Osverdoses (ODs) involving the synthetic opioid fentanyl annually contribute to thousands of deaths in the United States and are largely attributed to opioid-induced respiratory depression (OIRD). However, fentanyl OD negatively affect several aspects of cardiorespiratory function, including respiratory muscle rigidity and pulmonary gas exchange. Here, we tested the hypothesis that high doses of intravenous fentanyl (50-750 µg/kg) cause unique, time- and dose-dependent changes in physiologic functions and behaviors consistent with OIRD, muscle rigidity, impaired gas exchange, and acute withdrawal in a large animal model (adult female goats; n = 12). Within the first 15 min after injection, intravenous fentanyl dose-dependently reduced minute ventilation (V̇i; P < 0.0001), breathing frequency (Fb; P < 0.0001), swallowing (P < 0.001), and [Formula: see text] (P < 0.0001) but increased tidal volume (Vt; P = 0.0029), [Formula: see text] (P < 0.0001), the alveolar-arterial O2 gradient (A-a; P = 0.0024), and respiratory pump and airway muscle electromyographic (EMG) activity. Thereafter (30-90 min postfentanyl), swallowing and [Formula: see text] remained reduced, whereas the A-a O2 gradient and muscle EMG activity remained increased. Beginning 90 min postfentanyl, overall movement (cumulative vector magnitude, g; P < 0.0001) and the total behavioral response (sum of withdrawal behaviors; P < 0.001) were dose-dependently increased for 4 h. Thus, high-dose fentanyl caused significant and transient OIRD, sustained respiratory muscle activation, impaired gas exchange, and dose-dependent acute withdrawal-like behaviors. We conclude that fentanyl elicits dose- and time-dependent impairments of vital physiologic systems in adult goats similar to those reported in humans, suggesting that the goat is a unique and valuable model for translational hypothesis testing for future novel countermeasure development in vivo.NEW & NOTEWORTHY Here, we defined the time course of integrated physiologic effects of high doses of fentanyl in adult goats. High-dose fentanyl caused hypoventilation and sustained increases in tonic respiratory-related chest wall and airway muscle activity, increased the alveolar-to-arterial O2 gradient, and caused dose-dependent increases in acute withdrawal-like behaviors. These data suggest that the integrated physiologic responses to fentanyl in adult goats closely resemble those in humans.

Ultrapotent synthetic opioids such as fentanyl are highly addictive and at high doses can cause life-threatening cardiorespiratory depression, hypoxemia and death. Fentanyl is the primary driver of the high rates of opioid-involved overdose (OD) deaths (~80K/year in US) through at least three mechanisms: 1) its extreme inhibitory effects on brain centers controlling breathing (Opioid-induced Respiratory Depression; OIRD), 2) reductions in Alveolar-arterial oxygen exchange (increased A-a O 2 gradient), and 3) increased chest wall rigidity and upper airway dysfunction via tonic muscle activation (Wooden Chest Syndrome; WCS). However, opioid-involved OD deaths from fentanyl alone are less common than those that include additional drugs. Among the fastest growing are opioid-involved OD deaths from fentanyl combined with the α2 receptor agonists xylazine or medetomidine. Here we tested in adult female goats (n=5) the hypothesis that fentanyl/xylazine combinations have greater than additive (supra-additive) physiological effects compared to fentanyl alone which are resistant to µ-opioid receptor (MOR) antagonism (naloxone; NAL). IV fentanyl (50-150 µg/kg) caused transient (~10 min) reduction in ventilation (-20%) and modest hypoventilation (PaCO 2 ; +5 mmHg) and hypoxemia (PaO 2 ; -18 mmHg) with a sustained (>90 min) increase in tonic respiratory muscle activation (consistent with WCS) and an increased A-a O 2 gradient (+20 mmHg). Xylazine alone (10-20 µg/kg; IV) also reduced ventilation (-20%) for 30-45 min but did not alter arterial blood gases and had no effect on respiratory muscle activity or the A-a O 2 gradient. However, xylazine/fentanyl combinations had a more severe and long-lasting respiratory suppression (-50% for ~45 min), increasing PaCO 2 by +12 mmHg, reducing PaO 2 by 40 mmHg and increasing the A-a O 2 gradient by ~40 mmHg. In addition, cardiorespiratory coupling (measured by the respiratory sinus arrhythmia (RSA) amplitude) was not appreciably affected with high dose fentanyl or xylazine alone, but xylazine/fentanyl combinations increased the RSA amplitude >2-fold. Finally, IV NAL only briefly (~5 min) reversed the severe breathing suppression during xylazine/fentanyl combinations even after giving twice standard doses (6-12 µg/kg). While IV administration of the α2 receptor antagonist atipamezole (20 µg/kg) increased ventilation, the goats developed severe tachycardia and hypertension and showed adverse behavioral responses. Thus, these preliminary data support the hypothesis that xylazine/fentanyl combinations elicit a supra-additive and lasting cardiorespiratory suppression greater than that predicted from each drug alone, and that the enhanced cardiorespiratory suppression appears resistant to reversal by NAL. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Fentanyl is the leading contributor to opioid-involved overdose (OD) mortality in the United States. Despite the demonstrated efficacy, safety, and wide availability of naloxone (NAL), opioid-involved OD fatalities remain high. This suggests our understanding of the negative integrated physiological effects of fentanyl remains incomplete, and highlights the need to develop additional novel countermeasures. Here we tested whether the physiological and behavioral effects of intravenous fentanyl in adult female goats (n = 12) could be mitigated with NAL or the potential countermeasure d-cysteine ethyl ester (d-CYSee). As hypothesized, intravenous injections of high doses (HD) fentanyl caused immediate ventilatory suppression via reduced breathing frequency leading to hypoventilation and hypoxemia (≥10 min). HD fentanyl elicited immediate and sustained (≥90 min) increases in diaphragm, intercostal, abdominal, and laryngeal constrictor muscle activity along with an increased alveolar to arterial oxygen (A-a O2) gradient and hypertension. Intravenous NAL administered immediately following HD fentanyl mitigated most of these effects except the increased activation of respiratory pump and airway muscles. In contrast, d-CYSee administration immediately following HD fentanyl countered the initial hypoventilation but did not mitigate the increases in muscle activation and persistent hypoxemia. Neither treatment prevented acute withdrawal-like behaviors emerging >90 min after fentanyl administration. The data suggest that there are physiological effects of HD fentanyl that are NAL-insensitive, and d-CYSee can transiently normalize blood gases and counter opioid-induced respiratory depression (OIRD) in adult goats.NEW & NOTEWORTHY Here we determined whether any of the deleterious physiological effects of high-dose fentanyl could be countered by naloxone and/or d-cysteine ethyl ester (d-CYSee) in adult goats. Fentanyl induced hypoventilation and sustained increases in respiratory and airway muscle activity and hypoxemia. Naloxone reversed all fentanyl effects except tonic muscle activation, and d-CYSee reversed fentanyl-induced hypoventilation. These data suggest some effects of fentanyl are naloxone-insensitive and that d-CYSee may be a valuable countermeasure for OIRD.

Opioids such as fentanyl are addictive and at high doses compromise multiple physiologic functions including breathing leading to life-threatening hypoxemia and death. Opioid-involved overdose deaths (ODs), driven primarily by fentanyl, are currently thought to arise from the extreme inhibitory effects on brain centers that control breathing, leading to Opioid-induced Respiratory Depression (OIRD). However, fentanyl also impairs the respiratory system via alveolar-capillary gas exchange (increased A-a O 2 gradient), causes upper airway (UAW) dysfunction, and increases chest wall rigidity via the synchronous activation of normally reciprocal respiratory muscles. In addition, opioid-involved ODs from fentanyl alone are less common than those that include 1 or more additional drugs (~75% of opioid-involved ODs; fentanyl+) such as the non-opioid sedative xylazine (α2-adrenergic receptor agonist). The opioid receptor antagonist Narcan (naloxone; NAL) is a highly effective “rescue” agent for severe OIRD but has a short half-life and may not be as effective during fentanyl+ situations. To date, few studies have systematically tested whether fentanyl in combination with xylazine has more severe physiological effects than fentanyl alone. Given this, we hypothesize that combinations of fentanyl and xylazine will elicit enhanced physiologic dysfunction and hypoventilation-induced hypoxemia more than fentanyl alone. Adult female goats (n=4) were surgically instrumented with electromyographic (EMG) wires in upper airway and respiratory pump muscles and the carotid arteries were relocated subcutaneously for arterial blood sampling via catheters. Goats were allowed 2 weeks for recovery before the onset of studies and all injections were made via jugular catheter. Following a 30-minute control period, we injected saline (vehicle) or fentanyl alone (50 µg/kg; IV) or co-administration of Xylazine (0.01 µg/kg; IV) + fentanyl (50 µg/kg; IV) and measured physiologic effects for 60 min thereafter. Fentanyl alone (50 µg/kg; IV) lead to a transient reduction in breathing frequency (F B ) (~25% reduction from baseline), but a sustained increase in tidal volume (V T ) and total ventilation (V E ) over the duration of the study. Combinations of xylazine (0.01 µg/kg; IV) and fentanyl (50 µg/kg; IV) reduced both F B (40% from baseline) and V E (60% from baseline), with sustained reduction persisting ≥ 15min post-injection. Arterial oxygen (PaO 2 ) decreased substantially (40 mmHg), and arterial CO 2 (PaCO 2 ) increased to as high as 50mmHg. This preliminary data suggests that fentanyl in combination with xylazine further exacerbates the life-threatening effects on ventilation when compared to fentanyl alone. Funded by NIH DA050571 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Since 1999, the opioid epidemic in the United States has claimed more than 300,000 lives, and data from more recent years indicate annual increases exceeding 80,000 fatalities. Synthetic opioids like fentanyl contribute to these opioid-related deaths through multiple physiologic effects including Opioid-induced respiratory depression (OIRD), impairment of the alveolar to arterial oxygen gradient (A-a), and rigidity of upper airway and pump muscles causing airway dysfunction, or Wooden Chest Syndrome (WCS). We have previously shown (PMID: 37885800) that sub-lethal doses of fentanyl (25-125 μg/kg; IV) cause in healthy adult goats a transient time and dose dependent effect on breathing frequency and tidal volume, WCS and A-a which varied greatly between goats. We hypothesize that high dose fentanyl will exacerbate the effects seen in sub-lethal doses in both a time and dose-dependent manner. Adult female goats (n=3) were surgically instrumented with electromyographic (EMG) wires in both the airway (genioglossus and thyropharyngeal) and respiratory pump muscles (diaphragm, transverse abdominus and diaphragm) and the carotid arteries were relocated to a subcutaneous location. Goats were allowed 2 weeks for recovery before the onset of studies. Arterial blood samples were obtained through carotid catheters and fentanyl injections were made through acute placement of jugular venous catheters. Following a 30-minute control period, we injected saline (vehicle) or high-dose fentanyl (250-500μg/kg). In a time dependent manner, high doses of IV fentanyl injections decreased breathing frequency (FB), increased expiratory time (TE) and increased tidal volume (VT). Arterial oxygen (PaO2) decreased substantially (~35 mmHg; and arterial CO2 (PaCO2) increased to as high as 70mmHg. From about 15 to 90 minutes after fentanyl injections, the overall effect on breathing and respiratory muscle activity was consistent with a stimulatory effect of fentanyl. These data suggest high dose fentanyl increases OIRD and exacerbate life-threatening effects that cause ventilatory suppression, hypoxemia, and hypercapnia. Funded by NIH DA050571. 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.

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft (DFG) Background The hormone and neurotransmitter serotonin regulates multiple processes, including immune responses and inflammatory signalling. After a cardiac injury, such as myocardial infarction (MI), a precise regulation of the local inflammation is determining the healing process and therefore functional outcome. Immune cells invading the infarcted tissue can be critical modulators, and various immune cells, including macrophages, express different serotonin receptors. Purpose We characterized the importance of serotonin receptor 7 (5-HT7R) signalling in the regulation of macrophage function, which infiltrate the heart after MI and thus can determine the course of cardiac remodelling processes. Methods For investigation of 5-HT7R signalling on a cellular level, we used two in vitro models: primary murine bone marrow-derived macrophages (BMDM) and secondary human THP-1-derived macrophages. To validate the impact of macrophages on intact cardiac tissue, we pre-treated pro-inflammatory M1-like BMDMs with a selective 5-HT7R agonist and then co-cultured them with living cardiac rat slices for up to 48 h (ex vivo) followed by functional analysis. Results We detected 5-HT7R expression in both models during all stages of macrophage polarization at mRNA as well as protein levels. Pro-inflammatory THP-1-derived macrophages displayed significantly enhanced mRNA expression levels of IL-1β and CD80, while pro-inflammatory differentiated M1-like BMDMs showed increased levels of CD38 and TNFα. Pharmacological activation of 5-HT7R with LP-211 reduced phagocytic activity up to 4-fold in THP-1-derived pro-inflammatory macrophages, altered their cytokine secretion profile and inhibited their migration ability (recovered area in scratch assay 1.3 % after LP-211 treatment compared to 6.1 % in control). A reduction in phagocytic activity was also verified in pro-inflammatory BMDMs. Our results also indicate reduced ability of LP-211 treated M1-like BMDMs to infiltrate living cardiac slices 3, 12, 24 and 48 h after seeding. Conclusion 5-HT7R signalling affects the functional properties of pro-inflammatory macrophages, including phagocytic activity, motility and secretion profile. Targeted manipulation of 5-HT7R activity in macrophages represents thus a possibility to modulate their inflammatory profile, which might be a reasonable approach to guide recovery post MI.

The worsening opioid epidemic in the United States continues to claim >80,000 lives each year. Synthetic opioids such as fentanyl are highly useful in the clinical management of pain but at high doses are thought to cause death through opioid-induced respiratory depression (OIRD), impairment of the alveolar to arterial oxygen (A-a O2) gradient and though upper airway and respiratory pump muscles activation causing airway dysfunction and/or Wooden Chest Syndrome (WCS). These negative effects of high dose opioids may be exacerbated when individuals use additional drugs such as ketamine or by an existing hypoxia. We have shown previously that low (sub-lethal) doses of fentanyl (25-125 μg/kg; IV) cause transient apnea (<30 sec), a brief suppression of breathing frequency (FB; <5 min), increased tidal volume (VT), upper airway and pump muscle activity, and an increased A-a O2 gradient with an overall stimulatory effect on ventilation for up to 90 min post-injection (PMID: 37885800). Here we tested the hypothesis that the physiological effects of high doses of fentanyl will be exacerbated when combined with pre-treatment of either mild hypoxia (FIO2=0.12) or IV ketamine (10 mg/kg). Adult female goats (n=3) were surgically instrumented with electromyographic (EMG) wires in airway (genioglossus and thyropharyngeal) and respiratory pump (diaphragm, transverse abdominus and intercostal) muscles and the carotid arteries relocated subcutaneously and allowed 2 weeks for recovery. Arterial blood samples were obtained through a carotid catheter and all injections were via a catheter in the jugular. Following a 30-minute control period, goats were given IV saline (vehicle) or fentanyl (50–500 μg/kg). In most goats, fentanyl doses of 200-250 μg/kg cause immediate apnea up to 60 sec in duration and decreased FB (~40%) and PaO2 (~35 mmHg) while increasing PaCO2 (~70mmHg) along with an EMG activation and increased A-a O2 gradient. These effects tended to be less severe at a given dose when injections were performed during mild hypoxia (15 min; n=2) but were exacerbated by pretreatment with ketamine (n=1). These data suggest high doses of fentanyl in goats cause similar life-threatening features as in humans, and that these effects seem to be mitigated by hypoxic ventilatory stimulation but exacerbated by polypharmacy with ketamine. NIH DA050571. 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.

Chronic hypercapnia (CH, PaCO 2 > 45mmHg) is commonly reported in chronic obstructive pulmonary disease (COPD). The severity of CH can increase acutely during frequent acute-on-chronic exacerbations and/or chronically as COPD progresses. We have shown (Buchholz et al., 2022) in awake goats that mild (PaCO 2 ~55mmHg) and moderate (~65mmHg) CH elicits similar time-dependent steady-state physiologic adaptations. However, during acute-on-chronic CO2 challenges, moderate CH chronically suppressed ventilatory CO2 sensitivity whereas mild CH only transiently decreased it suggesting limitations in adaptation to CH in respiratory control. To gain mechanistic insight, we previously identified transient changes in markers of glutamate-receptor plasticity, interleukin-1 Beta signaling, and serotonergic neuromodulation within key nodes of cardiorespiratory control in mild CH. However, these changes did not appear to account for the observed physiologic changes, indicating a broader unbiased approach is needed to identify molecular shifts in pathways altered by CH. Here, utilizing bulk tissue RNA-Sequencing (RNAseq), we tested the hypothesis that mild CH alters gene expression and signaling pathways that are distinct from moderate CH in key nodes of cardiorespiratory control. Female goats were randomly assigned into 3 experimental groups (n=6/group): Group 1 (control) was exposed to room air for 14 days (d), Group 2 (Mild CH) was exposed to 6% inhaled CO 2 (InCO 2 ) for 14d and Group 3 (Moderate CH) was exposed to 6% InCO 2 for 7d followed by 8% InCO2 for an additional 7d. Total RNA was extracted from flash-frozen tissue biopsies of the retrotrapezoid nucleus (RTN), nucleus tractus solitarius (NTS), and ventral respiratory column (VRC). cDNA libraries were generated for paired-end whole transcriptome sequencing. When compared to room air control, we found that sites of cardiorespiratory control in mild CH were highly enriched with upregulated genes involving innate immunity, cytokine activity, T-cell activation, and vascular function. Alternatively, moderate CH downregulates genes related to adaptive immunity, cellular function and structure, and vascular function. Both mild and moderate CH enriched pathways of antigen processing and presentation and immune response. However, the genes and their transcriptional activity in these pathways greatly differ between mild and moderate CH. RNAseq and pathway analyses reveal that varying severities of CH result in different gene expression profiles, primarily related to immune function. These contrasting findings between mild and moderate CH suggests that neuroinflammation may influence the respiratory network’s ability to adjust to physiologic challenges of varying severities to chronic and acute-on-chronic hypercapnia. National Heart, Lung, and Blood Institute Grant F31HL159908; Department of Veterans Affairs Grant BX003284. This is the full abstract presented at the American Physiology Summit 2023 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.

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.

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.

Synthetic opioids alter several vital physiological functions and discontinuation of its use can cause acute withdrawal symptoms. In this and three adjoining posters we present data on the dose-dependent physiologic and behavioral effects of intravenous (IV) fentanyl in adult awake goats. Here, we tested two hypotheses: 1) that fentanyl will lead to acute, dose-dependent withdrawal symptoms, and 2) that co-administration of a known reversal agent (naloxone (NAL)) or novel respiratory stimulant (D-cysteine ethyl ester; D-CYSee) can mitigate the acute physiological and behavioral effects of fentanyl. Acute withdrawal was measured for 4 hrs. beginning 90 min post-injection of IV vehicle or fentanyl (n=5-7) using a wearable activity tracking device (Biopac) and subjective videographic observations. Animal movement (Vector Magnitude; g) was lowest with vehicle across all time points but was increased (p<0.05) in a dose-dependent manner from 90-210 min after fentanyl administration. Additional behaviors such as pawing, vocalization, hyperphagia, itching and rearing also appeared dose-dependent and were increased from 90-210 min post-injection but waned across the 4h observation period (n=4). Based on the demonstrated efficacy of D-CYSee in reversing OIRD in rats (Getsy et al., 2022), we next tested if fentanyl-induced physiological dysfunction or withdrawal symptoms were mitigated by co-administration of either D-CYSee (500 μmol/kg) or NAL (0.06 mg/kg). Transient suppression of minute ventilation (VI) and breathing frequency was followed by a secondary increase >10 min after 50 μg/kg fentanyl, where co-administration of D-CYSee stimulated VI and NAL prevented the secondary increase in VI (n=5-6). Fentanyl alone moderately decreased HR where D-CYSee prevented this effect, whereas NAL exacerbated the fentanyl-induced bradycardia for up to 45 min. Mean, systolic and diastolic blood pressures were increased with fentanyl for >90 min post-injection, where D-CYSee had no additional effect but NAL prevented the hypertension. Finally, NAL but not D-CYSee reversed sedation (n=7), but neither NAL or D-CYSee appeared to mitigate withdrawal behaviors. These preliminary data suggest that: 1) fentanyl causes withdrawal behaviors in goats, and 2) NAL and D-CYSee have differential effects on fentanyl-induced physiological dysfunction suggesting D-CYSee has a distinctly different mechanism of action as a potential OIRD countermeasure. Supported by NIH DA050571. This is the full abstract presented at the American Physiology Summit 2023 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.

Synthetic opioids, such as fentanyl, typically suppress breathing frequency (f) and tidal volume (VT) in rodents but decrease and increase f and VT in humans, respectively. In this and the three adjoining posters we present data on the physiologic and behavioral dose-dependent effects of intravenous (IV) fentanyl in adult awake female goats. Here we tested the hypothesis that like humans, goats would show dose-dependent decreases in f and increases in VT and corresponding changes in blood gases and pH. Pulmonary ventilation (VI), f, VT and arterial blood gases were measured for 30 minutes (min) before, during and up to 90 min after IV injection (2 min) of 0, 25, 50, 75, 100, and 125 mcg/kg fentanyl. Ventilatory measures were expressed as a percent of control (room air breathing) in 30 sec intervals for 15 min pre- and post-injection and averaged over longer time intervals (5 or 15 min) thereafter. While we observed no changes in breathing measures over 30 min before and 90 min after saline injection, the responses to IV fentanyl were highly variable and individual- and dose-dependent. However, most goats showed transient decreases in f (20-30%) where the duration of f suppression was dose-dependent (2-10 min), whereas VT increased (25-50%) for 60 min or more without an apparent dose-dependence. As a result, VI was only transiently decreased (2-4 min) and instead was above control for up to 90 min post-injection. Arterial blood was drawn (~3 ml; 30 sec) in duplicate at ~15 and 25 min into the 30 min control period, and then individual samples were obtained continuously during and immediately following the injection and in duplicate at 10, 15, 30, 45, 60, 75 and 90 min post-injection. While some goats showed transient hypoventilation (decreased PaO2/O2 saturation) immediately after fentanyl injection, on average blood gas analyses suggested modest hyperventilation (decreased PaCO2/increased pH) 60-90 min post-injection consistent with the overall effects on VI. The calculated difference in the alveolar to arterial oxygen gradient (A-a gradient) was unaffected with saline or 25 mcg/kg fentanyl, but was increased with higher doses of fentanyl peaking 15-30 min after injection in a dose-dependent manner suggesting increased V/Q mismatch. We conclude that in awake goats, fentanyl has opposite effects on f and VT similar to that in humans. However, individual variation in timing and nature of the ventilatory responses to fentanyl over the dose range tested herein suggests that the compensatory f and VT changes allowed for the maintenance of arterial blood gas and pH status. NIH DA050571 This is the full abstract presented at the American Physiology Summit 2023 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.

Opioid induced respiratory depression (OIRD) occurs when respiratory pump and/or airway muscle activity are reduced ostensibly due to reduced output from the brainstem respiratory control system, resulting in central (CA) and/or obstructive apnea (OA) and reduced breathing frequency. Although less commonly reported in the literature, ORID may also result from an inappropriate activation of accessory muscles leading to the “Wooden Chest Syndrome” (WCS) -when airflow is limited due to simultaneous activation of inspiratory and expiratory pump and/or airway respiratory muscles to decrease chest wall compliance and inappropriately increase airway resistance. This phenomenon is difficult to directly study in humans, but here we present data from our large animal model (adult female goats). We tested the hypothesis that IV fentanyl would lead to transient dysfunction in respiratory accessory muscles indicative of WCS. We surgically instrumented goats (n=7) with electromyographic (EMG) wires in inspiratory (diaphragm; DIA) and expiratory (Transversus Abdominus; TA) pump muscles, as well as airway dilator (Thyropharyngeus; Thy) and constrictor (Genioglossus; GG) muscles. After >2 weeks of surgical recovery, responses to saline or various doses of fentanyl citrate (25-125 mcg/kg; IV) were tested for 30 min before and up to 90 min after injection. As described in an adjacent poster, particularly over the first several minutes after fentanyl injection, CA and OA were observed in some goats. However, disruption of airflow was primarily associated with increased simultaneous activation of pump and airway inspiratory and expiratory muscles resembling WCS. For most muscles, the rate of rise in muscle activity (an index of firing intensity) increased within 2 minutes following fentanyl injection and remained significantly (P<0.05) elevated up to 90 minutes. During WCS periods, airflow was oscillatory or interrupted suggesting transient closure of the airway. In addition, there were time- and dose-dependent effects on frequency, duration, and total activity of the pump and airway muscles. By 60 minutes post-injection, there were periods of transient return of muscle activity and airflow to at or below baseline values. There was no significant effect of saline on muscle activity. Overall, these data suggest that following fentanyl injection in awake goats, there was an increase in firing of pump and airway muscles which ultimately disrupts the coordinated reciprocal activity of inspiratory and expiratory muscles and contributes to OIRD. Supported by NIH DA050571 This is the full abstract presented at the American Physiology Summit 2023 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.

At high doses, synthetic opioids are thought to slow heart rate and reduce mean arterial blood pressures in humans and rodents and could create a life-threatening situation. Herein, we report dose-dependent effects of intravenous (IV) injection of fentanyl on systolic (SYS), diastolic (DIA), and mean (MAP) arterial blood pressure, heart rate (HR) metabolic rate and body temperatures in adult female goats (n=7). Variables were measured 30 min before, during and up to 90 min after IV injection of vehicle (saline) or 25, 50, 75, 100, and 125 mcg/kg fentanyl. Baseline averages for SYS, DIA, MAP was 102, 73, 83 mmHg, respectively, and HR was 84 beats/minute (bpm). There were no significant changes in these values over 90 minutes when saline was injected. Between 8 and 10 minutes after fentanyl injection, SYS, DIA, and MAP increased substantially from control by about 40, 25, and 30 mmHg, respectively. These increases did not appear dose independent, but by 90 minutes post-injection SYS, DIA, and MAP decreased toward control at the lowest dose (25 mcg/kg). The fentanyl induced arterial hypertension was partially compensated by a ~15 bpm decrease in heart rate which changed minimally between 10 and 90 minutes after fentanyl injection. The sustained increase in arterial blood pressure indicates a sustained excitatory effect of fentanyl consistent with the excitatory effect of fentanyl on tidal volume and respiratory muscle activity (see additional abstracts from our group). This excitatory effect is not due to changes in metabolic rate and/or body temperature as we found no consistent effect of fentanyl injections on these variables. This excitatory effect of fentanyl appears species-dependent as opioid agonists injected into rats and humans typically induces arterial hypotension. Species dependency of fentanyl may reflect species variation in effects of fentanyl on vagal nerve endings, brainstem control centers, and/or cardiac receptors (J. Car.Pharm, 1985), or alternatively may reflect differences in fentanyl metabolism whereby metabolites may have differential effects on blood pressure among these species. NIH DA050571 This is the full abstract presented at the American Physiology Summit 2023 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.

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, severity) in determining an individual's ability to tolerate hypercapnic exacerbations is unknown. Our primary objective herein was to test the hypothesis that mild-to-moderate CH (PaCO2 ~50-70mmHg) increases susceptibility to pathophysiologic responses to severe acute CO2 challenges. Three groups (GR) of adult female goats were studied during 14d exposure to room air (GR1; control) or 6% inspired CO2 (GR2; mild CH), or 7d of 6% InCO2 followed by 7d of 8% InCO2 (GR3; moderate CH). Consistent with previous reports, mild CH led to transient increases in steady state ventilation, a transient suppression of CO2/[H+] chemosensitivity, and there were no changes in physiologic parameters in GR1 (control). Further increasing InCO2 from 6% to 8% also further increased steady state PaCO2 and ventilation to levels greater than predicted like that during mild CH. 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 blunts physiological responses to acute severe exacerbations and provide evidence of recruitment of extra-pulmonary systems (i.e. gastric CO2 elimination) during times of moderate-severe hypercapnia.

CO2 retention (hypercapnia, CH) presents in a number of etiologies including sedative use, chronic lung disease, and central/peripheral neuromuscular disorders. Acute‐on‐chronic hypercapnia can develop in these disorders during times of acute stress (i.e., infection and postoperative states). Recurrent acute‐on‐chronic CH may lead to respiratory failure and transient, life‐threatening worsening of the hypercapnia. We previously established in awake goats that exposure to mild CH (PaCO2 ~55mmHg) elicits changes across multiple physiologic systems but does not impair the response to acute increases in inspired CO2(InCO2).Herein, we test the hypothesis that progressive exposure from mild‐to‐moderate CH (~55‐to‐65mmHg CO2) results in secondary adaptations across several physiologic systems but increases the risk of pathological responses during acute‐on‐chronic exacerbations. Utilizing our established large animal model of CH, female goats (n=7) were exposed to 7 days (d) of 6% InCO2 followed by 7d of 8% InCO2. Throughout the protocol, we regularly assessed steady‐state cardiorespiratory function (i.e., VI, f, VT, HR, and BP) and acute CO2/H+ chemosensitivity. During 6% InCO2, the temporal pattern of physiologic adaptations was consistent with that previously reported (J. Physiol. 2018). As expected, increasing steady‐state (SS) InCO2 resulted in a sustained 10 mmHg increase in PaCO2 and an increase in arterial [H+] that decreased over the 7d at 6%. During 6% InCO2 there was a transient decrease (6%‐Day 2) in acute chemosensitivity (DVI/DCO2) that returned to control values by Day 5 at 6%. Upon increasing SS InCO2from 6% to 8%, minute ventilation (VI) increased from 23±3.4L/min on Day 7 at 6% InCO2 to 34±2.2L/min on Day 1 at 8% InCO2,which was primarily driven by an increase in breathing frequency. By 24h at 8% InCO2, VIhad declined from the 8%‐Day 1 values and remained at or near that level thereafter. This temporal pattern of adaptation is comparable to that observed during 6% CO2. A similar pattern of adaptation in [H+] occurred during 8% as occurred at 6%, while [HCO3‐] continually increased. PaCO2remained elevated throughout the 8% InCO2 exposure, compared to 6% InCO2. Finally, when SS InCO2 was increased to 8% the acute chemoreflex remained suppressed throughout the 7d. Our results suggest that progressive worsening of chronic hypercapnia results in similar SS adaptations compared to initial hypercapnic exposure. However, more severe levels of CH, such as 8% InCO2, perturb the acute chemoreflex, creating a risk for consequences such as CO2 narcosis and cardiorespiratory failure in patients with pre‐existing CH.

A hallmark of respiratory-related diseases, such as chronic obstructive pulmonary disease (COPD), is chronic hypercapnia (CH). Common therapeutic intervention for patients experiencing CH is mechanical ventilation to restore blood-gas homeostasis. While many neurophysiological consequences of CH have been characterized, little is known about the effects reversing CH. The primary goal of the present study was to test the hypothesis that abrupt deacclimatization from CH would significantly alter markers of neuroplasticity and tryptophan metabolism, but not levels of excitatory neuromodulators. Utilizing our goat model of increased inspired CO2 (InCO2)-induced CH, adult female goats were exposed to an InCO2 of 6% for 30 days (d) followed by a return to room air for 24 hours (h). Following 24h of recovery, goats were euthanized, and brainstems were rapidly extracted. Tissue punches of key nuclei throughout the brainstem respiratory network (hypoglossal motor nucleus (XII), nucleus tractus solitarius (NTS)/dorsal motor nucleus of the vagus (DMV), ventral respiratory column (VRC), medullary raphe (MR), ventrolateral medulla (VLM), retrotrapezoid nucleus (RTN), and cuneate nucleus (CN)) were obtained and used for either western blot or HPLC analysis. Changes in glutamatergic signaling, neuroinflammation, tryptophan metabolism, and neuromodulators concentration, following recovery from CH, were assessed. Following 24h of recovery, there were no significant differences in AMPA or NMDA receptor expression or phosphorylation, compared to room air control goats, across all nuclei investigated. Similarly, there were no significant differences in the inflammatory cytokine IL1B following 24h recovery across all nuclei investigated, compared to room air control goats. However, there was significantly (P<0.05) lower expression of key enzymes of tryptophan metabolism (Indolamine 2,3-dioxygenase (IDO); Tryptophan Hydroxylase (TPH) and neuronal markers (NeuN) compared to control within the rostral VLM and MR. Within the solitary complex (NTS & DMV), there was significantly (P>0.05) greater concentrations in norepinephrine (+132%±49), serotonin (+348%±142), and dopamine (+86%±53) at 24h of recovery compared to 30d of CH. We conclude that although specific markers of neuroplasticity and neuroinflammation were unchanged from control during deacclimatization, there were brainstem-site dependent changes in excitatory neuromodulators during deacclimatization from CH. These data suggest that the mechanisms of neuroplasticity during acclimatization to- and deacclimatization from- CH are fundamentally different.

It is well known that opioid analgesics induce life-threatening respiratory depression. A few studies have also documented additional deleterious effects of opioids on multiple physiologic functions. Moreover, there are reports of interindividual variation in susceptibility, magnitude, and duration of physiologic effects of opioids such as fentanyl. Here we tested the dose-dependent effects of intravenous injected fentanyl on physiologic functions in 7 awake, adult goats. Goats were instrumented for recording of multiple cardiorespiratory variables including respiratory pump and airway muscle activity. After a 30-minute control period, 0, 10, 20, or 50 µg/kg fentanyl was infused intravenously. We found that rapid IV infusion of fentanyl over 2 min caused a dose-dependent decrease in minute ventilation due to dramatic suppression of breathing frequency within 5 min after IV injection. These effects were coupled with dose-dependent increases in VT, which represents a more human like ventilatory response to opioids. The major suppression of ventilation occurred in 5 of the 7 goats, whereas in 2 of the goats IV fentanyl injections increased breathing frequency in a dose-dependent manner. This variation in ventilatory response to opioids agrees with findings that in a subset of humans, opioids have an excitatory effect on physiologic functions (Br. J. Anaesth 81, 1998). The suppression of ventilation seen in the majority of goats is transient, lasting for 3-10 minutes post-injection and is accompanied by an increased or normal VT up to 30-90 minutes. Over the initial 10 minutes after injection, there were obstructive and central apneas often exceeding one minute resulting in arterial hypoxemia (PaO2<50 mmHg) and hypercapnia (PaCO2>50mmHg). Heart rate was decreased for up to 15 minutes following 50µg/kg injection, whereas arterial blood pressure was increased for at least 90 minutes post-injection. Particularly over the initial minutes after 50µg/kg, fentanyl infusion decreased the duration but increased the intensity of diaphragm and intercostal muscle activity while increasing tonic activity of abdominal and airway muscles. Infusion of naloxone before or after infusion of fentanyl prevented or reversed the acute and delayed physiologic effects induced by fentanyl. Injection of Naloxone alone had no effect on physiological measures of goats. We conclude that goats have multiple dose- and individual-dependent physiologic responses to systemic infusion of fentanyl. Furthermore, our reversal data suggests the effects of fentanyl in goats are likely mediated by activation of µ-opioid receptors.

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.

Respiratory disturbances, such as chronic obstructive pulmonary disease (COPD), are often associated with chronically elevated arterial CO2 (PaCO2 >45mmHg), which has been linked to an increase risk of all‐cause mortality. Additionally, acute exacerbations of respiratory symptoms may lead to greater levels of PaCO2 that require mechanical intervention to restore PaCO2 to control levels.Many physiological effects of chronic hypercapnia have been previously reported, and include robust adaptations in acid/base, and respiratory control mechanisms. However, the effects of restoring PaCO2 to control levels following chronically elevated PaCO2 are largely unknown. Thus, the goal of my project is to gain insight into the physiological and ventilatory control adaptations during the acclimatization to‐ and deacclimatization from‐chronic hypercapnia. Based on studies focusing on the deacclimatization from chronic hypoxia, I hypothesize that deacclimatization from chronic hypercapnia would not be completed within 24 hours of return to normoxia.To assess the effects of deacclimatization from chronic hypercapnia, I measured the temporal pattern of ventilation, ventilatory CO2/[H+] chemoreflex, and acid‐base status during acclimatization to‐ and deacclimatization from‐chronic exposure to elevated inspired CO2 (InCO2) in goats. Goats were chronically housed in environmental chambers that allowed for modification of ambient CO2 levels. Following a room air control period, goats were exposed to an InCO2 of 6% for 30 days, followed by returned to room air for 24 hours (hrs). Within 1 hour of 6% InCO2, VI increased to 322% above control, but decreased slightly to 293% above control after 24 hours. By Day 2 of exposure, PaCO2 increased 15 Torr and changed minimally thereafter. pH decreased by 0.06 units upon initial exposure but was partially compensated by Day 5 due to an 8mEq/L increase in arterial [HCO3−]. Within 4hrs of deacclimatization, VI decreased to 123% above control, and continued to decrease to near control levels by 24hrs. PaCO2 decreased 13mmHg by 24hrs, while pH increased 0.04 units. The CO2/[H+] chemoreflex was assessed by increasing InCO2 to 3, 5, and 7% at 4 and 24 hours of deacclimatization. The chemoreflex was above control at 4 and 24 hours of deacclimatization. The steady‐state ventilation/[H+] relationship was also slightly elevated at 4 and 24hrs.Taken together, these data suggest that deacclimatization from chronic hypercapnia is nearly complete by 24 hours. Further studies will extend the recovery time to 5 days to see if the deacclimatization is complete. Additional studies will aim to use western blotting to investigate neurological adaptations underlying the deacclimatization processSupport or Funding InformationDepartment of Veteran Affairs