Carotid Sinus Nerve Research Articles

Periodontitis accelerates the onset of hypertension in spontaneously hypertensive rats, while the electrical activation of the carotid sinus nerve delays the beginning of the increase in blood pressure.

We have previously demonstrated that electrical stimulation of the carotid sinus nerve (CSN) protects the development of periodontitis. In the current study, we evaluated whether periodontitis accelerates the onset of hypertension in spontaneously hypertensive rats (SHR); and whether electrical stimulation of the CSN would delay the onset of hypertension. Three-week old SHR were implanted with electrodes around the CSN for electrical stimulation for 13 days. Bilateral ligation of the first molar and oral administration of Porphyromonas gingivalis induced periodontitis. The femoral artery of the SHR was cannulated, and 24 h later, in a conscious state, the blood pressure was recorded. Five-week old sham SHR (subjects without electrical stimulation of the CSN) did not demonstrate hypertension. However, when the SHR were submitted to periodontitis they exhibited hypertension at 5 weeks of age. Nevertheless, the stimulation of the CSN prevented the onset of hypertension. Periodontitis promoted alveolar bone loss in SHR; but, electrical stimulation of the CSN prevented this undesirable outcome following the exposure to periodontitis. Periodontitis accelerated the onset of hypertension in SHR; while the electrical stimulation of the CSN delayed the onset of hypertension.

Low-intensity focused ultrasound combined with microbubbles for non-invasive downregulation of rabbit carotid body activity in the treatment of hypertension.

Targeting the carotid body (CB) is a new approach in treating hypertension. This study investigates the efficacy and safety of ultrasound combined with microbubbles in targeting CB to treat hypertension. Twenty-seven hypertensive rabbits were randomly assigned to three groups: microbubbles only (sham group, n = 11), ultrasound plus microbubbles (LIFU group, n = 11), and bilateral carotid sinus nerve denervation (CSND group, n = 5). Four weeks post-intervention, blood pressure, hypoxic ventilatory response (HVR), blood pressure variability (BPV), heart rate variability (HRV), biochemical indicators, neurohormones, and histopathology were assessed in all groups. The results indicated significant reductions in systolic and diastolic blood pressure in the LIFU and CSND groups post-intervention, along with decreases in BPV, HRV, and catecholamines. HVR results showed a 35.10% reduction in CB activity in the LIFU group compared to the sham group, which was significantly lower than the reduction in the CSND group compared to the sham group (73.85%). Histopathology and transmission electron microscopy confirmed CB damage and cell apoptosis, with immunofluorescence showing a reduction in type I and II cells. In conclusion, LIFU combined with microbubbles can reduce blood pressure by lowering CB and sympathetic nerve activity.

Abstract P388: Electrical Stimulation of the Porcine Carotid Sinus Nerve Reliably Decreases Blood Pressure and Heart Rate

Introduction: Neuromodulation of the baroafferent pathway via direct carotid sinus nerve (CSN) stimulation is recognized as a potential therapeutic modality for treating uncontrolled hypertension. Although proof of concept has been demonstrated acutely in humans, there are many questions that remain in both acute and chronic applications. A translational non-companion large animal model is vital for safe development of this modality. Here we present data on the effects of CSN and baroafferent stimulation in farm pigs. Hypothesis: We hypothesized that an effective CSN-bioelectode interface in the pig would demonstrate significant and reproducible reductions in blood pressure in tandem with pulse rate (PR). Methods: Acute CSN stimulation was investigated in 9 anesthetized Yorkshire pigs. Following general anesthesia, CSNs were exposed and an application-specific electrode was placed. A fixed current paradigm was employed at constant 100Hz and 100µs pulse width. Following contact mapping to identify the baroafferent neurointerface, electrical current was escalated to establish dose-response curves. The primary outcome was systolic blood pressure (SBP) and secondary outcomes were diastolic pressure (DBP) and PR. Data were analyzed in Matlab to extract the baseline BP and PR, during and following stimulation. Statistical calculations used a one-sample Wilcoxon signed rank test. Explanted electrodes with surrounding tissue were evaluated using micro-computed tomography (μCT) and histologically. Results: Six of 9 animals were responders (n=7, 2 sides were stimulated in one animal), CSN stimulation caused average (mean±SD, p<0.001) decreases in SBP (6.8±7.2mmHg) and DBP (3.9±3.7mmHg), which tracked closely with decreases in PR (6.2±5.7bpm). Average maximum decreases were: SBP, 9.3±9.9mmHg; DBP, 7.7±6.4mmHg; and PR, 9.7±8.3bpm. Upon stimulation withdrawal, there was near-immediate recovery of both BP and HR. μCT and histological analysis of tissue samples demonstrated inter-animal anatomic heterogeneity. Conclusions: CSN stimulation causes reproducible reduction in BP synchronous with PR drops followed by near immediate recovery of both with stimulation withdrawal. Variations in animal response may be due to anatomical variation in CSN anatomy that can be overcome with further anatomical study. Pigs provide a robust large-animal model for investigating CSN stimulation, offering a platform to better understand baroafferent stimulation as treatment for uncontrolled hypertension.

Three-Dimensional Ultrastructure of Flower-Spray Nerve Endings in the Rat Carotid Sinus.

The flower-spray nerve endings are afferent nerve terminals in the carotid sinus that arise from carotid sinus nerve of glossopharyngeal nerve. However, the three-dimensional ultrastructural characteristics of flower-spray nerve endings and spatial relationships between the terminal parts and other cellular elements have not been fully understood. To elucidate their detailed relationship, backscattered electron imaging of serial sections was performed with a scanning electron microscope to produce a three-dimensional reconstruction of the flower-spray endings. The terminal parts of flower-spray endings were distributed horizontally approximately 5µm outside the external elastic membrane in the tunica adventitia of the internal carotid artery. The three-dimensional reconstruction showed that the terminal parts of flower-spray endings were flat with irregular contours and were partially covered by the thin cytoplasmic processes of Schwann cells. The complex consisting of the nerve terminals and associated Schwann cells was surrounded by a multilayered basement membrane. The terminal parts of the endings were also surrounded by fibroblasts with elastic fibers and collagen fibrils. Secretory vesicles without an electron-dense core were observed in the terminal parts of the endings. The accumulation of vesicles just below the axonal membrane was observed in terminal parts not covered by Schwann cell cytoplasmic processes on both the luminal and basal sides. Swollen mitochondria, concentric membranous structures, and glycogen granule-like electron-dense materials were often noted in some of the terminal parts of the endings and the parent axon. Collectively, the present results suggest that flower-spray endings are baroreceptors because their morphology was similar to other mechanoreceptors. Furthermore, flower-spray endings may be affected by glutamate secreted in an autocrine manner.

The Accelerator and Brake Modulatory System of Carotid Body Sensitivity

The carotid body (CB) is the primary sensor of arterial gases in the human body that through reflex pathways maintains cardiovascular homeostasis. It is known that CB chemosensitivity increases upon repeated stimulation as well as disease states; however, the molecular basis for this remains elusive. Objectives: To investigate the effect of excitatory and inhibitory amino acids in mediating CB afferent activity. Hypothesis: We hypothesised that glutamate and γ-aminobutyric acid (GABA) act to modulate CB sensitivity. Methods: Electrophysiological recordings of carotid sinus nerve (CSN) afferent discharge were made from an ex vivo arterially perfused common carotid artery bifurcation preparation. Effects of agents on CB sensitivity were assessed from responses evoked by cyanide (CN−) (1.23 μmol bolus). Results: The CSN response to CN− consisted of a rapid high amplitude discharge that was quenched abruptly, leading to a low amplitude tail. Glutamate administration augmented the CSN response to CN− by 2-fold compared to baseline ( p=0.0005). In contrast, GABA administration suppressed the CN− response by 2-fold ( p = 3.71 x 10−5). An N-methyl-D-aspartic acid (NMDA) receptor antagonist (MK801; 100 μM) increased the response amplitude resulting in one single high amplitude discharge (160% increase from baseline), abolishing the low amplitude tail. Such an effect was also observed following GABAA receptor blockade with bicuculline (500 μM), when an 80% increase was observed. The CN− response was blocked by PPADS (a P2X receptor antagonist; 100 μM) (242 ± 154 vs 4.82 ± 2.23 % change from baseline; p=0.03), suggesting it to be solely mediated by purinergic transmission. Summary of the data: We propose that CN-evoked excitation of CB chemosensory cells leads to the co-release of ATP and glutamate. ATP activates directly P2X receptors on petrosal chemosensory afferents, while glutamate acting via NMDA receptors on chemosensory cells leads to activity-induced potentiation of ATP release and co-release of GABA to quench ATP-mediated CSN discharge in a time-controlled manner. This is supported by our finding that blockade of either excitatory (NMDAR) or inhibitory (GABAA) receptors both heighten the CN− evoked CB response. A statement of the conclusions: Our results suggest an intrinsic ‘accelerator and brake’ paracrine mechanism within the CB involving glutamate and GABA controlling CB afferent sensitivity. Research was supported by the Health Research Council of New Zealand and the Sidney Taylor Trust. 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.

A comparative study of carotid body chemosensitivity in lean and obese mice: Implication for obesity induced hypertension

Obesity is associated with increased circulating level of the hormone leptin. Previously we have shown in mice that, leptin induces hypertension acting on transient receptor potential melastatin 7 (Trpm7) channels in the carotid body (CB), a multi-modal peripheral chemoreceptor organ that controls breathing and drives sympathetic tone. Further, in mice leptin augments hypoxic ventilatory response which is abolished by CB denervation. This suggests a potential involvement of CB chemosensitivity. However, no comparative study of CB chemosensory responses has been conducted in lean and obese mice to support this notion. Based on these premises, we hypothesize that obese mice should have higher leptin induced CB chemosensory responses during normoxia and hypoxia compared to lean mice, and Trpm7 receptor antagonist should abolish/reduce these responses. To prove this, we measured afferent carotid sinus nerve (CSN) activity, using a novel ex vivo perfused mouse (male) CB preparation from lean (b.w.; 35 ± 0.5 gm) and diet induced obese (DIO) mice (b.w.; 53 ± 1.5gm) with C57BL/6J background. We found that compared to lean mice; (a) CSN response to hypoxia (PO2 =60 Torr) was 50% more in obese mice, (b) in presence of leptin (50nM), CSN responses to normoxia (PO2=100Torr) and hypoxia were augmented 50% and 150% more respectively in obese mice. Finally, leptin induced increase in CSN responses were partially reduced by Trpm7 antagonist FTY720 (3μM) in obese compared to lean mice. In conclusion, CB chemosensitivity is enhanced in obese compared to lean mice and CB Trpm7 channel could be a therapeutic target for obesity related hypertension. Grant support: NIH R01 HL1331000. 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.

Studies on Membrane Potential and Ca2+ Oscillations in Rat Carotid Body Type I Cells

Carotid body (CB) glomus cells sense changes in arterial O2 pressure (PO2) and pH, and adjust the secretory and carotid sinus nerve activity and ventilation to help maintain normal levels of blood PO2 and pH. Our recent studies showed that isolated CB cell clusters perfused with physiological buffer solution generate spontaneous Ca2+ oscillations at low frequency in normoxia. Mild and moderate levels of hypoxia (2-5%O2) and acidosis (pHo7.2-7.3) increased the frequency and amplitude of Ca2+ oscillations. Inhibitors of voltage-dependent Ca2+ channels and removal of external Ca2+ abolished Ca2+ oscillations, indicating that Ca2+ influx was critical for generating Ca2+ oscillations. To better understand the phenomenon of Ca2+oscillations in glomus cells, we examined the potential role of oscillations in cell membrane potential (Em) in CB cells in triggering Ca2+ influx and Ca2+ oscillations. Using the cell-attached patch configuration, we assessed cell Em by recording TASK single channels, because a linear relationship exists between TASK amplitude and cell Em. Recording of TASK in CB cells in normoxia showed that many CB cells exhibit oscillations in TASK amplitude, indicating the presence of spontaneous oscillations in cell Em. The oscillation frequency of cell Em was similar to that of Ca2+ oscillations. Oscillations in TASK single channel amplitude were blocked by nifedipine (Ca2+ channel antagonist), by removal of extracellular Ca2+, and by 2-APB (an inhibitor of ER Ca2+ channel and store-operated Ca2+ entry). Mild hypoxia increased the frequency of oscillations of TASK amplitude, indicating that mild hypoxia increased the frequency of cell Em oscillations. These findings suggest that cell Em oscillations (that open voltage-dependent Ca2+ channels and increase Ca2+ influx) are an integral component of the cellular signaling mechanism by which Ca2+ oscillations are produced in CB cells. This work was funded by National Institutes of Health (NIH) grants to D.K. (HL111497) and C.W. (HL142906), and an award from Rosalind Franklin University of Medicine and Science. 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.

Mechanisms Underlying Long-Term Facilitation in the Carotid Body

Glutamate and γ-aminobutyric acid (GABA) are major modulators of excitatory and inhibitory transmission in the mammalian brain. Glutamate transmission is critical for neural plasticity, learning and memory, whereas GABA plays a fundamental role in regulating neuronal excitability and facilitating the generation of neural oscillations. Our data indicates that both glutamate and GABA are released in the carotid body (CB) to modulates its response to hypoxia. Objectives: We set out to investigate whether repeated stimulation with glutamate and GABA induces lasting effects on CB afferent discharge. Hypothesis: We hypothesised that glutamate/GABA mediates long-term facilitation (LTF) of CB afferent activity. Methods: We mined high-throughput RNA sequence data and used immunohistochemistry to map components underlying neuroplasticity in the CB in Wistar and Spontaneously-hypertensive rats (SHR). CSN discharge was measured as a functional readout of CB function from an ex vivo arterially perfused carotid artery-CB preparation. Data: We catalogue the expression of glutamate receptors (R) and transporters in the rat and mouse CB. We found N-methyl-D-aspartic acid receptor (NMDAR), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPAR) and transporters to be localised to the CB chemosensory cells. Targeted administration of 15 mM glutamate activated the carotid sinus nerve ( p=0.003). Repetitive [5 times, 2-minute duration, 5-minute intervals] application of 15 mM glutamate evoked LTF of CB afferent discharge as demonstrated by both a 12-fold increase in basal firing frequency (0.78 ± 0.37 vs 1232 ± 596 spikes 1s−1; p = 0.004) over 60 minutes ( p = 0.004) and amplification of the evoked response to chemical hypoxia (CN−; 1.23 μmol bolus) by 2-fold ( p = 0.009). The stimulation paradigm was specific as neither application of glutamate, 5 times for 1-minute at 5-minute intervals, nor a single glutamate exposure evoked LTF. In contrast, repeated application of 15mM GABA [3 times for 1-minute at 5-minute intervals] evoked long-term depression (LTD) of CB afferent activity as seen by a reduced basal discharge from 10.5 to 3.5 spikes 1s−1 ( p = 0.0004) and a 2-fold suppression of the response to chemical hypoxia ( p = 3.71 x 10−5). Summary of the data: Repetitive exposure to glutamate using a specific protocol increased basal discharge of CB afferent output akin to LTF and caused sensitisation of evoked responses to CN−. Repeated exposures of GABA depressed the CB basal discharge akin to LTD. A statement of the conclusions: It is conceivable that processes such as LTP and LTD operate in the CB to modulate the set point of peripheral chemoreceptor sensitivity and its state-dependent tonicity; the role of LTP in peripheral chemoreflex sensitisation in disease remains to be determined. Research was supported by the Health Research Council of New Zealand and the Sidney Taylor Trust. 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.

Bioelectronic modulation of carotid sinus nerve to treat type 2 diabetes: current knowledge and future perspectives.

Bioelectronic medicine are an emerging class of treatments aiming to modulate body nervous activity to correct pathological conditions and restore health. Recently, it was shown that the high frequency electrical neuromodulation of the carotid sinus nerve (CSN), a small branch of the glossopharyngeal nerve that connects the carotid body (CB) to the brain, restores metabolic function in type 2 diabetes (T2D) animal models highlighting its potential as a new therapeutic modality to treat metabolic diseases in humans. In this manuscript, we review the current knowledge supporting the use of neuromodulation of the CSN to treat T2D and discuss the future perspectives for its clinical application. Firstly, we review in a concise manner the role of CB chemoreceptors and of CSN in the pathogenesis of metabolic diseases. Secondly, we describe the findings supporting the potential therapeutic use of the neuromodulation of CSN to treat T2D, as well as the feasibility and reversibility of this approach. A third section is devoted to point up the advances in the neural decoding of CSN activity, in particular in metabolic disease states, that will allow the development of closed-loop approaches to deliver personalized and adjustable treatments with minimal side effects. And finally, we discuss the findings supporting the assessment of CB activity in metabolic disease patients to screen the individuals that will benefit therapeutically from this bioelectronic approach in the future.

The effect of carotid sinus neurectomy for carotid restenosis: a study protocol for a double-blinded and randomized controlled trial

BackgroundPatients undergoing carotid endarterectomy (CEA) have a high restenosis rate, which increases the risk of stroke, and there is still a lack of effective treatment for restenosis. The cause of stenosis is related to local inflammatory reactions. Some basic studies have shown that the inflammatory response causing arterial stenosis is closely related to the nerve axons distributed in its outer membrane, and that removal of the nerve is effective in reducing the inflammatory response to prevent arterial stenosis. Therefore, we propose to design a randomized controlled trial to study whether disconnecting the carotid sinus nerve during a CEA operation can reduce carotid arterial restenosis.Method/designThis study is a randomized, double-blind, single-center study. We will recruit 276 patients, who will be randomly divided into the experimental group and the control group. Based on the standard CEA operation, the operator will search for the carotid sinus nerve on the surface of the internal carotid artery and will entirely transect it in the experimental group. Both groups will be guided with the same postoperative treatment and will be followed up every 3 months for 3 years after the operation. The main indices observed will be the carotid restenosis rate, incidence and nature of carotid plaque, and carotid blood flow velocity. Other indices will be arrhythmia, blood pressure variability, and biomarkers of atherosclerosis, such as blood lipids, hypersensitive C-reactive protein (hs-CRP), homocysteine, and total bilirubin.DiscussionIt is expected that carotid sinus nerve transection will significantly reduce the occurrence of restenosis after CEA, decrease the incidence of ischemic stroke, and realize the effective primary prevention of stroke.Trial registrationChiCTR2300073652. Registered on July 18, 2023.

Carotid body denervation improves hyperglycemia in obese mice.

The carotid bodies (CBs) have been implicated in glucose abnormalities in obesity via elevation of activity of the sympathetic nervous system. Obesity-induced hypertension is mediated by insulin receptor (INSR) signaling and by leptin, which binds to the leptin receptor (LEPRb) in CB and activates transient receptor potential channel subfamily M member 7 (TRPM7). We hypothesize that in mice with diet-induced obesity, hyperglycemia, glucose intolerance, and insulin resistance will be attenuated by the CB denervation (carotid sinus nerve dissection, CSND) and by knockdown of Leprb, Trpm7, and Insr gene expression in CB. In series of experiments in 75 male diet-induced obese (DIO) mice, we performed either CSND (vs. sham) surgeries or shRNA-induced suppression of Leprb, Trpm7, or Insr gene expression in CB, followed by blood pressure telemetry, intraperitoneal glucose tolerance and insulin tolerance tests, and measurements of fasting plasma insulin, leptin, corticosterone, glucagon and free fatty acids (FFAs) levels, hepatic expression of gluconeogenesis enzymes phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G-6-Pase) mRNA and liver glycogen levels. CSND decreased blood pressure, fasting blood glucose levels and improved glucose tolerance without any effect on insulin resistance. CSND did not affect any hormone levels and gluconeogenesis enzymes, but increased liver glycogen level. Genetic knockdown of CB Leprb, Trpm7, and Insr had no effect on glucose metabolism. We conclude that CB contributes to hyperglycemia of obesity, probably by modulation of the glycogen-glucose equilibrium. Diabetogenic effects of obesity on CB in mice do not occur via activation of CB Leprb, Trpm7, and Insr.NEW & NOTEWORTHY This paper provides first evidence that carotid body denervation abolishes hypertension and improves fasting blood glucose levels and glucose tolerance in mice with diet-induced obesity. Furthermore, we have shown that this phenomenon is associated with increased liver glycogen content, whereas insulin sensitivity and enzymes of gluconeogenesis were not affected.

Electrical carotid sinus nerve stimulation attenuates experimental colitis induced by acetic acid in rats

AimsThe carotid bodies are sensors that detect physiological signals and convey them to the central nervous system, where the stimuli are processed inducing reflexes through efferent pathways. Recent studies have demonstrated that electrical stimulation of the carotid sinus nerve (CSN) triggers the anti-inflammatory reflex under different conditions. However, whether this electrical stimulation attenuates colitis was never examined. This study aimed to evaluate if the electrical CSN stimulation attenuates the experimental colitis induced by intrarectal administration of acetic acid in rats. MethodsElectrodes were implanted around the CSN to stimulate the CSN, and a catheter was inserted into the left femoral artery to record the arterial pressure. The observation of hypotensive responses confirmed the effectiveness of the electrical CNS stimulation. This maneuver was followed by a 4 % acetic acid or saline administered intrarectally. After 24 h, colons were segmented into distal and proximal parts for macroscopy, histological and biochemical assessment. Key findingsAs expected, the electrical CSN stimulation was effective in decreasing arterial pressure in saline and colitis rats. Moreover, electrical CSN stimulation effectively reduced colonic tissue lesions, colitis scores, and histopathologic parameters associated with colitis. In addition, the CSN stimulation also reduced the colonic mucosa pro-inflammatory cytokine interleukin-1 beta, and increased the anti-inflammatory interleukin-10, in rats submitted to colitis. SignificanceThese findings indicated that electrical CSN stimulation breaks the vicious cycle of local colon inflammation in colitis, which might contribute to its better outcome.

Abstract 14807: Direct Carotid Sinus Nerve Stimulation in Anesthetized Human Subjects

Introduction: Drug resistant hypertension is a growing clinical phenomenon across the world and there is a critical need for alternative therapeutic modalities. Hypothesis: A novel multi-contact electrode designed to interface with the carotid sinus nerve in conjunction with a unique implantation technique can selectively activate the baroreflex causing reversible drops in blood pressure (BP) & heart rate (HR) in anesthetized humans. Methods: Patients undergoing therapeutic operations that necessitated carotid bulb exposure were prospectively enrolled in this IRB approved trial. Using a novel surgical approach, an application specific electrode was placed around the CSN followed by contact interface mapping to identify presumed baroafferent fibers which were selectively stimulated. Outcome measures included change in systolic BP (SBP), diastolic BP (DBP), and HR during and after stimulation. Results: 14 subjects met eligibility criteria. In 13, following correct contact identification, stimulation caused a 23±20mmHg (mean±SD) maximum drop in SBP with associated drops in DPB (11±9mmHg), and HR (10±9bpm). All subjects recovered immediately following stimulation withdrawal. All drops were significant (p<0.001, t-test). In subjects with baseline SBP >120mmHg (n=8), maximum SBP drop was 30±23mmHg, while subjects with baseline SBP <120mmHg (n=5) exhibited a maximum SBP drop of 11±3mmHg. There was evidence of a positive dose (current) response relationship. Conclusions: Using a novel surgical approach and electrode, CSN stimulation in anesthetized human subjects caused a marked drop in BP/HR followed by rapid recovery with stimulation withdrawal. There was evidence of dose dependency.

Hypoxia releases S-nitrosocysteine from carotid body glomus cells-relevance to expression of the hypoxic ventilatory response.

We have provided indirect pharmacological evidence that hypoxia may trigger release of the S-nitrosothiol, S-nitroso-L-cysteine (L-CSNO), from primary carotid body glomus cells (PGCs) of rats that then activates chemosensory afferents of the carotid sinus nerve to elicit the hypoxic ventilatory response (HVR). The objective of this study was to provide direct evidence, using our capacitive S-nitrosothiol sensor, that L-CSNO is stored and released from PGCs extracted from male Sprague Dawley rat carotid bodies, and thus further pharmacological evidence for the role of S-nitrosothiols in mediating the HVR. Key findings of this study were that 1) lysates of PGCs contained an S-nitrosothiol with physico-chemical properties similar to L-CSNO rather than S-nitroso-L-glutathione (L-GSNO), 2) exposure of PGCs to a hypoxic challenge caused a significant increase in S-nitrosothiol concentrations in the perfusate to levels approaching 100fM via mechanisms that required extracellular Ca2+, 3) the dose-dependent increases in minute ventilation elicited by arterial injections of L-CSNO and L-GSNO were likely due to activation of small diameter unmyelinated C-fiber carotid body chemoafferents, 4) L-CSNO, but not L-GSNO, responses were markedly reduced in rats receiving continuous infusion (10μmol/kg/min, IV) of both S-methyl-L-cysteine (L-SMC) and S-ethyl-L-cysteine (L-SEC), 5) ventilatory responses to hypoxic gas challenge (10% O2, 90% N2) were also due to the activation of small diameter unmyelinated C-fiber carotid body chemoafferents, and 6) the HVR was markedly diminished in rats receiving L-SMC plus L-SEC. This data provides evidence that rat PGCs synthesize an S-nitrosothiol with similar properties to L-CSNO that is released in an extracellular Ca2+-dependent manner by hypoxia.

Histology and immunohistochemistry of the human carotid sinus nerve.

The carotid sinus nerve (CSN) is well known as mediating baroreflexes. However, studies of its detailed histological analysis are scant in the literature. Therefore, the current anatomical study sought to better elucidate the microanatomy of the CSN. Ten fresh frozen adult cadavers underwent dissection of the CSN. Then, it was harvested and submitted for histological and immunohistochemical staining. Specimens were all shown to be nerve fibers on histology and immunohistochemistry. We identified tyrosine hydroxylase positive fibers in all CSN specimens. These fibers were always found to be within the CSN and not on its surface i.e., epineurium. Based on our findings, the majority of fibers contained in the CSN are tyrosine positive in nature. Further studies are necessary to understand the true function of this autonomic nerve fibers.

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.

Effect of Carotid Body Denervation on Systemic Endothelial Function in a Diabetic Animal Model.

Endothelial dysfunction is an essential intermediary for development of cardiovascular diseases associated with diabetes and hypertension (HT). The carotid body (CB) dysfunction contributes to dysmetabolic states, and the resection of carotid sinus nerve (CSN) prevents and reverts dysmetabolism and HT. Herein, we investigated if CSN denervation ameliorates systemic endothelial dysfunction in an animal model of type 2 diabetes mellitus (T2DM).We used Wistar male rats submitted to HFHSu diet during 25weeks and the correspondent age-matched controls fed with a standard diet. CSN resection was performed in half of the groups after 14weeks of diet. In vivo insulin sensitivity, glucose tolerance and blood pressure, ex vivo aortic artery contraction and relaxation and nitric oxide (NO) levels in plasma and aorta, aorta nitric oxide synthase (NOS) isoforms, and PGF2αR levels were evaluated.We demonstrated that, alongside to dysmetabolism and HT reversion, CSN resection restores endothelial function in the aorta and decreases the NO levels in plasma and aorta at the same time that restores normal levels of iNOS in aorta without changing eNOS or PGF2αR levels.These results suggest that the modulation of CB activity can be important for the treatment of HT and endothelial dysfunction related with T2DM.

Chronic Metformin Administration Does Not Alter Carotid Sinus Nerve Activity in Control Rats.

Metformin is a glucose-lowering, insulin-sensitizing drug that is commonly used in the treatment of type 2 diabetes (T2D). In the last decade, the carotid body (CB) has been described as a metabolic sensor implicated in the regulation of glucose homeostasis, being CB dysfunction crucial for the development of metabolic diseases, such as T2D. Knowing that metformin could activate AMP-activated protein kinase (AMPK) and that AMPK has been described to havean important role in CB hypoxic chemotransduction, herein we have investigated the effect of chronic metformin administration on carotid sinus nerve (CSN) chemosensory activity in basal and hypoxic and hypercapnic conditions in control animals. Experiments were performed in male Wistar rats subjected to 3weeks of metformin (200mg/kg) administration in the drinking water. The effect of chronic metformin administration was tested in spontaneous and hypoxic (0% and 5% O2) and hypercapnic (10% CO2) evoked CSN chemosensory activity. Metformin administration for 3weeks did not modify the basal CSN chemosensory activity in control animals. Moreover, the CSN chemosensory response to intense and moderate hypoxia and hypercapnia was not altered by the chronic metformin administration. In conclusion, chronic metformin administration did not modify chemosensory activity in control animals.

Carotid Body Resection Prevents Short-Term Spatial Memory Decline in Prediabetic Rats Without Changing Insulin Signaling in the Hippocampus and Prefrontal Cortex.

Individuals who develop type 2 diabetes (T2D) at an early age are at higher risk of developing neurodegenerative disorders such as Alzheimer's and Parkinson's disease. A shared dysfunctional characteristic between T2D and these neurodegenerative disorders is insulin resistance. Recently, it was shown that prediabetes animals and patients exhibited increased carotid body (CB) activity. Moreover, these organs are deeply involved in the development of metabolic diseases, since upon abolishment of their activity via carotid sinus nerve (CSN) resection, several dysmetabolic features of T2D were reverted. Herein, we investigated if CSN resection may also prevent cognitive impairment associated with brain insulin resistance. We explored a diet-induced prediabetes animal model where Wistar rats are kept in a high fat-high sucrose (HFHSu) diet for 20weeks. We evaluated CSN resection effects on behavioral parameters and on insulin signaling-related proteins levels, in the prefrontal cortex and the hippocampus. HFHSu animals exhibited impaired short-term memory evaluated by the y-maze test. Remarkably, CSN resection prevented the development of this phenotype. HFHSu diet or CSN resection did not promote significant alterations in insulin signaling-associated proteins levels. Our findings suggest that CBs modulation might have a role in preventing short-term spatial memory deficits associated with peripheral dysmetabolic states.

Acute effects of insulin and insulin-induced hypoglycaemia on carotid body chemoreceptor activity and cardiorespiratory responses in dogs.

What is the central question of this study? What are the effects of insulin and insulin-induced hypoglycaemia on carotid body chemoreceptor activity in vivo and how do carotid body chemoreceptor stimulation-mediated cardiorespiratory responses in beagle dogs compare during euglycaemia and insulin-induced hypoglycaemia? What is the main finding and its importance? Intracarotid insulin administration leads to sustained increase in carotid body chemoreceptor activity and respiratory response with significant cardiovascular effects. Insulin-induced hypoglycaemia exacerbated NaCN-mediated carotid body chemoreceptor activity and respiratory response with enhanced cardiovascular reflex response. These findings suggest that insulin-induced hypoglycaemia augments the carotid body chemoreceptors to initiate the adaptive counter-regulatory responses to restore the normoglycaemic condition. The carotid body chemoreceptors (CBC) play an important role in the adaptive counter-regulatory response to hypoglycaemia by evoking the CBC-mediated sympathetic neuronal system to restore normoglycaemia. Ex vivo studies have shown varied responses of insulin-induced hypoglycaemia on CBC function, and several in vivo studies have indirectly established the role of CBCs in restoring normoglycaemia in both animals and humans. However, a direct effect of insulin and/or insulin-induced hypoglycaemia on CBC activity is not established in animal models. Therefore, the aim of this study was to evaluate in vivo effects of insulin and insulin-induced hypoglycaemia on CBC activity and cardiorespiration in a preclinical large animal model. The carotid sinus nerve (CSN) activity and cardiorespiratory responses to sodium cyanide (NaCN; 25µg/kg) were compared before (euglycaemic) and after (hypoglycaemic) intracarotid administration of insulin (12.5-100µU/dogs) in beagle dogs. Insulin administration increased CSN activity and minute ventilation ( E ) with significant (P<0.0001) effects on heart rate and blood pressure. Insulin-mediated effects on CSN and cardiorespiration were sustained and the change in E was driven by tidal volume only. Insulin significantly (P<0.0001) lowered blood glucose level. NaCN-mediated CSN activity and E were significantly (P<0.0001) augmented during insulin-induced hypoglycaemia. The augmented E was primarily driven by respiratory frequency and partially by tidal volume. The cardiovascular reflex response mediated through CBC stimulation was significantly (P<0.0001) exacerbated during insulin-induced hypoglycaemia. Collectively, these results demonstrate direct effects of insulin and insulin-induced hypoglycaemia on CBC chemosensitivity to potentiate CBC-mediated neuroregulatory pathways to initiate adaptive neuroendocrine and cardiorespiratory counter-regulatory responses to restore normoglycaemia.