Fernando A. Moraga

Fetal and maternal blood oxygen affinity: A comparative study in llamas and sheep☆

We compared blood oxygen affinity (P50) and hemoglobin concentration among fetal and maternal llamas and sheep, as respective examples of species native to high and low altitudes. P50, hemoglobin concentration and blood oxygen content were determined at sea level in 16 pregnant llamas, 6 pregnant sheep and their respective fetuses. P50 was similar in fetal llamas and sheep, but maternal llamas had higher blood oxygen affinity than maternal sheep. As a consequence, the P50 difference between mother and fetus was less in llamas than in sheep. Fetal llamas had higher hemoglobin concentrations than fetal sheep. In contrast, the maternal hemoglobin concentrations were similar. The blood oxygen content was higher in fetal and maternal llamas than in fetal and maternal sheep. We conclude that the llama, a species native to the altiplano, has a higher blood oxygen content than the sheep, as determined in the fetus by a high hemoglobin concentration and in the mother by a low P50.

Store-operated channels in the pulmonary circulation of high- and low-altitude neonatal lambs

We determined whether store-operated channels (SOC) are involved in neonatal pulmonary artery function under conditions of acute and chronic hypoxia, using newborn sheep gestated and born either at high altitude (HA, 3,600 m) or low altitude (LA, 520 m). Cardiopulmonary variables were recorded in vivo, with and without SOC blockade by 2-aminoethyldiphenylborinate (2-APB), during basal or acute hypoxic conditions. 2-APB did not have effects on basal mean pulmonary arterial pressure (mPAP), cardiac output, systemic arterial blood pressure, or systemic vascular resistance in both groups of neonates. During acute hypoxia 2-APB reduced mPAP and pulmonary vascular resistance in LA and HA, but this reduction was greater in HA. In addition, isolated pulmonary arteries mounted in a wire myograph were assessed for vascular reactivity. HA arteries showed a greater relaxation and sensitivity to SOC blockers than LA arteries. The pulmonary expression of two SOC-forming subunits, TRPC4 and STIM1, was upregulated in HA. Taken together, our results show that SOC contribute to hypoxic pulmonary vasoconstriction in newborn sheep and that SOC are upregulated by chronic hypoxia. Therefore, SOC may contribute to the development of neonatal pulmonary hypertension. We propose SOC channels could be potential targets to treat neonatal pulmonary hypertension.

Role of the α-adrenergic system in femoral vascular reactivity in neonatal llamas and sheep: a comparative study between highland and lowland species

Using an integrative approach at the whole animal, isolated vessels, and molecular levels, we tested the hypothesis that the llama, a species that undergoes pregnancy under the influence of the chronic hypoxia of high altitude, delivers offspring with an increased α-adrenergic peripheral vascular reactivity compared with neonates from lowland species. We studied the femoral vascular response to acute hypoxia in vivo, the reactivity of femoral vessels ex vivo, and the expression of femoral α1-adrenergic receptor subtypes using RT-PCR in vitro. The increase in femoral resistance during hypoxia was 3.6 times greater in newborn llamas than newborn sheep (P < 0.05). The sensitivity of the contractile response to noradrenaline (pD2 = 5.18 ± 0.06 vs. 4.84 ± 0.05, P < 0.05) and the maximal response (Rmax = 101.3 ± 1.4 vs. 52.4 ± 1.4% K+max, P < 0.05) and sensitivity (pD2 = 5.47 ± 0.03 vs. 4.57 ± 0.05, P < 0.05) to phenylephrine were higher in femoral vessels from newborn llamas than newborn sheep. Competitive inhibition with prazosin of noradrenaline-induced contraction followed by Schild analysis showed higher affinity in the llama than the sheep (pA2 = 10.08 ± 0.093 vs. 8.98 ± 0.263, respectively, P < 0.05), consistent with greater α1B-adrenergic receptor transcript expression observed in small femoral arteries from neonatal llama. The llama newborn demonstrates significantly greater α-adrenergic peripheral vascular reactivity compared with neonates from lowland species that could be partially explained by preferential expression of α1B-adrenergic receptor subtype.

Role of the RhoA/ROCK pathway in high-altitude associated neonatal pulmonary hypertension in lambs

Exposure to high-altitude chronic hypoxia during pregnancy may cause pulmonary hypertension in neonates, as a result of vasoconstriction and vascular remodeling. We hypothesized that susceptibility to pulmonary hypertension, due to an augmented expression and activity of the RhoA/Rho-kinase (ROCK) pathway in these neonates, can be reduced by daily administration of fasudil, a ROCK inhibitor. We studied 10 highland newborn lambs with conception, gestation, and birth at 3,600 m in Putre, Chile. Five highland controls (HLC) were compared with 5 highland lambs treated with fasudil (HL-FAS; 3 mg·kg(-1)·day(-1) iv for 10 days). Ten lowland controls were studied in Lluta (50 m; LLC). During the 10 days of fasudil daily administration, the drug decreased pulmonary arterial pressure (PAP) and resistance (PVR), basally and during a superimposed episode of acute hypoxia. HL-FAS small pulmonary arteries showed diminished muscular area and a reduced contractile response to the thromboxane analog U46619 compared with HLC. Hypoxia, but not fasudil, changed the protein expression pattern of the RhoA/ROCKII pathway. Moreover, HL-FAS lungs expressed less pMYPT1(T850) and pMYPT1T(696) than HLC, with a potential increase of the myosin light chain phosphatase activity. Finally, hypoxia induced RhoA, ROCKII, and PKG mRNA expression in PASMCs of HLC, but fasudil reduced them (HL-FAS) similarly to LLC. We conclude that fasudil decreases the function of the RhoA/ROCK pathway, reducing the PAP and PVR in chronically hypoxic highland neonatal lambs. The inhibition of ROCKs by fasudil may offer a possible therapeutic tool for the pulmonary hypertension of the neonates.

Counterpoint: High Altitude is not for the Birds!

The best evidence for the adaptation of a species to its environment is that it reproduces efficiently and passes genes to future generations. This has occurred in mammals and birds resident at high altitude, despite the low oxygen availability, which is challenging at all stages of life. High

Repeated doses of the perfluorocarbon FC-100 improve lung function of preterm lambs

Intratracheal administration of a single dose of the perfluorocarbon FC-100 improves lung function in surfactant-deficient animals. In this study we compared the response to repeated doses of FC-100 (3 mL/kg 3% solution.n = 5) with that observed after administration of Exosurf (5 mL/kg,n = 5) to mechanically ventilated preterm lambs of 125 d of gestation. The initial dose of FC-100 rapidly increased arterial Po2, decreased arterial Pco2, and improved arterial pH. Also dynamic lung compliance markedly improved with this agent. Administration of an additional dose of FC-100 resulted in relatively similar changes, albeit of lesser magnitude than those observed with the initial dose. In contrast, Exosurf did not improve these variables even after three doses. All lambs treated with FC-100 survived the 6-h study period, whereas one of the five Exosurf-treated lambs survived (p < 0.05). Mean arterial blood pressure and heart rate decreased in those lambs that received FC-100, but not in surviving lambs that received Exosurf. Our data demonstrate that repeated intratracheal administration of the perfluorocarbon FC-100 improves lung function and survival of surfactant-deficient lambs better than the synthetic surfactant Exosurf. We speculate that tensio-active agents with properties different from surfactant, such as FC-100, might improve lung function in preterm neonates with diseases due to surfactant deficiency.

The Effect of Oxygen Enrichment on Cardiorespiratory and Neuropsychological Responses in Workers With Chronic Intermittent Exposure to High Altitude (ALMA, 5,050 m)

It is estimated that labor activity at high altitudes in Chile will increase from 60,000 to 120,000 workers by the year 2020. Oxygenation of spaces improves the quality of life for workers at high geographic altitudes (<5,000 m). The aim of this study was to determine the effect of a mobile oxygen module system on cardiorespiratory and neuropsychological performance in a population of workers from Atacama Large Millimeter/submillimeter Array (ALMA, 5,050 m) radiotelescope in the Chajnantor Valley, Chile. We evaluated pulse oximetry, systolic and diastolic arterial pressure (SAP/DAP), and performed neuropsychological tests (Mini-Mental State examination, Rey-Osterrieth Complex Figure test) at environmental oxygen conditions (5,050 m), and subsequently in a mobile oxygenation module that increases the fraction of oxygen in order to mimic the higher oxygen partial pressure of lower altitudes (2,900 m). The use of module oxygenation at an altitude of 5,050 m, simulating an altitude of 2,900 m, increased oxygen saturation from 84 ± 0.8 to 91 ± 0.8% (p < 0.00001), decreased heart rate from 90 ± 8 to 77 ± 12 bpm (p < 0.01) and DAP from 96 ± 3 to 87 ± 5 mmHg (p < 0.01). In addition, mental cognitive state of workers (Mini-Mental State Examination) shown an increased from 19 to 31 points (p < 0.02). Furthermore, the Rey-Osterrieth Complex Figure test (memory) shown a significant increase from 35 to 70 (p < 0.0001). The results demonstrate that the use of an oxygen module system at 5,050 m, simulating an altitude equivalent to 2,900 m, by increasing FiO2 at 28%, significantly improves cardiorespiratory response and enhances neuropsychological performance in workers exposed to an altitude of 5,050 m.

Cerebral Oxygen Uptake (VO2cer) Decreases DUring Acute Hypoxemia in The Llama Fetus

Cerebral blood flow (CBF) in the llama fetus does not augment during acute hypoxemia, decreasing cerebral oxygen delivery. To investigate whether VO2 cer could be maintained during hypoxemia by increasing cerebral oxygen extraction, we placed catheters in ascending and descending aorta, inferior vena cava and sagital sinus in 7 llama fetuses (0.6-0.7 of gestation). At 4th days post-surgery, measurements were made of hemoglobin saturation, carotid (Ca O2) and sagital (CsO2) oxygen content, CBF (radiolabelled micropheres) basally and after 60 min of hypoxemia) (15% Hb sat in ascending aorta). Results: means ± SEM; *p<0.05 Basal vs. Hypoxemia. Table

MATERNAL ADMINISTRATION OF THYROTROPIN-RELEASING HORMONE |[lpar]|TRH|[rpar]| AND BETAMETHASONE |[lpar]|B|[rpar]| INCREASES LUNG COMPLIANCE IN PREMATURE LAMBS

Administration of TRH and B to mothers decreases the incidence of bronchopulmonary dysplasia in premature babies. Fetal administration of TRH and Cortisol at 0.85 of gestation increases lung compliance in premature lambs. Since it is not known whether maternal administration of TRH plus B in sheep (in similar doses as in humans) modifies fetal lung compliance, we measured the changes of fetal lung volume induced “in vitro” by pressures of 40, 10, and 5 cm of H2O into the fetal trachea (V40, V10, V5). Twenty one fetal sheep at 0.83 of gestation were divided in three groups: Group TRH + B (n=5) the mothers received TRH (400 ug q 8 h for 6 doses, i.v.) plus B (12 mg q day for 2 doses, i.m.), mothers in Group B (n=5) received only B (same doses), while Group C were 11 control fetuses. The results are expressed as ml air/g of wet fetal lung (ml/g).Administration of TRH and B to mothers has greater effects on fetal lung compliance than B alone when given to the pregnant ewe at similar doses to those used in humans. Our results are consistent with the hypothesis that TRH and B act synergistically on fetal lung maturation.

Plasmatic Concentrations of ADMA and Homocystein in Llama (Lama glama) and Regulation of Arginase Type II: An Animal Resistent to the Development of Pulmonary Hypertension Induced by Hypoxia

There are animal species that have adapted to life at high altitude and hypobaric hypoxia conditions in the Andean highlands. One such species is the llama (Lama glama), which seem to have developed efficient protective mechanisms to avoid maladaptation resulting from chronic hypoxia, such as a resistance to the development of hypoxia -induced pulmonary hypertension. On the other hand, it is widely known that different models of hypertension can arise as a result of changes in endothelial function. The respect, one of the common causes of deregulation in endothelial vasodilator function have been associated with down-regulation of the NO synthesis and an increase in plasma levels of asymmetric dimethylarginine (ADMA) and homocysteine. Additionally, it is also known that NO production can be regulated by plasma levels of L-arginine as a result of the competition between nitric oxide synthase (NOS) and arginase. The objective of this study, was to determine the baseline concentrations of ADMA and homocysteine in llama, and to evaluate their effect on the arginase pathway and their involvement in the resistance to the development of altitude-induced pulmonary hypertension. METHOD: Lowland and highland newborn sheep and llama were investigated near sea level and at high altitude. Blood determinations of arterial blood gases, ADMA and homocysteíne are made and the effect of these on the arginase activity was evaluated. RESULTS: The basal concentrations of ADMA and homocysteine were determined in llama, and they were found to be significantly lower than those found in other species and in addition, the exposure to hypoxia is unable to increase its concentration. On the other hand, it was observed that the llama exhibited 10 times less arginase II activity as compared to sheep, and the expression was not induced by hypoxia. Finally, ADMA y Hcy, has no effect on the type II arginase pathway. CONCLUSION: Based on our results, we propose that low concentrations of ADMA and homocysteine found in llamas, the low expression of arginase type II, DDAH-2 and CBS, as well as its insensitivity to activation by homocysteine could constitute an adaptation mechanism of these animals to the hypoxia.