within the ventilatory control network may contribute to the difference between measured vs. predicted ventilation and the elevation in the acute ventilatory [H+] chemoreflex; and 3) neuroplasticity is fundamentally distinct from acclimatization to CH.NEW & NOTEWORTHY In healthy adult goats, steady-state ventilation and most physiological measures return to control within 24 h after termination of chronic hypercapnia (CH). However, the acute [H+] chemoreflex is increased, and measured ventilation exceeds predicted ventilation. At 24 h of recovery, excitatory neuromodulators are above control, but other measured markers of neuroplasticity are unchanged from control. Our data suggest that CH elicits persistent physiological and neurochemical changes for up to 24 h after termination of CH.

Plasticity of respiratory rhythm-generating mechanisms in adult goats.

Abrupt destruction of >70% of the pre-Bötzinger complex (preBötzC) in awake goats results in terminal apnea (Wenninger et al. 2004b). Herein we report data on awake and sleeping goats in which the preBötzC was incrementally destroyed by injection of ibotenic acid (IBO) in increasing volumes at weekly intervals. All injections resulted in an acute tachypnea and dysrhythmia featuring apneas and increased variation in breathing. In studies at night, 10-15 hours after the injections, apneas were nearly all central and occurred during the awake state and variation in breathing was greater while awake than during NREM sleep. However, one week after the final IBO injection, the breathing pattern, breath-to-breath variation, and arterial blood gases were unchanged from baseline, indicating recovery. Histology revealed more than 90% destruction of the preBötzC region, and greater than 80% destruction of the surrounding area. We conclude: (1) the dysrhythmic effects on breathing acutely after the injection are state-dependent, and (2) after incremental, near-complete destruction of the preBötzC region, time-dependent plasticity within the respiratory network provides a normal respiratory rhythm that sustains normal arterial blood gases.

Midbrain and cerebral inflammatory and glutamatergic adaptations during chronic hypercapnia in goats.

Cognitive impairment is associated with multiple human diseases that have in common chronic hypercapnia. However, the mechanisms leading to chronic hypercapnia-induced cognitive decline are not known. We have previously shown chronic hypercapnia through exposure to increased inspired CO2 (6% InCO2) in conscious goats caused an immediate (within hours) and sustained decline in cognitive performance during a shape discrimination test. Herein, within the same goats, we assessed markers of neuroinflammation and glutamate receptor expression/phosphorylation within CNS regions important for cognitive function following 24 hours (h) or 30 days (d) of chronic hypercapnia. Within 24 h, chronic hypercapnia increased expression of the inflammatory cytokine IL-1ß in the orbitofrontal cortex and medial prefrontal cortex, but at 30d IL-1ß levels were not different relative to time-matched goats exposed to room-air. Additionally, Iba1 expression (a marker of microglial activation) was unaltered by chronic hypercapnia in all regions tested. Finally, levels of the total and phosphorylated AMPA receptor subunit GluR2 were reduced within the hippocampus at both 24 h and 30 d of hypercapnia, and reduced following 30 d within the anterior insular cortex. These data suggest that chronic hypercapnia leads to CNS site-dependent acute inflammatory responses and shifts in select glutamate receptor expression/phosphorylation in brain regions contributing to cognitive function. Such changes may be indicative of alterations in glutamatergic receptor-mediated signaling and neuronal dysfunction that contribute to declines in cognitive function associated with human diseases defined or marked by chronic CO2 retention.

Glutamate receptor plasticity in brainstem respiratory nuclei following chronic hypercapnia in goats.

Patients that retain CO2 in respiratory diseases such as chronic obstructive pulmonary disease (COPD) have worse prognoses and higher mortality rates than those with equal impairment of lung function without hypercapnia. We recently characterized the time-dependent physiologic effects of chronic hypercapnia in goats, which suggested potential neuroplastic shifts in ventilatory control mechanisms. However, little is known about how chronic hypercapnia affects brainstem respiratory nuclei (BRN) that control multiple physiologic functions including breathing. Since many CNS neuroplastic mechanisms include changes in glutamate (AMPA (GluR) and NMDA (GluN)) receptor expression and/or phosphorylation state to modulate synaptic strength and network excitability, herein we tested the hypothesis that changes occur in glutamatergic signaling within BRN during chronically elevated inspired CO2 (InCO2 )-hypercapnia. Healthy goats were euthanized after either 24 h or 30 days of chronic exposure to 6% InCO2 or room air, and brainstems were rapidly extracted for western blot analyses to assess GluR and GluN receptor expression within BRN. Following 24-hr exposure to 6% InCO2 , GluR or GluN receptor expression were changed from control (P < 0.05) in the solitary complex (NTS & DMV),ventrolateral medulla (VLM), medullary raphe (MR), ventral respiratory column (VRC), hypoglossal motor nucleus (HMN), and retrotrapezoid nucleus (RTN). These neuroplastic changes were not found following 30 days of chronic hypercapnia. However, at 30 days of chronic hypercapnia, there was overall increased (P < 0.05) expression of glutamate receptors in the VRC and RTN. We conclude that time- and site-specific glutamate receptor neuroplasticity may contribute to the concomitant physiologic changes that occur during chronic hypercapnia.

Ventilation and neurochemical changes during µ-opioid receptor activation or blockade of excitatory receptors in the hypoglossal motor nucleus of goats.

Neuromodulator interdependence posits that changes in one or more neuromodulators are compensated by changes in other modulators to maintain stability in the respiratory control network. Herein, we studied compensatory neuromodulation in the hypoglossal motor nucleus (HMN) after chronic implantation of microtubules unilaterally ( n = 5) or bilaterally ( n = 5) into the HMN. After recovery, receptor agonists or antagonists in mock cerebrospinal fluid (mCSF) were dialyzed during the awake and non-rapid eye movement (NREM) sleep states. During day studies, dialysis of the µ-opioid inhibitory receptor agonist [d-Ala2, N-MePhe4, Gly-ol]enkephalin (DAMGO; 100 µM) decreased pulmonary ventilation (V̇i), breathing frequency ( f), and genioglossus (GG) muscle activity but did not alter neuromodulators measured in the effluent mCSF. However, neither unilateral dialysis of a broad spectrum muscarinic receptor antagonist (atropine; 50 mM) nor unilateral or bilateral dialysis of a mixture of excitatory receptor antagonists altered V̇i or GG activity, but all of these did increase HMN serotonin (5-HT) levels. Finally, during night studies, DAMGO and excitatory receptor antagonist decreased ventilatory variables during NREM sleep but not during wakefulness. These findings contrast with previous dialysis studies in the ventral respiratory column (VRC) where unilateral DAMGO or atropine dialysis had no effects on breathing and bilateral DAMGO or unilateral atropine increased V̇i and f and decreased GABA or increased 5-HT, respectively. Thus we conclude that the mechanisms of compensatory neuromodulation are less robust in the HMN than in the VRC under physiological conditions in adult goats, possibly because of site differences in the underlying mechanisms governing neuromodulator release and consequently neuronal activity, and/or responsiveness of receptors to compensatory neuromodulators. NEW & NOTEWORTHY Activation of inhibitory µ-opioid receptors in the hypoglossal motor nucleus decreased ventilation under physiological conditions and did not affect neurochemicals in effluent dialyzed mock cerebral spinal fluid. These findings contrast with studies in the ventral respiratory column where unilateral [d-Ala2, N-MePhe4, Gly-ol]enkephalin (DAMGO) had no effects on ventilation and bilateral DAMGO or unilateral atropine increased ventilation and decreased GABA or increased serotonin, respectively. Our data support the hypothesis that mechanisms that govern local compensatory neuromodulation within the brain stem are site specific under physiological conditions.

Effects on breathing of agonists to µ-opioid or GABAA receptors dialyzed into the ventral respiratory column of awake and sleeping goats.

Pulmonary ventilation (V̇I) in awake and sleeping goats does not change when antagonists to several excitatory G protein-coupled receptors are dialyzed unilaterally into the ventral respiratory column (VRC). Concomitant changes in excitatory neuromodulators in the effluent mock cerebral spinal fluid (mCSF) suggest neuromodulatory compensation. Herein, we studied neuromodulatory compensation during dialysis of agonists to inhibitory G protein-coupled or ionotropic receptors into the VRC. Microtubules were implanted into the VRC of goats for dialysis of mCSF mixed with agonists to either µ-opioid (DAMGO) or GABAA (muscimol) receptors. We found: (1) V̇I decreased during unilateral but increased during bilateral dialysis of DAMGO, (2) dialyses of DAMGO destabilized breathing, (3) unilateral dialysis of muscimol increased V̇I, and (4) dialysis of DAMGO decreased GABA in the effluent mCSF. We conclude: (1) neuromodulatory compensation can occur during altered inhibitory neuromodulator receptor activity, and (2) the mechanism of compensation differs between G protein-coupled excitatory and inhibitory receptors and between G protein-coupled and inotropic inhibitory receptors.

State-dependent and -independent effects of dialyzing excitatory neuromodulator receptor antagonists into the ventral respiratory column.

Unilateral dialysis of the broad-spectrum muscarinic receptor antagonist atropine (50 mM) into the ventral respiratory column [(VRC) including the pre-Bötzinger complex region] of awake goats increased pulmonary ventilation (V̇i) and breathing frequency (f), conceivably due to local compensatory increases in serotonin (5-HT) and substance P (SP) measured in effluent mock cerebral spinal fluid (mCSF). In contrast, unilateral dialysis of a triple cocktail of antagonists to muscarinic (atropine; 5 mM), neurokinin-1, and 5-HT receptors does not alter V̇i or f, but increases local SP. Herein, we tested hypotheses that 1) local compensatory 5-HT and SP responses to 50 mM atropine dialyzed into the VRC of goats will not differ between anesthetized and awake states; and 2) bilateral dialysis of the triple cocktail of antagonists into the VRC of awake goats will not alter V̇i or f, but will increase local excitatory neuromodulators. Through microtubules implanted into the VRC of goats, probes were inserted to dialyze mCSF alone (time control), 50 mM atropine, or the triple cocktail of antagonists. We found 1) equivalent increases in local 5-HT and SP with 50 mM atropine dialysis during wakefulness compared with isoflurane anesthesia, but V̇i and f only increased while awake; and 2) dialyses of the triple cocktail of antagonists increased V̇i, f, 5-HT, and SP (<0.05) during both day and night studies. We conclude that the mechanisms governing local neuromodulator levels are state independent, and that bilateral excitatory receptor blockade elicits an increase in breathing, presumably due to a local, (over)compensatory neuromodulator response.NEW & NOTEWORTHY The two major findings are as follows: 1) during unilateral dialysis of 50 mM atropine into the ventral respiratory column to block excitatory muscarinic receptor activity, a compensatory increase in other neuromodulators was state independent, but the ventilatory response appears to be state dependent; and 2) the hypothesis that absence of decreased V̇i and f during unilateral dialysis of excitatory receptor antagonists was due to compensation by the contralateral VRC was not supported by findings herein.