Chemoreflex Responses Research Articles

Chemoreflex sensitization occurs in both male and female rats during recovery from acute lung injury.

Sex-specific patterns in respiratory conditions, such as asthma, COPD, cystic fibrosis, obstructive sleep apnea, and idiopathic pulmonary fibrosis, have been previously documented. Animal models of acute lung injury (ALI) have offered insights into sex differences, with male mice exhibiting distinct lung edema and vascular leakage compared to female mice. Our lab has provided evidence that the chemoreflex is sensitized in male rats during the recovery from bleomycin-induced ALI, but whether sex-based chemoreflex changes occur post-ALI is not known. To bridge this gap, the current study employed the bleomycin-induced ALI animal model to investigate sex-based differences in chemoreflex activation during the recovery from ALI. ALI was induced using a single intra-tracheal instillation of bleomycin (bleo, 2.5mg/Kg) (day 1). Resting respiratory frequency (fR) was measured at 1-2 days pre-bleo, day 7 (D7) post-bleo, and 1 month (1 mth) post-bleo. The chemoreflex responses to hypoxia (10% O2, 0% CO2) and normoxic-hypercapnia (21% O2, 5% CO2) were measured before bleo administration (pre-bleo) and 1 mth post-bleo using whole-body plethysmography. The apnea-hypopnea Index (AHI), post-sigh apneas, and sighs were measured at each time point. There were no significant differences in resting fR between male and female rats at the pre-bleo time point or in the increase in resting fR at D7 post-bleo. At 1 mth post-bleo, the resting fR was partially restored in both sexes but the recovery towards normal ranges of resting fR was significantly lower in male rats. The AHI, post-sigh apneas, and sighs were not different between male and female rats pre-bleo and 1 mth post-bleo. However, at D7 post-bleo, the male rats exhibited a higher AHI than female rats. Both male and female rats exhibited a sensitized chemoreflex in response to hypoxia and normoxic-hypercapnia with no significant differences between sexes. A sex difference in resting ventilatory parameters occurs post ALI with a prolonged increase in resting fR and larger AHI in male rats. On the other hand, we did not find any sex differences in the chemoreflex sensitization that occurs at 1 mth post-bleo. This work contributes to a better understanding of sex-based variations in lung disorders.

Carotid body ablation restores astrocyte/microglia morphology in the NTS and reduces hypertension following long-term intermittent hypoxia

Chronic intermittent hypoxia (CIH) heightened carotid body (CB) chemosensory discharges, which contributes to hypertension development. Previously, we reported that CB ablation significantly reduced blood pressure after 21 days of CIH; however, whether CB-mediated glial cell activation in the caudal Nucleus of the Solitary Tract (cNTS) following long-term CIH contribute to maintain high blood pressure and enhanced ventilatory chemoreflex remains unknown. To test this hypothesis, we exposed male C57BL6 mice to CIH (5% FIO2, 12 times/h, 8 h/day) for 60 days. At 45 days of CIH, CBs were selectively denervated, and animals returned to CIH for 15 more days. At the end of the experimental protocol, we measured arterial blood pressure, hypoxic ventilatory response (HVR) in awake mice and characterized astrocyte and microglia morphological features using Sholl analysis. CIH induces hypertension (MABP: 83.47±1.39 vs. 95.00±2.18 mmHg; Sham vs. CIH), increases the HVR (1.69±0.17 vs. 4.31±0.87 ΔVE/min; Sham vs. CIH), modify cNTS astrocytes phenotype (N° of branches: 13.01±0.66 vs. 11.31±0.52; cable length: 181.00±8.90 vs 148.10±1.48 μm, convex hull: 788.10±37.60 vs. 584.30±23.75 μm3; Sham vs. CIH), and microglia arborization (N° of branches: 196.10±8.35 vs. 376.30±16.80; cable length: 667.4±29.63 vs. 1267±60.47 μm, convex hull: 6177±362 vs. 9721±564 μm3; Sham vs. CIH). Importantly, CB denervation normalized the elevated blood pressure (MABP: 83.47±1.38 mmHg; CIHd), the altered peripheral chemoreflex response (HVR: 1.63±0.43 ΔVE/min; CIHd) and morphological changes in astrocytes (N °of branches: 17.23±0.88, cable length: 231.70±12.87, convex hull: 982.70±55.55 μm3) and microglia located in the cNTS (N° of branches 126.20±13.34; cable length: 126.2±13.3, Convex hull size: 3584±380 μm3; CIHd). Present results suggest that CBs play a crucial role in the maintenance of high arterial pressure and respiratory alterations induced by long-term CIH and may contribute to the formation of a neuroinflammatory niche at the cNTS by modifying glial cell activity. Supported by Fondecyt Grants 1211443 and 1220950. 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.

The Sex-based Differences in Chemoreflex during Recovery from Acute Lung Injury

Introduction- Sex-specific patterns in respiratory conditions, such as asthma, COPD, cystic fibrosis, obstructive sleep apnea, and idiopathic pulmonary fibrosis, have been previously documented. Animal models of acute lung injury (ALI) have offered insights into sex differences, with male mice exhibiting distinct lung edema and vascular leakage compared to female mice. Our lab has provided evidence that the peripheral (or carotid body) chemoreflex is sensitized in male rats during the recovery from bleomycin-induced ALI, but whether sex-based chemoreflex changes occur post-ALI in not known. To bridge this gap, our current study employed the bleomycin-induced ALI animal model to investigate sex-based differences in chemoreflex activation during the recovery of ALI. Methods- ALI was induced using a single intra-tracheal instillation of bleomycin (bleo) (day 1). Resting fR (respiratory frequency) was measured at pre-bleo, day 7 (D7) post-bleo, and 1 month (1 mth) post-bleo time points. The chemoreflex response to hypoxia (10% O2, 0% CO2) and normoxic-hypercapnia (21% O2, 5% CO2) were measured before bleo administration (pre-bleo) time point and 1 month (1 mth) post-bleo using whole-body plethysmography. Apnea-hypopnea Index (AHI), post-sigh apneas, and sighs were measured at each time point. Results- Resting fR: There were no significant differences in resting fR, either at pre-bleo time point or at day 7 (D7) post-bleo between male and female rats. At 1 mth post-bleo, the resting fR was partially restored in both sexes but the recovery towards normal ranges of resting fR was significantly slower in male rats compared to female rats. AHI, post-sigh apneas, and sighs: At the pre-bleo and 1 mth post-bleo time points, there were no significant differences in AHI, post-sigh apneas, and sighs between male and female rats. However, at D7 post-bleo, the male rats exhibited a higher AHI than female rats; Chemoreflex sensitivity: Both male and female rats exhibited a sensitized chemoreflex activation to hypoxia and normoxic-hypercapnia with no significant differences between sexes. Discussion- Our study showed a sex difference in resting ventilatory parameters including resting fR and AHI post ALI. On the other hand, we did not find any sex difference in chemoreflex sensitization at 1 mth post-bleo. This research contributes to a better understanding of sex-based variations in lung disorders. This study was supported by NIH grant R01 HL-152160 and in part, by NIH grants R01 HL-121012 and R01 HL126796. H-JW was also supported by Margaret R. Larson Professorship in Anesthesiology. IZ was partially supported by the Theodore F. Hubbard Foundation. KK was supported by the AHA predoctoral fellowship -AHA award #903872 by the American Heart Association. 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.

Galaninergic and hypercapnia-activated neuronal projections to the ventral respiratory column

In mammals, the ventral respiratory column (VRC) plays a pivotal role in integrating neurochemically diverse inputs from brainstem and forebrain regions to generate respiratory motor patterns. VRC microinjection of the neuropeptide galanin has been reported to dampen carbon dioxide (CO2)-mediated chemoreflex responses. Additionally, we previously demonstrated that galaninergic neurons in the retrotrapezoid nucleus (RTN) are implicated in the adaptive response to hypercapnic stimuli, suggesting a link between RTN neuroplasticity and increased neuronal drive to the VRC. VRC neurons express galanin receptor 1, suggesting potential regulatory action by galanin, however, the precise galaninergic chemoreceptor-VRC circuitry remains to be determined. This study aimed to identify sources of galaninergic input to the VRC that contribute to central respiratory chemoreception. We employed a combination of retrograde neuronal tracing, in situ hybridisation and immunohistochemistry to investigate VRC-projecting neurons that synthesise galanin mRNA. In an additional series of experiments, we used acute hypercapnia exposure (10% CO2, 1 h) and c-Fos immunohistochemistry to ascertain which galaninergic nuclei projecting to the VRC are activated. Our findings reveal that a total of 30 brain nuclei and 51 subnuclei project to the VRC, with 12 of these containing galaninergic neurons, including the RTN. Among these galaninergic populations, only a subset of the RTN neurons (approximately 55%) exhibited activation in response to acute hypercapnia. Our findings highlight that the RTN is the likely source of galaninergic transmission to the VRC in response to hypercapnic stimuli.

Inhibition of Pro-Inflammatory Microglia with Minocycline Improves Cognitive and Sleep-Wake Dysfunction Under Respiratory Stress in a Sporadic Model for Alzheimer's Disease.

Neuroinflammation in Alzheimer's disease (AD) can occur due to excessive activation of microglia in response to the accumulation of amyloid-β peptide (Aβ). Previously, we demonstrated an increased expression of this peptide in the locus coeruleus (LC) in a sporadic model for AD (streptozotocin, STZ; 2 mg/kg, ICV). We hypothesized that the STZ-AD model exhibits neuroinflammation, and treatment with an inhibitor of microglia (minocycline) can reverse the cognitive, respiratory, sleep, and molecular disorders of this model. To evaluate the effect of minocycline treatment in STZ model disorders. We treated control and STZ-treated rats for five days with minocycline (30 mg/kg, IP) and evaluated cognitive performance, chemoreflex response to hypercapnia and hypoxia, and total sleep time. Additionally, quantification of Aβ, microglia analyses, and relative expression of cytokines in the LC were performed. Minocycline treatment improved learning and memory, which was concomitant with a decrease in microglial cell density and re-establishment of morphological changes induced by STZ in the LC region. Minocycline did not reverse the STZ-induced increase in CO2 sensitivity during wakefulness. However, it restored the daytime sleep-wake cycle in STZ-treated animals to the same levels as those observed in control animals. In the LC, levels of A and expression of Il10, Il1b, and Mcp1 mRNA remained unaffected by minocycline, but we found a strong trend of minocycline effect on Tnf- α. Our findings suggest that minocycline effectively reduces microglial recruitment and the inflammatory morphological profile in the LC, while it recovers cognitive performance and restores the sleep-wake pattern impaired by STZ.

Paraventricular nucleus projections to the nucleus tractus solitarii are essential for full expression of hypoxia-induced peripheral chemoreflex responses.

The hypothalamic paraventricular nucleus (PVN) is essential to peripheral chemoreflex neurocircuitry, but the specific efferent pathways utilized are not well defined. The PVN sends dense projections to the nucleus tractus solitarii (nTS), which exhibits neuronal activation following a hypoxic challenge. We hypothesized that nTS-projecting PVN (PVN-nTS) neurons contribute to hypoxia-induced nTS neuronal activation and cardiorespiratory responses. To selectively target PVN-nTS neurons, rats underwent bilateral nTS nanoinjection of retrogradely transported adeno-associated virus (AAV) driving Cre recombinase expression. We then nanoinjected into PVN AAVs driving Cre-dependent expression of Gq or Gi designer receptors exclusively activated by designer drugs (DREADDs) to test the degree that selective activation or inhibition, respectively, of the PVN-nTS pathway affects the hypoxic ventilatory response (HVR) of conscious rats. We used immunohistochemistry for Fos and extracellular recordings to examine how DREADD activation influences PVN-nTS neuronal activation by hypoxia. Pathway activation enhanced the HVR at moderate hypoxic intensities and increased PVN and nTS Fos immunoreactivity in normoxia and hypoxia. In contrast, PVN-nTS inhibition reduced both the HVR and PVN and nTS neuronal activation following hypoxia. To further confirm selective pathway effects on central cardiorespiratory output, rats underwent hypoxia before and after bilateral nTS nanoinjections of C21 to activate or inhibit PVN-nTS terminals. PVN terminal activation within the nTS enhanced tachycardic, sympathetic and phrenic (PhrNA) nerve activity responses to hypoxia whereas inhibition attenuated hypoxia-induced increases in nTS neuronal action potential discharge and PhrNA. The results demonstrate the PVN-nTS pathway enhances nTS neuronal activation and is necessary for full cardiorespiratory responses to hypoxia. KEY POINTS: The hypothalamic paraventricular nucleus (PVN) contributes to peripheral chemoreflex cardiorespiratory responses, but specific PVN efferent pathways are not known. The nucleus tractus solitarii (nTS) is the first integration site of the peripheral chemoreflex, and the nTS receives dense projections from the PVN. Selective GqDREADD activation of the PVN-nTS pathway was shown to enhance ventilatory responses to hypoxia and activation (Fos immunoreactivity (IR)) of nTS neurons in conscious rats, augmenting the sympathetic and phrenic nerve activity (SSNA and PhrNA) responses to hypoxia in anaesthetized rats. Selective GiDREADD inhibition of PVN-nTS neurons attenuates ventilatory responses, nTS neuronal Fos-IR, action potential discharge and PhrNA responses to hypoxia. These results demonstrate that a projection from the PVN to the nTS is critical for full chemoreflex responses to hypoxia.

A test of the interaction between central and peripheral respiratory chemoreflexes in humans.

How central and peripheral chemoreceptor drives to breathe interact in humans remains contentious. We measured the peripheral chemoreflex sensitivity to hypoxia (PChS) at various isocapnic CO2 tensions ( ) to determine the form of the relationship between PChS and central . Twenty participants (10F) completed three repetitions of modified rebreathing tests with end-tidal ( ) clamped at 150, 70, 60 and 45mmHg. End-tidal ( ), , ventilation ( E ) and calculated oxygen saturation (SC O2 ) were measured breath-by-breath by gas-analyser and pneumotach. The E - relationship of repeat-trials were linear-interpolated, combined, averaged into 1mmHg bins, and fitted with a double-linear function ( E S, L min-1 mmHg-1 ). PChS was computed at intervals of 1mmHg of as follows: the difference in E between the three hypoxic profiles and the hyperoxic profile (∆ E ) was calculated; three ∆ E values were plotted against corresponding SC O2 ; and linear regression determined PChS (Lmin-1 mmHg-1 %SC O2 -1 ). These processing steps were repeated at each to produce the PChS vs. isocapnic relationship. These were fitted with linear and polynomial functions, and Akaike information criterion identified the best-fit model. One-way repeated measures analysis of variance assessed between-condition differences. E S increased (P<0.0001) with isoxic from 3.7±1.5 L min-1 mmHg-1 at 150mmHg to 4.4±1.8, 5.0±1.6 and 6.0±2.2 Lmin-1 mmHg-1 at 70, 60 and 45mmHg, respectively. Mean SC O2 fell progressively (99.3±0%, 93.7±0.1%, 90.4±0.1% and 80.5±0.1%; P<0.0001). In all individuals, PChS increased with , and this relationship was best described by a linear model in 75%. Despite increasing central chemoreflex activation, PChS increased linearly with indicative of an additive central-peripheral chemoreflex response. KEY POINTS: How central and peripheral chemoreceptor drives to breathe interact in humans remains contentious. We measured peripheral chemoreflex sensitivity to hypoxia (PChS) at various isocapnic carbon dioxide tensions ( ) to determine the form of the relationship between PChS and central . Participants performed three repetitions of modified rebreathing with end-tidal fixed at 150, 70, 60 and 45mmHg. PChS was computed at intervals of 1mmHg of end-tidal ( ) as follows: the difference in E between the three hypoxic profiles and the hyperoxic profile (∆ E ) was calculated; three ∆ E values were plotted against corresponding calculated oxygen saturation (SC O2 ); and linear regression determined PChS (Lmin-1 mmHg-1 %SC O2 -1 ). In all individuals, PChS increased with , and this relationship was best described by a linear (rather than polynomial) model in 15 of 20. Most participants did not exhibit a hypo- or hyper-additive effect of central chemoreceptors on the peripheral chemoreflex indicating that the interaction was additive.

DIFFERENCES IN SYMPATHETIC OUTFLOW PROFILES AMONG CANDIDATES TO RENAL DENERVATION

Objective: Renal denervation (RDN) is increasingly used to reduce sympathetic outflow and improve blood pressure (BP) control in patients with resistant or difficult-to-treat arterial hypertension. However, the BP response to the procedure is variable, and factors influencing this variability remain largely unknown. Sympathetic outflow recorded before the procedure might predict the BP response to RDN, although results from different studies remain conflicting. These discrepancies might depend on the limited characterization of the sympathetic outflow before RDN, making its better definition an important goal to refine the patient selection. Design and method: We prospectively enrolled patients with difficult-to-treat arterial hypertension undergoing RDN. Patients underwent an extensive clinical evaluation, and the muscle sympathetic nerve activity (MSNA) in resting conditions and during respiratory maneuvers (controlled inspiratory apneas, evaluation of chemoreflex sensitivity to hypoxia and hypercapnia with the rebreathing technique) was recorded through microneurography. Sympathetic burst frequency, incidence, amplitude, duration and integral, as well as inter-burst interval, were calculated with a semi-automated in-house software. Results: Table 1 reports the clinical characteristics of the 10 patients that were enrolled and underwent MSNA recording before RDN. Only in 6 patients the MSNA signal was clearly interpretable and, of these, 5 subjects (Figure 1) completed the whole battery of the respiratory stimuli (2 patients couldn’t perform chemoreflex testing due to panic attack and loss of neural signal). Apnea increased the burst frequency and incidence in all patients, although the magnitude of the changes from the resting acquisition was variable. The MSNA responses to the other respiratory maneuvers were jeopardized. Burst duration remained stable, whereas an increased MSNA could be observed (subjects 1 and 3) in terms of risen burst integral and reduced inter-burst interval compared to baseline. The sympathetic response to inspiratory apneas seems mediated by chemoreflex responses in these patients. The other subjects did not show such variations. Conclusions: Respiratory maneuvers are feasible during MSNA acquisitions and unveil different patterns of sympathetic responses in patients with hypertension undergoing RDN. Such differences might be missed at rest but could represent novel predictors of the BP response to the procedure.

Effect of Central Chemoreceptors on the Peripheral Chemoreflex Response to Hypoxia

In the absence of central chemoreceptor input, the slope of the hypoxic ventilatory response (HVR) to progressive oxyhemoglobin desaturation (L·min-1·mmHg-1·%SaO2-1) – a measure of peripheral chemoreflex sensitivity – increases linearly with carbon dioxide tension (PCO2). We assessed whether this relationship remains linear with increasing central PCO2 in humans. Over four days, 20 participants (10 females; mean±SD age: 24±4 years) completed three repetitions of modified rebreathing with end-tidal PO2 (PETO2) clamped at 150, 70, 60, and 45 mmHg. End-tidal PCO2 (PETCO2), PETO2, ventilation (V̇E), and SaO2 were measured breath-by-breath by dual gas analyser, pneumotach, and pulse oximetry. The V̇E vs PETCO2 relationship in the hyperoxic test gave the central chemoreflex response to PCO2 and the hypoxic tests provided a combined central-peripheral chemoreflex response. The V̇E vs PETCO2 relationship of repeated trials were linear-interpolated, combined, averaged into 1 mmHg bins, and fit with a linear function (V̇ES, L·min-1·mmHg-1) to provide hyperoxic and hypoxic profiles. HVR was computed at 1 mmHg intervals of PETCO2 as follows: the difference in V̇E between the three hypoxic profiles and the hyperoxic profile (ΔV̇E) was calculated; these three ΔV̇E values were plotted against their corresponding SaO2; and linear regression determined HVR (L·min-1·mmHg-1·%SaO2-1). Steps were repeated at each PETCO2 to produce the HVR vs PCO2 relationship. Each participant’s HVR vs PETCO2 relationship was fit with a linear and polynomial function and Akaike Information Criterion identified the best-fit model. One-way repeated measures analysis of variance assessed between-condition differences. V̇ES rose (p&lt;0.001) with isoxic PO2 from 3.7±1.5 L·min-1·mmHg-1 at 150 mmHg to 4.4±1.8, 5.0±1.6, and 6.0±2.2 L·min-1·mmHg-1 at 70, 60, and 45 mmHg, respectively. Mean SaO2 fell progressively (99.3%, 93.7%, 90.4%, and 80.5% for 150, 70, 60, and 45 mmHg, respectively; p&lt;0.001). In all individuals, HVR increased with PETCO2 and this relationship was best described by a linear model in 75% of participants. In most participants, the peripheral chemoreflex-mediated HVR maintained a linear relationship with PETCO2 despite increasing central chemoreflex activation. Data suggest that central chemoreceptors did not alter the peripheral chemoreflex response to low O2. Natural Sciences and Engineering Research Council of Canada 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.

The Role of Superior Cervical Ganglion in the Chronic Sensitization of Carotid Bodies Post-Acute Lung Injury

Background/hypothesis of the study: Acute lung injury (ALI) increases respiratory rate (RR), induces hypoxemia. This stimulates carotid body (CB) chemoreflex to maintain oxygen homeostasis. Our previous study indicates a sensitized chemoreflex during the recovery of ALI. Chronic chemoreflex hyperactivity negatively affects the overall quality of life. The neural mechanisms underlying the sensitized CB chemoreflex during the recovery from ALI remain a gap in knowledge. Electrical stimulation of the superior cervical ganglion (SCG) sensitizes the CB chemoreflex in rats. We hypothesized that the SCG causes the CB chemoreflex sensitization post-ALI. Methods and Results: We performed a bilateral SCG ganglionectomy (SCGx) or sham-SCGx (Sx) in our male SD rats 2 weeks before inducing ALI (W-2). After 2 weeks of recovery, ALI was induced using a single intra-tracheal instillation of bleomycin (day 1) and measured the resting respiratory variables- RR, TV (Tidal Volume), and MV (Minute Ventilation). The chemoreflex response to 10% hypoxia and 5% normoxic hypercapnia were measured on W(-3) (pre-SCGx), W0 (post-SCGx/pre-ALI) and W4 (post-ALI) using whole-body plethysmography (WBP). Ganglionectomy did not alter the resting lung parameters and the chemoreflex response to either gas challenge in normal, healthy rats. An increase in resting RR at W1 post-ALI was not significantly different between Sx and SCGx rats. At W4 post-ALI, there were no significant differences in resting lung parameters between Sx and SCGx rats. Consistent with our previous study, the CB chemoreflex response to hypoxia and normoxic hypercapnia in Sx rats at W4 post-ALI was sensitized (p&lt;0.01). However, the chemoreflex sensitivity significantly lowered in SCGx rats in response to hypoxia (p=0.01) and normoxic-hypercapnia (p=0.002). Conclusion(s): The CB chemoreflex sensitization during the recovery from the ALI may be mediated by the SCG. Further understanding of the underlying mechanism will provide important information to develop therapeutic approaches to pulmonary disease to improve clinical outcomes. This study was supported by: NIH Grant- RO1 HL-152160 and RO1 HL-126796; Lieberman Grant 2021- Dept. of Anesthesiology, UNMC; American Heart Association- Pre-doctoral fellowship, AHA. 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.

Identifying the building blocks for learning and memory in the carotid body

Glutamate is a major excitatory neurotransmitter with well-established roles for inducing neural plasticity, learning and memory in the central nervous system. In the carotid body, an organ derived in part from the neural crest, evidence suggests glutamatergic signalling is involved in its sensing of hypoxia. We hypothesized that altered glutamatergic signalling may underpin enhanced chemoreflex responses in carotid body in disease states. Therefore, we have initiated an investigation to assess the presence and location of glutamate receptors expressed in the carotid body of Wistar rats. We mined two independent RNA-sequencing (RNAseq) datasets to identify glutamate signalling components. Digital droplet PCR (ddPCR) was performed to validate the expression of selected glutamatergic target genes. Protein localisation was detailed using immunofluorescence and confocal microscopy in 12–16-week-old normotensive male Wistar rats. Neuronal phenotypes were identified using tyrosine hydroxylase (type I or glomus cells) and glial fibrillary acidic protein antibodies (type II cells). In carotid body extracts, RNAseq data revealed the presence of novel glutamatergic signalling components, not previously described. ddPCR analysis confirmed the expression of AMPA, NMDA and excitatory amino acid transporters. Glutamate ionotropic receptor AMPA type subunit 1 ( Gria1) showed co-localisation with the membrane of type I glomus cells. Glutamate ionotropic receptor NMDA type 1 ( Grin1) showed co-localisation with the nuclei membrane [JP1] of glomus cells. NMDA receptor type subunit 2C ( GRIN2C) showed co-localisation with the membranes of both glomus cells and type II cells. Expression of these receptors was not found on all glomus cells within a cluster suggesting functional heterogeneity within the carotid body. Synaptic Ras GTPase-activating protein 1 ( Syngap), a protein that interacts with glutamate synapses involved in learning, was expressed in both plasma and nuclear membranes of glomus cells. The excitatory amino acid transporter 5 ( EAAT5) showed co-localisation with both type I and type II cells. Additionally, we have observed that a local arterial injection of glutamate (via the internal carotid artery) evoked excitatory activity, followed by an inhibitory period as recorded from the carotid sinus nerve. Our results identified expression and location of glutamatergic AMPA and NMDA receptors in the carotid body[JP2]. We also found the presence of excitatory amino acid transporters, and proteins involved in chaperoning glutamate receptors for learning and memory, All told, we have identified the substrate for glutamate as a transmitter in the carotid body. Whether it plays a role in the learning and memory of carotid body function and/or sensitisation in disease remains to be elucidated. Funding: Health Research Council of New Zealand. Nothing to disclose. 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.

Perinatal ethanol exposure alters respiratory chemoreflex responses in anesthetized rats

In the USA 10-15% of pregnant women drink alcohol (ethanol) leading to a variable compilation of developmental abnormalities, including deficits in critical homeostatic functions such as poor coordination of suckling, breathing, and swallowing. Here, we test the hypothesis that perinatal ethanol exposure alters respiratory chemoreflex responses in neonatal rats. Pregnant rats were exposed to 5% ethanol through their drinking water starting on embryonic day 5, which exposes the fetuses to ethanol during gestation and then after birth through the breast milk. Pups of both sexes were studied in the first week of life (postnatal days (P) 1-5). First, in awake pups, we used head-out plethysmography to assess the effects of ethanol exposure on the respiratory response to a brief, 5- minute episode of hypoxic hypercapnia (12% O2/5% CO2, balance N2, delivered through a nose cone). We found no significant differences in the frequency (P = 0.6667), tidal volume (P = 0.3627), or minute ventilation (P = 0.1582) responses to the challenge between control and ethanol exposed pups (2-way ANOVA, n = 6 control and 6 ethanol pups). In a second experiment, we used electromyography in anesthetized pups to study the diaphragm and tongue muscle responses to nasal occlusion, which results in strong muscle efforts but an absence of lung inflation, as well as hypoxia, hypercapnia, and acidosis. Here, we found that both diaphragm and tongue muscle EMG responses were significantly blunted in ethanol exposed animals compared to control. This included a decrease in the peak amplitude responses of both muscles (2-way ANOVA: diaphragm, P = 0.0175; tongue, P= 0.0317, n = 8 control and 8 ethanol pups), as well as a delay in the onset of the tongue muscle activation (Student’s unpaired T-test, P = 0.0219, n = 8 control and 8 ethanol pups). Together, these data indicate that perinatal ethanol exposure causes impairments in respiratory chemoreflex control, which appear to be highly dependent on level of arousal. NIH 5R01HD071302-08 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.

Central and peripheral chemoreflexes in humans with treated hypertension.

Increased peripheral chemoreflex sensitivity is a pathogenic feature of human hypertension (HTN), while both central and peripheral chemoreflex sensitivities are reportedly augmented in animal models of HTN. Herein, we tested the hypothesis that both central and combined central and peripheral chemoreflex sensitivities are augmented in HTN. Fifteen HTN participants (68±5years; mean±SD) and 13 normotensives (NT; 65±6years) performed two modified rebreathing protocols in which the partial pressure of end-tidal carbon dioxide ( ) progressively increased while the partial pressure of end-tidal oxygen was clamped at either 150mmHg (isoxic hyperoxia; central chemoreflex activation) or 50mmHg (isoxic hypoxia; combined central and peripheral chemoreflex activation). Ventilation ( ; pneumotachometer) and muscle sympathetic nerve activity (MSNA; microneurography) were recorded, and ventilatory ( vs. slope) and sympathetic (MSNA vs. slope) chemoreflex sensitivities and recruitment thresholds (breakpoint) were calculated. Global cerebral blood flow (gCBF; duplex Doppler) was measured, and the association with chemoreflex responses was examined. Central ventilatory and sympathetic chemoreflex sensitivities were greater in HTN than NT (2.48±1.33 vs. 1.58±0.42Lmin-1 mmHg-1 , P=0.030: 3.32±1.90 vs. 1.77±0.62a.u.mmHg-1 , P=0.034, respectively), while recruitment thresholds were not different between groups. HTN and NT had similar combined central and peripheral ventilatory and sympathetic chemoreflex sensitivities and recruitment thresholds. A lower gCBF was associated with an earlier recruitment threshold for (R2 =0.666, P<0.0001) and MSNA (R2 =0.698, P=0.004) during isoxic hyperoxic rebreathing. These findings indicate that central ventilatory and sympathetic chemoreflex sensitivities are augmented in human HTN and perhaps suggest that targeting the central chemoreflex may help some forms of HTN. KEY POINTS: In human hypertension (HTN) increased peripheral chemoreflex sensitivity has been identified as a pathogenic feature, and in animal models of HTN, both central and peripheral chemoreflex sensitivities are reportedly augmented. In this study, the hypothesis was tested that both central and combined central and peripheral chemoreflex sensitivities are augmented in human HTN. We observed that both central ventilatory and sympathetic chemoreflex sensitivities were augmented in HTN compared to age-matched normotensive controls, but no difference was found in the combined central and peripheral ventilatory and sympathetic chemoreflex sensitivities. During central chemoreflex activation, the ventilatory and sympathetic recruitment thresholds were lower in those with lower total cerebral blood flow. These results indicate a potential contributory role of the central chemoreceptors in the pathogenesis of human HTN and support the possibility that therapeutic targeting of the central chemoreflex may help some forms of HTN.

The superior cervical ganglion is involved in chronic chemoreflex sensitization during recovery from acute lung injury.

Introduction: Acute lung injury (ALI) initiates an inflammatory cascade that impairs gas exchange, induces hypoxemia, and causes an increase in respiratory rate (fR). This stimulates the carotid body (CB) chemoreflex, a fundamental protective reflex that maintains oxygen homeostasis. Our previous study indicated that the chemoreflex is sensitized during the recovery from ALI. The superior cervical ganglion (SCG) is known to innervate the CB, and its electrical stimulation has been shown to significantly sensitize the chemoreflex in hypertensive and normotensive rats. We hypothesized that the SCG is involved in the chemoreflex sensitization post-ALI. Methods: We performed a bilateral SCG ganglionectomy (SCGx) or sham-SCGx (Sx) in male Sprague Dawley rats 2weeks before inducing ALI (Week -2 i.e., W-2). ALI was induced using a single intra-tracheal instillation of bleomycin (bleo) (day 1). Resting-fR, Vt (Tidal Volume), and V̇ E (Minute Ventilation) were measured. The chemoreflex response to hypoxia (10% O2, 0% CO2) and normoxic-hypercapnia (21% O2, 5% CO2) were measured before surgery on W (-3), before bleo administration on W0 and on W4 post-bleo using whole-body plethysmography (WBP). Results: SCGx did not affect resting fR, Vt and V̇E as well as the chemoreflex responses to hypoxia and normoxic hypercapnia in either group prior to bleo. There was no significant difference in ALI-induced increase in resting fR between Sx and SCGx rats at W1 post-bleo. At W4 post-bleo, there were no significant differences in resting fR, Vt, and V̇E between Sx and SCGx rats. Consistent with our previous study, we observed a sensitized chemoreflex (delta fR) in response to hypoxia and normoxic hypercapnia in Sx rats at W4 post-bleo. However, at the same time, compared to Sx rats, the chemoreflex sensitivity was significantly less in SCGx rats in response to either hypoxia or normoxic hypercapnia. Discussion: These data suggest that SCG is involved in the chemoreflex sensitization during ALI recovery. Further understanding of the underlying mechanism will provide important information for the long-term goal of developing novel targeted therapeutic approaches to pulmonary diseases to improve clinical outcomes.

Role of Peripheral Chemoreceptors on Enhanced Central Chemoreflex Drive in Nonischemic Heart Failure.

Heart failure (HF) is a prevalent disease in elderly population. Potentiation of the ventilatory chemoreflex drive plays a pivotal role in disease progression, at least in part, through their contribution to the generation/maintenance of breathing disorders. Peripheral and central chemoreflexes are mainly regulated by carotid body (CB) and the retrotrapezoid nuclei (RTN), respectively. Recent evidence showed an enhanced central chemoreflex drive in rats with nonischemic HF along with breathing disorders. Importantly, increase activity from RTN chemoreceptors contribute to the potentiation of central chemoreflex response to hypercapnia. The precise mechanism driving RTN potentiation in HF is still elusive. Since interdependency of RTN and CB chemoreceptors has been described, we hypothesized that CB afferent activity is required to increase RTN chemosensitivity in the setting of HF. Accordingly, we studied central/peripheral chemoreflex drive and breathing disorders in HF rats with and without functional CBs (CB denervation). We found that CB afferent activity was required to increase central chemoreflex drive in HF. Indeed, CB denervation restored normal central chemoreflex drive and reduced the incidence of apneas by twofold. Our results support the notion that CB afferent activity plays an important role in central chemoreflex potentiation in rats with HF.

Carotid Body-Mediated Chemoreflex Function in Aging and the Role of Receptor-Interacting Protein Kinase.

Ventilatory impairment during aging has been linked to carotid body (CB) dysfunction. Anatomical/morphological studies evidenced CB degeneration and reductions in the number of CB chemoreceptor cells during aging. The mechanism(s) related to CB degeneration in aging remains elusive. Programmed cell death encompasses both apoptosis and necroptosis. Interestingly, necroptosis can be driven by molecular pathways related to low-grade inflammation, one hallmark of the aging process. Accordingly, we hypothesized that necrotic cell death dependent on receptor-interacting protein kinase-3 (RIPK3) may contribute, at least in part, to impair CB function during aging. Adult (3months) and aged (24months) wild type (WT) and RIPK3-/- mice were used to study chemoreflex function. Aging results in significant reductions in both the hypoxic (HVR) and hypercapnic ventilatory responses (HCVR). Adult RIPK3-/- mice showed normal HVR and HCVR compared to adult WT mice. Remarkable, aged RIPK3-/- mice displayed no reductions in HVR nor in HCVR. Indeed, chemoreflex responses obtained in aged RIPK3-/- KO mice were undistinguishable from the ones obtained in adult WT mice. Lastly, we found high prevalence of breathing disorders during aging and this was absent in aged RIPK3-/- mice. Together our results support a role for RIPK3-mediated necroptosis in CB dysfunction during aging.

The HMOX2 polymorphism contributes to the carotid body chemoreflex in European sea-level residents by regulating hypoxic ventilatory responses.

This study investigates whether a functional single nucleotide polymorphism of HMOX2 (heme oxygenase-2) (rs4786504 T>C) is involved in individual chemosensitivity to acute hypoxia, as assessed by ventilatory responses, in European individuals. These responses were obtained at rest and during submaximal exercise, using a standardized and validated protocol for exposure to acute normobaric hypoxia. Carriers of the ancestral T allele (n = 44) have significantly lower resting and exercise hypoxic ventilatory responses than C/C homozygous carriers (n = 40). In the literature, a hypoxic ventilatory response threshold to exercise has been identified as an independent predictor of severe high altitude-illness (SHAI). Our study shows that carriers of the T allele have a higher risk of SHAI than carriers of the mutated C/C genotype. Secondarily, we were also interested in COMT (rs4680 G > A) polymorphism, which may be indirectly involved in the chemoreflex response through modulation of autonomic nervous system activity. Significant differences are present between COMT genotypes for oxygen saturation and ventilatory responses to hypoxia at rest. In conclusion, this study adds information on genetic factors involved in individual vulnerability to acute hypoxia and supports the critical role of the ≪ O2 sensor ≫ - heme oxygenase-2 - in the chemosensitivity of carotid bodies in Humans.

Nucleus Tractus Solitarius Neurons Activated by Hypercapnia and Hypoxia Lack Mu Opioid Receptor Expression.

Impaired chemoreflex responses are a central feature of opioid-induced respiratory depression, however, the mechanism through which mu opioid receptor agonists lead to diminished chemoreflexes is not fully understood. One brainstem structure involved in opioid-induced impairment of chemoreflexes is the nucleus of the solitary tract (NTS), which contains a population of neurons that express mu opioid receptors. Here, we tested whether caudal NTS neurons activated during the chemoreflex challenge express mu opioid receptors and overlap with neurons activated by opioids. Using genetic labeling of mu opioid receptor-expressing neurons and cFos immunohistochemistry as a proxy for neuronal activation, we examined the distribution of activated NTS neurons following hypercapnia, hypoxia, and morphine administration. The main finding was that hypoxia and hypercapnia primarily activated NTS neurons that did not express mu opioid receptors. Furthermore, concurrent administration of morphine with hypercapnia induced cFos expression in non-overlapping populations of neurons. Together these results suggest an indirect effect of opioids within the NTS, which could be mediated through mu opioid receptors on afferents and/or inhibitory interneurons.

Generation, breakdown and responses of ATP in the carotid body

Purinergic signaling involving adenosine triphosphate (ATP) acting on P2X2/3 receptors modulates physiological carotid body (CB) afferent discharge and chemoreflex activation however, the upregulation of P2X3 receptors on petrosal sensory neurons is partly responsible for aberrant CB tonicity and hyperreflexia, and the increase in blood pressure in the Spontaneously Hypertensive rat (SHR). The enhanced chemoreflex occurs prior to the onset of hypertension and elevated CB activity is thought to contribute to the underlying pathophysiology of the disease but the exact mechanisms remain unclear. We hypothesize that there is greater release of ATP and/or decreased ATP breakdown in the pre‐hypertensive SHR CB compared with that in the Wistar rat, and this evokes an increase in the chemoreflex response and subsequent activation of the sympathetic nervous system. We performed three studies. (i) Using digital droplet PCR (ddPCR), we measured mRNA expression levels of enzymes involved in the breakdown of ATP in CBs extracted from male 4‐week‐old prehypertensive SHR and Wistar rats; these included: ectonucleotide pyrophosphatase/phosphodiesterase 1‐3 (Enpp1‐3), ectonucleoside triphosphate diphosphohydrolase 2‐3 (Entpd2‐3), and ecto‐5’‐nucleotidase (Nt5e). (ii) We quantified ATP release from CBs in vitro during baseline conditions and during cyanide‐evoked ATP release using a colorimetric assay (213‐579‐1 ‐ Millipore), comparing between male 4‐week‐old pre‐hypertensive SHR and Wistar rats. (iii) In an in situ working heart‐brainstem preparation of 4‐week‐old Wistar rats, simultaneous recordings were made from phrenic (PN), hypoglossal (HN), recurrent laryngeal (RLN), abdominal (ABN), and thoracic sympathetic nerves (tSNA). The peripheral chemoreceptors were stimulated with either potassium cyanide (KCN 20‐100 μL, 0.04%) or ATP at varying concentrations (10‐100 µL, 50 µM ‐ 2 mM) delivered via the internal carotid artery. In whole CB extracts, ddPCR revealed increased Enpp2‐3 and Nt5e expression (p&lt;0.05) in pre‐hypertensive SHRs relative to Wistar rats suggesting greater capacity to break down ATP in the pre‐hypertensive SHR CB. There were no differences in the expressions of Enpp1 or Entpd2‐3 between rat strains. However, the amount of ATP released from the CB in both baseline and during cyanide stimulation was two‐fold greater in the pre‐hypertensive SHR (P&lt;0.05). Stimulation of the CB with KCN evoked hyperpnoea, bradycardia and sympathoexcitation at all doses. In contrast, CB injection of low dose ATP increased PN rate with little effect on tSNA, whereas higher doses induced apnea accompanied by repetitive burst discharge of post‐inspiratory activity in the RLN with tonic elevations in the discharge of both ABN and tSNA. In sum, despite the upregulation of several ATP degrading enzymes in the CBs of the SHR, CB release of ATP is higher in this species. We hypothesize that the increase in ATP release in the SHR CB drives the CB hyperexcitability and that the shift towards an upregulation of ATP degrading enzymes even prior to the development of hypertension is compensatory to oppose the increase in ATP signaling. The dose‐dependent effect of exogenous ATP at supraphysiological levels evoking greater CB‐mediated expiratory and sympathetic outflow in the Wistar rat mimics aspects of pathophysiological CB chemoreflex signaling in the SHR.

Acute and Chronic Effects of Seizures on Cardiorespiratory Control in the SSKcnj16‐/‐Rat

Epilepsy is a common neurological disorder in which 1/3rd of patients experiences repeated seizures, which puts them at greater risk for Sudden Unexpected Death in Epilepsy (SUDEP). The pathophysiological consequences of repeated seizures resulting in SUDEP, including the effects of repeated seizures on cardiorespiratory function, remain unclear. We previously established a rat model (SSKcnj16‐/‐rats) in which 10 days of repeated daily audiogenic generalized tonic‐clonic seizures (GTCSs) lead to ictal apnea, post‐ictal respiratory suppression, blunted chemoreflexes and high spontaneous mortality (PMID: 33232300). Here we tested the hypotheses that a single GTCS is sufficient to blunt ventilatory chemoreflexes and that repeated seizures lead to progressively greater post‐ictal heart rate (HR) suppression and/or increases in blood pressure (BP). In our pilot experiment, a male SSKcnj16‐/‐rat was implanted with a pressure telemeter and placed in a chronic monitoring plethysmograph to measure breathing and BP/HR continuously for 10 days. The rat was exposed to a 10 kHz sound (2 mins) to elicit a seizure once/day for 10 days. We found that audiogenic seizures led to ictal apneas of variable lengths (4‐25 sec) and ictal asystole (~1‐2 sec), followed by a 31% increase in BP and 30% decrease in HR on days 1‐4. On days 9‐10, there was a 45% increase in BP and 50% decrease in HR, suggesting a progressive effect on HR and BP with repeated seizures. Ongoing studies aim to determine if the ventilatory responses to hypoxia (12% O2) or hypercapnia (7% CO2) are affected 1 hr after recovery from a single GTCS. Naïve SSKcnj16‐/‐rats (n=4, female) were subjected to pre‐ and post‐seizure hypercapnic chemoreflex testing. Relative to room air breathing, minute ventilation (expressed as % of control) increased during hypercapnia before (262% ± 12%) and 1 hr post‐seizure (240% ± 21%). However, breathing frequency responses to hypercapnia 1 hr post‐seizure were reduced (126% ± 6%) compared to before (164% ± 14%), and tidal volume increased to a greater amount post‐seizure (192% ± 13%) compared to before (168% ± 7%), suggesting a significant shift away from a frequency‐mediated to a tidal volume‐mediated chemoreflex response in the post‐ictal period. Our preliminary data suggest that like breathing, repeated seizures may have a progressively negative and acute effect on BP and HR, and that solitary seizures are sufficient to fundamentally alter the hypercapnic ventilatory responses in this rat model, which may have high relevance to human SUDEP.