Emilia M. Sanhueza

Adrenergic and vasopressinergic contributions to the cardiovascular response to acute hypoxaemia in the llama fetus

1 The effects of fetal intravenous treatment with phentolamine or a vasopressinergic V1‐receptor antagonist on the fetal cardiovascular responses to acute hypoxaemia in the llama were investigated. 2 Six llama fetuses were surgically prepared between 60 and 70% of gestation under general halothane anaesthesia with vascular catheters and transit‐time ultrasonic flow probes around a carotid artery and a femoral artery. At least 4 days after surgery all fetuses were subjected to a 3 h experiment: 1 h of normoxia, 1 h of hypoxaemia and 1 h of recovery while on slow i.v. infusion with saline. On separate days this experiment was repeated with fetal i.v. treatment with either phentolamine or a V1‐receptor antagonist dissolved in saline. 3 During saline infusion all llama fetuses responded to acute hypoxaemia with intense femoral vasoconstriction. Phentolamine during normoxia produced hypotension, tachycardia and vasodilatation in both the carotid and the femoral circulations. During hypoxaemia, fetuses treated with phentolamine did not elicit the pronounced femoral vasoconstriction and all died within 20 min of the onset of hypoxaemia. A V1‐receptor antagonist produced a femoral vasodilatation during normoxia but did not affect the fetal cardiovascular responses to acute hypoxaemia. 4 In conclusion, α‐adrenergic and V1‐vasopressinergic mechanisms contribute to a basal vasoconstrictor tone in the femoral circulation in the llama fetus. The enhanced femoral vasoconstriction during acute hypoxaemia in the llama fetus is not mediated by stimulation of V1‐vasopressin receptors, but is dependent on α‐adrenergic receptor stimulation. Such α‐adrenergic efferent mechanisms are indispensable to fetal survival during hypoxaemia in the llama since their abolition leads to cardiovascular collapse and death.

The fetal llama versus the fetal sheep: different strategies to withstand hypoxia

The pregnant llama (Lama glama) has walked for millions of years through the thin oxygen trail of the Andean altiplano. We hypothesize that a pool of genes has been selected in the llama that express efficient mechanisms to withstand this low-oxygen milieu. The llama fetus responds to acute hypoxia with an intense peripheral vasoconstriction that is not affected by bilateral section of the carotid sinus nerves. Moreover, the increase in fetal plasma concentrations of vasoconstrictor hormones, such as catecholamines, neuropeptide Y, and vasopressin, is much greater in the llama than in the sheep fetus. Furthermore, treatment of fetal llamas with an alpha-adrenergic antagonist abolished the peripheral vasoconstriction and resulted in fetal cardiovascular collapse and death during acute hypoxia, suggesting an indispensable upregulation of alpha-adrenergic mechanisms in this high altitude species. Local endothelial factors such as nitric oxide (NO) also play a key role in the regulation of fetal adrenal blood flow and in the adrenal secretion of catecholamines and cortisol. Interestingly, in contrast to the human or sheep fetus, the llama fetus showed a small increase in brain blood flow during acute hypoxia, with no increase in oxygen extraction across the brain, and thereby a decrease in brain oxygen consumption. These results suggest that the llama fetus responds to acute hypoxia with hypometabolism. How this reduction in metabolism is produced and how the cells are preserved during this condition remain to be elucidated.

Low-Dose Inhaled Carbon Monoxide Reduces Pulmonary Vascular Resistance During Acute Hypoxemia in Adult Sheep

Carbon monoxide (CO) is produced by the action of the heme oxygenase (HO) complex through the oxidation of heme. CO, like nitric oxide (NO), is a molecular gas that among other actions stimulates guanylyl cyclase and increases cGMP levels in smooth muscle cells, regulating the vascular tone. Acute hypoxia generates pulmonary hypertension and increases the expression of inducible HO isoform (HO-1) in the vascular endothelium. Inhaled NO causes a potent pulmonary vasodilation. We hypothesized that inhaled CO might produce similar actions as NO on pulmonary vascular resistance (PVR). To test our contention, we studied the effects of inhaled CO (40 ppm) in the augmented PVR observed during hypoxemia. Five chronically instrumented German Merino sheep were submitted to a protocol consisting of 20 min of normoxemia (N), 20 min of isocapnic hypoxemia (H20), 20 min of isocapnic hypoxemia plus CO 40 ppm (H40), and 20 min of recovery (R). In the control protocol, we did not administer inhaled CO. Arterial gases and pH, percentage of carboxyhemoglobin (COHb), systemic and pulmonary arterial pressure, systemic and pulmonary vascular resistance, and cardiac output were measured during each period. During H20 period, there was a significant increase in cardiac output and PVR in sheep submitted to both protocols. The sheep treated with inhaled CO (H40 + CO) showed a modest but significant decrease (16%) in the elevated PVR. Our data indicate that inhaled CO decreases pulmonary vascular resistance associated with acute hypoxemia in adult sheep.

Fetal brain hypometabolism during prolonged hypoxaemia in the llama

In this study we looked for additional evidence to support the hypothesis that fetal llama reacts to hypoxaemia with adaptive brain hypometabolism. We determined fetal llama brain temperature, Na+ and K+ channel density and Na+–K+‐ATPase activity. Additionally, we looked to see whether there were signs of cell death in the brain cortex of llama fetuses submitted to prolonged hypoxaemia. Ten fetal llamas were instrumented under general anaesthesia to measure pH, arterial blood gases, mean arterial pressure, heart rate, and brain and core temperatures. Measurements were made 1 h before and every hour during 24 h of hypoxaemia (n= 5), which was imposed by reducing maternal inspired oxygen fraction to reach a fetal arterial partial pressure of oxygen (P  a,O 2 ) of about 12 mmHg. A normoxaemic group was the control (n= 5). After 24 h of hypoxaemia, we determined brain cortex Na+–K+‐ATPase activity, ouabain binding, and the expression of NaV1.1, NaV1.2, NaV1.3, NaV1.6, TREK1, TRAAK and KATP channels. The lack of brain cortex damage was assessed as poly ADP‐ribose polymerase (PARP) proteolysis. We found a mean decrease of 0.56°C in brain cortex temperature during prolonged hypoxaemia, which was accompanied by a 51% decrease in brain cortex Na+–K+‐ATPase activity, and by a 44% decrease in protein content of NaV1.1, a voltage‐gated Na+ channel. These changes occurred in absence of changes in PARP protein degradation, suggesting that the cell death of the brain was not enhanced in the fetal llama during hypoxaemia. Taken together, these results provide further evidence to support the hypothesis that the fetal llama responds to prolonged hypoxaemia with adaptive brain hypometabolism, partly mediated by decreases in Na+–K+‐ATPase activity and expression of NaV channels.

Evolving in thin air : Lessons from the llama fetus in the altiplano

Compared with lowland species, fetal life for mammalian species whose mothers live in high altitude is demanding. For instance, fetal llamas have to cope with the low fetal arterial PO2 of all species, but also the likely superimposition of hypoxia as a result of the decreased oxygen environment in which the mother lives in the Andean altiplano. When subjected to acute hypoxia the llama fetus responds with an intense peripheral vasoconstriction mediated by alpha-adrenergic mechanisms plus high plasma concentrations of catecholamines and neuropeptide Y (NPY). Endothelial factors such as NO and endothelin-1 also play a role in the regulation of local blood flows. Unlike fetuses of lowland species such as the sheep, the llama fetus shows a profound cerebral hypometabolic response to hypoxia, decreasing cerebral oxygen consumption, Na-K-ATPase activity and temperature, and resulting in an absence of seizures and apoptosis in neural cells. These strategies may have evolved to prevent hypoxic injury to the brain or other organs in the face of the persistent hypobaric hypoxia of life in the Andean altiplano.

Regional brain blood flow and cerebral hemispheric oxygen consumption during acute hypoxaemia in the llama fetus

Unlike fetal animals of lowland species, the llama fetus does not increase its cerebral blood flow during an episode of acute hypoxaemia. This study tested the hypothesis that the fetal llama brain maintains cerebral hemispheric O2 consumption by increasing cerebral O2 extraction rather than decreasing cerebral oxygen utilisation during acute hypoxaemia. Six llama fetuses were surgically instrumented under general anaesthesia at 217 days of gestation (term ca 350 days) with vascular and amniotic catheters in order to carry out cardiorespiratory studies. Following a control period of 1 h, the llama fetuses underwent 3 × 20 min episodes of progressive hypoxaemia, induced by maternal inhalational hypoxia. During basal conditions and during each of the 20 min of hypoxaemia, fetal cerebral blood flow was measured with radioactive microspheres, cerebral oxygen extraction was calculated, and fetal cerebral hemispheric O2 consumption was determined by the modified Fick principle. During hypoxaemia, fetal arterial O2 tension and fetal pH decreased progressively from 24 ± 1 to 20 ± 1 Torr and from 7.36 ± 0.01 to 7.33 ± 0.01, respectively, during the first 20 min episode, to 16 ± 1 Torr and 7.25 ± 0.05 during the second 20 min episode and to 14 ± 1 Torr and 7.21 ± 0.04 during the final 20 min episode. Fetal arterial partial pressure of CO2 (Pa,CO2, 42 ± 2 Torr) remained unaltered from baseline throughout the experiment. Fetal cerebral hemispheric blood flow and cerebral hemispheric oxygen extraction were unaltered from baseline during progressive hypoxaemia. In contrast, a progressive fall in fetal cerebral hemispheric oxygen consumption occurred during the hypoxaemic challenge. In conclusion, these data do not support the hypothesis that the fetal llama brain maintains cerebral hemispheric O2 consumption by increasing cerebral hemispheric O2 extraction. Rather, the data show that in the llama fetus, a reduction in cerebral hemispheric metabolism occurs during acute hypoxaemia.

Nitric oxide plays a role in the regulation of adrenal blood flow and adrenocorticomedullary functions in the llama fetus

The hypothesis that nitric oxide plays a key role in the regulation of adrenal blood flow and plasma concentrations of cortisol and catecholamines under basal and hypoxaemic conditions in the llama fetus was tested. At 0.6‐0.8 of gestation, 11 llama fetuses were surgically prepared for long‐term recording under anaesthesia with vascular and amniotic catheters. Following recovery all fetuses underwent an experimental protocol based on 1 h of normoxaemia, 1 h of hypoxaemia and 1 h of recovery. In nine fetuses, the protocol occurred during fetal i.v. infusion with saline and in five fetuses during fetal i.v. treatment with the nitric oxide synthase inhibitor l‐NAME. Adrenal blood flow was determined by the radiolabelled microsphere method during each of the experimental periods during saline infusion and treatment with l‐NAME. Treatment with l‐NAME during normoxaemia led to a marked fall in adrenal blood flow and a pronounced increase in plasma catecholamine concentrations, but it did not affect plasma ACTH or cortisol levels. In saline‐infused fetuses, acute hypoxaemia elicited an increase in adrenal blood flow and in plasma ACTH, cortisol, adrenaline and noradrenaline concentrations. Treatment with l‐NAME did not affect the increase in fetal plasma ACTH, but prevented the increments in adrenal blood flow and in plasma cortisol and adrenaline concentrations during hypoxaemia in the llama fetus. In contrast, l‐NAME further enhanced the increase in fetal plasma noradrenaline. These data support the hypothesis that nitric oxide has important roles in the regulation of adrenal blood flow and adrenal corticomedullary functions during normoxaemia and hypoxaemia functions in the late gestation llama fetus.

Cardiovascular Responses to Arginine Vasopressin Blockade During Acute Hypoxemia in the Llama Fetus

The fetal llama has a marked increase in the peripheral vascular resistance and no augmentation of brain blood flow during hypoxemia. In spite of the substantial plasma arginine-vasopressin (AVP) increase during hypoxemia, up to 8 times greater than in fetal sheep, there are no changes of carotid and femoral blood flows during hypoxemia with a V1 receptor blockade, as is seen in the fetal sheep. The aim of this study was to assess the role of AVP function in mediating the combined ventricular output and organ blood flow in the hypoxemic llama fetus. Six fetal llamas at 0.65 of gestation were instrumented under general anesthesia, and cardiorespiratory responses and blood flows determined under normoxemic and hypoxemic conditions. The AVP effect was determined using a V1 antagonist during normoxemic and hypoxemic conditions. Organ blood flows were measured with the radioactive microsphere technique. No significant differences in organ blood flow or in their vascular resistances were seen between the control and treated fetuses during hypoxemia. We conclude that V1 blockade did not have any important role in the cardiovascular response to acute hypoxemia in the llama fetus, in contrast with lowland fetuses. AVP may be playing a role in other regions, possibly in kidney or lung, during hypoxemia.

Cardiorespiratory Responses to Acute Hypoxemia in the Chronically Catheterized Fetal Llama at 0.7–0.9 of Gestation

The adult llama (Lama glama) has several compensatory mechanisms that allow it to successfully survive at high altitude. Llama fetuses at 0.6-0.7 of gestation, and near-term llama fetuses studied close to surgery, did not increase cerebral blood flow and decreased cerebral oxygen delivery during acute hypoxemia. It is not known whether these responses were the result of immaturity or surgical stress. The aim of this study was to determine whether the lack of increase in cerebral blood flow and the decrease in cerebral oxygen delivery during hypoxemia in the fetal llama is characteristic of this high-altitude species near term, and under nonstressed conditions. We chronically catheterized 7 llamas and their fetuses near to term, at 0.7-0.9 of gestation. Fetal cardiac output, cerebral and regional blood flows, systemic blood pressure, heart rate, pH, and blood gases, organ vascular resistances and organ oxygen deliveries were determined at least 4 days after surgery, both during the basal state and after 1 hr of acute fetal hypoxemia. During hypoxemia the llama fetus did not increase cerebral blood flow and markedly decreased its cerebral oxygen delivery. There was also a marked decrease in kidney blood flow and oxygen delivery. These results indicate that, in contrast to fetuses of lowland species, the fetal llama does not increase the cerebral blood flow during hypoxemia, suggesting specific cellular mechanisms to preserve brain integrity during oxygen limitation.

The role of neuropeptide Y in the ovine fetal cardiovascular response to reduced oxygenation

This study investigated the role of neuropeptide Y (NPY) in mediating cardiovascular responses to reduced oxygenation in the late gestation ovine fetus by: (1) comparing the effects on the cardiovascular system of an exogenous infusion of NPY with those elicited by moderate or severe reductions in fetal oxygenation; and (2) determining the effect of fetal i.v. treatment with a selective NPY‐Y1 receptor antagonist on the fetal cardiovascular responses to acute moderate hypoxaemia. Under general anaesthesia, 14 sheep fetuses (0.8‐0.9 of gestation) were surgically prepared with vascular and amniotic catheters. In 5 of these fetuses, a Transonic flow probe was also implanted around a femoral artery. Following at least 5 days of recovery, one group of fetuses (n= 9) was subjected to a 30 min treatment period with exogenous NPY (17 μg kg−1 bolus plus 0.85 μg kg−1 min−1 infusion). In this group, fetal blood pressure and heart rate were monitored continuously and the distribution of the fetal combined ventricular output was assessed via injection of radiolabelled microspheres before and during treatment. The second group of fetuses instrumented with the femoral flow probe (n= 5) were subjected to a 3 h experiment consisting of 1 h of normoxia, 1 h of hypoxaemia, and 1 h of recovery during a slow i.v. infusion of vehicle. One or two days later, the acute hypoxaemia protocol was repeated during fetal i.v. treatment with a selective NPY‐Y1 receptor antagonist (50 μg kg−1bolus + 1.5μg kg−1 min−1 infusion). In these fetuses, fetal arterial blood pressure, heart rate and femoral vascular resistance were recorded continuously. The results show that fetal treatment with exogenous NPY mimics the fetal cardiovascular responses to asphyxia, and that treatment of the sheep fetus with a selective NPY‐Y1 receptor antagonist does not affect the fetal cardiovascular response to acute moderate hypoxaemia. These results support a greater role for NPY in mediating the fetal cardiovascular responses to acute asphyxia than to acute moderate hypoxaemia.