Efectos inducidos por la hipoxia crónica intermitente en rata y cobaya como modelos animales de la apnea obstructiva del sueño

Desde un enfoque integrador, que combina estudios funcionales in vivo e in vitro, se ha probado la hipótesis de que el efecto hipertensivo arterial sistémico de la hipoxia crónica intermitente, producido en la enfermedad respiratoria de la apnea obstructiva del sueño, se atenuaría o eliminaría debido a una respuesta quimiorreceptora menor en las ratas viejas y por falta de capacidad de respuesta del cuerpo carotídeo en los cobayas. Los datos muestran que la hipoxia crónica intermitente produce sensibilización del cuerpo carotídeo en la rata vieja sin modificaciones cardiovasculares aparentes. Por el contrario, la hipoxia crónica intermitente en el cobaya modifica parámetros cardiovasculares y metabólicos sin cambios en la funcionalidad del cuerpo carotídeo.Departamento de Bioquímica y Biología Molecular y FisiologíaDoctorado en Investigación Biomédic

Guinea Pig Oxygen-Sensing and Carotid Body Functional Properties

Mammals have developed different mechanisms to maintain oxygen supply to cells in response to hypoxia. One of those mechanisms, the carotid body (CB) chemoreceptors, is able to detect physiological hypoxia and generate homeostatic reflex responses, mainly ventilatory and cardiovascular. It has been reported that guinea pigs, originally from the Andes, have a reduced ventilatory response to hypoxia compared to other mammals, implying that CB are not completely functional, which has been related to genetically/epigenetically determined poor hypoxia-driven CB reflex. This study was performed to check the guinea pig CB response to hypoxia compared to the well-known rat hypoxic response. These experiments have explored ventilatory parameters breathing different gases mixtures, cardiovascular responses to acute hypoxia, in vitro CB response to hypoxia and other stimuli and isolated guinea pig chemoreceptor cells properties. Our findings show that guinea pigs are hypotensive and have lower arterial pO2 than rats, probably related to a low sympathetic tone and high hemoglobin affinity. Those characteristics could represent a higher tolerance to hypoxic environment than other rodents. We also find that although CB are hypo-functional not showing chronic hypoxia sensitization, a small percentage of isolated carotid body chemoreceptor cells contain tyrosine hydroxylase enzyme and voltage-dependent K+ currents and therefore can be depolarized. However hypoxia does not modify intracellular Ca2+ levels or catecholamine secretion. Guinea pigs are able to hyperventilate only in response to intense acute hypoxic stimulus, but hypercapnic response is similar to rats. Whether other brain areas are also activated by hypoxia in guinea pigs remains to be studied

Homeostasis de calcio y exocitosis de células quimiorreceptoras carotídeas

las células quimiorreceptoras de cuerpo carotídeo de rata poseen un activo intercambiador Na+/Ca2+ en su membrana plasmática puesto en evidencia mediante la eliminación de Na+ extracelular: la superfusión con ONa+ activa la entrada Ca2+ debido a la reversión del NCX promoviendo respuesta secretora. La activación del NCXREV potencia el aumento de ca+i y la liberación de CA inducido por despolarización con alto K+ extracelular. Este defectop parece ser debido al aumento de la entrada de Ca2 + por NCXREV como se observa tras su inhibición por KB-R7943.El intercambiador Na+/Ca2+participa en la recuperación de los niveles basales de ca2+i ya que su inhibición por KB-R7943 mantiene elevada la secreción de CA tras la estimulación de CQ por despolarización e hipoxia.Departamento de Bioquímica, Biología molecular y FisiologíaMáster en Investigación Biomédica2014-10-1

Guinea pig oxygen-sensing and carotid body functional properties

Mammals have developed different mechanisms to maintain oxygen supply to cells in response to hypoxia. One of those mechanisms, the carotid body (CB) chemoreceptors, is able to detect physiological hypoxia and generate homeostatic reflex responses, mainly ventilatory and cardiovascular. It has been reported that guinea pigs, originally from the Andes, have a reduced ventilatory response to hypoxia compared to other mammals, implying that CB are not completely functional, which has been related to genetically/epigenetically determined poor hypoxia-driven CB reflex. This study was performed to check the guinea pig CB response to hypoxia compared to the well-known rat hypoxic response. These experiments have explored ventilatory parameters breathing different gases mixtures, cardiovascular responses to acute hypoxia, in vitro CB response to hypoxia and other stimuli and isolated guinea pig chemoreceptor cells properties. Our findings show that guinea pigs are hypotensive and have lower arterial pO2 than rats, probably related to a low sympathetic tone and high hemoglobin affinity. Those characteristics could represent a higher tolerance to hypoxic environment than other rodents. We also find that although CB are hypo-functional not showing chronic hypoxia sensitization, a small percentage of isolated carotid body chemoreceptor cells contain tyrosine hydroxylase enzyme and voltage-dependent K+ currents and therefore can be depolarized. However hypoxia does not modify intracellular Ca2+ levels or catecholamine secretion. Guinea pigs are able to hyperventilate only in response to intense acute hypoxic stimulus, but hypercapnic response is similar to rats. Whether other brain areas are also activated by hypoxia in guinea pigs remains to be studied.This study was supported by grants BFU2015-70616R, SAF 2014-55399 and SAF 2016-77222-R (MINECO-FEDER), and CIBER CB06/06/0050 (ISCIII).We acknowledge support of the publication fee by the CSIC Open Access Support Initiative through its Unit of Information Resources for Research (URICI)Peer reviewedPeer Reviewe

Is the guinea pig a full negative model to study the carotid mediated chronic intermittent hipoxia effects?

Resumen del póster presentado al Joint Meeting of the American Physiological Society and the Physiological Society, celebrado en Dublin (Irlanda) del 29 al 31 de julio de 2016.Chronic Intermittent Hypoxia (CIH) is considered to be one of the main causes of systemic arterial hypertension observed in the obstructive sleep apnea syndrome. It is thought that repetitive episodes of hypoxia/re-oxygenation produce oxidative stress, inflammation and sympathetic hyperactivity, generating endothelial dysfunction and systemic hypertension. It has been proposed that the repeated carotid body (CB) stimulation produced by CIH would induce CB sensitization, increasing chemoreceptor input to the brainstem that originates an exaggerated sympathetic reflex with a rise of circulating catecholamine (CA) and hypertension. Unlike other rodents, preliminary data show a lack of CB sensitivity to acute hypoxia in guinea pigs, in which case, CIH would not induce CB sensitization and the effects derived from the CB hyperactivity would not be observed. Therefore, guinea pigs could be a negative control model to study the effects of CIH mediated by CB. In this study, experiments were performed on adult male Hartley guinea pigs (475±10 g; n=32) control or exposed to CIH (21% O2-80 s/5% O2-40s 8h/day; 30 days). Ventilatory parameters (tidal volume and respiratory rate) were measured in freely moving animals by whole body plethysmography; other measurements were performed on animals anaesthetized with a mixture of ketamine (100 mg/Kg) and diazepam (2 mg/Kg; ip.). Control and CIH guinea pig respiratory minute volume exhibited similar changes when acute hypoxic test was applied (399±5 vs 411±5 in air and 417±15 vs 456±17 ml/min/Kg in 10% O2; n=16). Values are means ± S.E.M. compared by ANOVA. There were no in vitro CB responses to acute hypoxia (CA secretion or Ca2+i changes) in either group of animals. No differences were found in mean arterial blood pressure (37±2 vs 43±3 mmHg; n=8), or in plasma and tissue CA levels (CB, adrenal medulla, renal artery) measured after exposure to CIH. The fact that the guinea pig CB is hypo-functional and is not sensitized by CIH would reinforce our working hypothesis: systemic effects associated to CIH in other species and absent in guinea pigs are due to the CB hyperactivity induced by CIH. However several unexpected results would indicate hypoxic activation of the sympathetic system during acute hypoxic test as increased arterial pulse pressure (20±1 mmHg in air and 28±2 mmHg in 10% O2; p<0.01; n=8), rise of heart rate (25%; p<0.01), renal artery CA synthesis (32%; p<0.05) and CB hypotrophy (50%; p<0.05) after CIH treatment. These results suggest that guinea pigs possess O2-sensitivity responsible for the sympathetic cardio-circulatory reflex, probably through CB stimulation, the main oxygen sensing structure mediating this reflex. The mechanisms are being studied.Supported by grants: MINECO BFU2015-70616R; ISCiii CIBER CB06/06/0050; JCyL BIO/VA11/15Peer Reviewe

T-type calcium channels in rat carotid body chemoreceptor cells. Molecular and functional hallmarks

Resumen del póster presentado al 14th International Meeting of the European Calcium Society, celebrado en Valladolid (España) del 25 al 29 de septiembre de 2016.An important path of extracellular calcium influx in carotid body chemoreceptor cells (CBCC) during hypoxic activation and neurotransmitter release is through voltage activated calcium channels of the plasma membrane. Both high (HVA) and low voltage-activated (LVA) Ca2+ channels are present in CBCC, yet no much is known about the relevance of the LVA T-type channels. Three different genes codify for T-type channels, Cav3.1, Cav3.2 and Cav3.3, but only the Cav3.2 isoform has been found in carotid body. It has been reported that chronic hypoxia (CH) up-regulates Cav3.2 channel expression in PC12, chromaffin and pulmonary artery smooth muscle cells and, more recently, in CBCC. In the present study, we investigate the expression and the role of T-type Ca2+ channels in rat CB responses to acute and chronic hypoxia using a combination of pharmacological and molecular approaches. By immunocytochemistry and RT-PCR experiments we provide molecular evidence for a main presence of Cav3.1 and minor Cav3.2 expression, but not Cav3.3, in CBCC. Rats exposure to CH during 7 days up-regulates both, Cav3.1 and Cav3.2 channels, but mainly the former. Hypoxia stimulated CB response can be 50% reduced by specific T-type inhibitors: low concentration of mibefradil, Ni2+ and TTA-A2 and TTA-P2 blockers. We demonstrated augmented responses in CH treated rat CB which also are 50% sensitive to Ni2+ and mibefradil. We conclude that in rat CBCC, Cav3.1 is the predominant T-type calcium channel contributing to basal and CH augmented CBCC excitability and secretory response.Supported by grants BFU2015-70616R from MINECO and ISCiii CIBER CB06/06/0050.Peer Reviewe

Guinea Pig as a Model to Study the Carotid Body Mediated Chronic Intermittent Hypoxia Effects

Clinical and experimental evidence indicates a positive correlation between chronic intermittent hypoxia (CIH), increased carotid body (CB) chemosensitivity, enhanced sympatho-respiratory coupling and arterial hypertension and cardiovascular disease. Several groups have reported that both the afferent and efferent arms of the CB chemo-reflex are enhanced in CIH animal models through the oscillatory CB activation by recurrent hypoxia/reoxygenation episodes. Accordingly, CB ablation or denervation results in the reduction of these effects. To date, no studies have determined the effects of CIH treatment in chemo-reflex sensitization in guinea pig, a rodent with a hypofunctional CB and lacking ventilatory responses to hypoxia. We hypothesized that the lack of CB hypoxia response in guinea pig would suppress chemo-reflex sensitization and thereby would attenuate or eliminate respiratory, sympathetic and cardiovascular effects of CIH treatment. The main purpose of this study was to assess if guinea pig CB undergoes overactivation by CIH and to correlate CIH effects on CB chemoreceptors with cardiovascular and respiratory responses to hypoxia. We measured CB secretory activity, ventilatory parameters, systemic arterial pressure and sympathetic activity, basal and in response to acute hypoxia in two groups of animals: control and 30 days CIH exposed male guinea pigs. Our results indicated that CIH guinea pig CB lacks activity elicited by acute hypoxia measured as catecholamine (CA) secretory response or intracellular calcium transients. Plethysmography data showed that only severe hypoxia (7% O2) and hypercapnia (5% CO2) induced a significant increased ventilatory response in CIH animals, together with higher oxygen consumption. Therefore, CIH exposure blunted hyperventilation to hypoxia and hypercapnia normalized to oxygen consumption. Increase in plasma CA and superior cervical ganglion CA content was found, implying a CIH induced sympathetic hyperactivity. CIH promoted cardiovascular adjustments by increasing heart rate and mean arterial blood pressure without cardiac ventricle hypertrophy. In conclusion, CIH does not sensitize CB chemoreceptor response to hypoxia but promotes cardiovascular adjustments probably not mediated by the CB. Guinea pigs could represent an interesting model to elucidate the mechanisms that underlie the long-term effects of CIH exposure to provide evidence for the role of the CB mediating pathological effects in sleep apnea diseases

Estrogens and age influence on pulmonary hypertension in female rats

Trabajo presentado en la 2ª Reunión de investigación en hipertensión pulmonar, celebrado en Madrid (España) el 2 de febrero de 2018

Chronic intermittent hypoxia effects are not mediated by guinea pig carotid body sensitization

Tranajo presentado en European Respiratory Society Annual Congress, celebrado en París(Francia) del 15 al 19 de septiembre de 2018

Peripheral Dopamine 2-Receptor Antagonist Reverses Hypertension in a Chronic Intermittent Hypoxia Rat Model

The sleep apnea-hypopnea syndrome (SAHS) involves periods of intermittent hypoxia, experimentally reproduced by exposing animal models to oscillatory PO2 patterns. In both situations, chronic intermittent hypoxia (CIH) exposure produces carotid body (CB) hyperactivation generating an increased input to the brainstem which originates sympathetic hyperactivity, followed by hypertension that is abolished by CB denervation. CB has dopamine (DA) receptors in chemoreceptor cells acting as DA-2 autoreceptors. The aim was to check if blocking DA-2 receptors could decrease the CB hypersensitivity produced by CIH, minimizing CIH-related effects. Domperidone (DOM), a selective peripheral DA-2 receptor antagonist that does not cross the blood-brain barrier, was used to examine its effect on CIH (30 days) exposed rats. Arterial pressure, CB secretory activity and whole-body plethysmography were measured. DOM, acute or chronically administered during the last 15 days of CIH, reversed the hypertension produced by CIH, an analogous effect to that obtained with CB denervation. DOM marginally decreased blood pressure in control animals and did not affect hypoxic ventilatory response in control or CIH animals. No adverse effects were observed. DOM, used as gastrokinetic and antiemetic drug, could be a therapeutic opportunity for hypertension in SAHS patients&rsquo; resistant to standard treatments