Purinergic transmission in the rostral but not caudal medullary raphe contributes to the hypercapnia-induced ventilatory response in unanesthetized rats

The medullary raphe (MR) is a putative central chemoreceptor site, contributing to hypercapnic respiratory responses elicited by changes in brain PCO2/pH. Purinergic mechanisms in the central nervous system appear to contribute to central chemosensitivity. To further explore the role of P2 receptors within the rostral and caudal MR in relation to respiratory control in room air and hypercapnic conditions, we performed microinjections of PPADS, a non-selective P2X antagonist, in conscious rats. Microinjections of PPADS into the rostral or caudal MR produced no changes in the respiratory frequency, tidal volume and ventilation in room air condition. The ventilatory response to hypercapnia was attenuated after microinjection of PPADS into the rostral but not in the caudal MR when compared to the control group (vehicle microinjection). These data suggest that P2X receptors in the rostral MR contribute to the ventilatory response to CO2, but do not participate in the tonic maintenance of ventilation under room air condition in conscious rats. (C) 2012 Elsevier B.V. All rights reserved.

Opioid mu-receptors in the rostral medullary raphe modulate hypoxia-induced hyperpnea in unanesthetized rats

Aim: It has been suggested that the medullary raphe (MR) plays a key role in the physiological responses to hypoxia. As opioid mu-receptors have been found in the MR, we studied the putative role of opioid mu-receptors in the rostral MR (rMR) region on ventilation in normal and 7% hypoxic conditions. Methods: We measured pulmonary ventilation ((V) over dotE) and the body temperatures (Tb) of male Wistar rats before and after the selective opioid l-receptor antagonist CTAP ( d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH2, cyclic, 0.1 mu g per 0.1 mu L) was microinjected into the rMR during normoxia or after 60 min of hypoxia. Results: The animals treated with intra-rMR CTAP exhibited an attenuation of the ventilatory response to hypoxia ( 430 +/- 86 mL kg) 1 min) 1) compared with the control group ( 790 +/- 82 mL kg) 1 min) 1) ( P < 0.05). No differences in the Tb were observed between groups during hypoxia. Conclusion: These data suggest that opioids acting on l-receptors in the rMR exert an excitatory modulation of hyperventilation induced by hypoxia.

POLYCHAETE ASSEMBLAGE OF AN IMPACTED ESTUARY, GUANABARA BAY, RIO DE JANEIRO, BRAZIL

Thirty-eight stations were sampled in Guanabara Bay, Rio de Janeiro, Brazil, to assess the spatio-temporal diversity and biomass of sublittoral polychaetes. Samples were collected during the dry (September 2000) and rainy season (May 2001) in shallow sublittoral sediments. The polychaete spatial composition showed a heterogeneous distribution throughout the bay. A negative gradient of diversity and biomass was observed towards the inner parts of the bay and sheltered areas. A wide azoic area was found inside the bay. Some high-biomass and low-diversity spots were found near a sewage-discharge point. In these areas, the polychaete biomass increased after the rainy season. A diversified polychaete community was identified around the bay mouth, with no dramatic changes of this pattern between the two sampling periods. Deposit-feeders were dominant in the entire study area. The relative importance of carnivores and omnivores increased towards the outer sector, at stations with coarse sediment fractions. Guanabara Bay can be divided into three main zones with respect to environmental conditions and polychaete diversity and biomass patterns: A) High polychaete diversity, hydrodynamically exposed areas composed of sandy, oxidized or moderately reduced sediments with normoxic conditions in the water column. B) Low diversity and high biomass of deposit and suspension-feeding polychaete species in the middle part of the bay near continental inflows, comprising stations sharing similar proportions of silt, clay and fine sands. C) Azoic area or an impoverished polychaete community in hydrodynamically low-energy areas of silt and clay with extremely reduced sediments, high total organic matter content and hypoxic conditions in the water column, located essentially from the mid-bay towards the north sector. High total organic matter content and hypoxic conditions combined with slow water renewal in the inner bay seemed to play a key role in the polychaete diversity and biomass. Sedimentation processes and organic load coming from untreated sewage into the bay may have negatively affected the survivorship of the fauna.

The breathing pattern and the ventilatory response to aquatic and aerial hypoxia and hypercarbia in the frog Pipa carvalhoi

Anuran amphibians are known to exhibit an intermittent pattern of pulmonary ventilation and to exhibit an increased ventilatory response to hypoxia and hypercarbia. However, only a few species have been studied to date. The aquatic frog Pipa carvalhoi inhabits lakes, ponds and marshes that are rich in nutrients but low in O-2. There are no studies of the respiratory pattern of this species and its ventilation during hypoxia or hypercarbia. Accordingly, the aim of the present study was to characterize the breathing pattern and the ventilatory response to aquatic and aerial hypoxia and hypercarbia in this species. With this purpose, pulmonary ventilation (V-1) was directly measured by the pneumotachograph method during normocapnic normoxia to determine the basal respiratory pattern and during aerial and aquatic hypercarbia (5% CO2) and hypoxia (5% O-2). Our data demonstrate that P. carvalhoi exhibits a periodic breathing pattern composed of single events (single breaths) of pulmonary ventilation separated by periods of apnea. The animals had an enhanced V-1 during aerial hypoxia, but not during aquatic hypoxia. This increase was strictly the result of an increase in the breathing frequency. A pronounced increase in V-1 was observed if the animals were simultaneously exposed to aerial and aquatic hypercarbia, whereas small or no ventilatory responses were observed during separately administered aerial or aquatic hypercarbia. P. carvalhoi primarily inhabits an aquatic environment. Nevertheless, it does not respond to low O-2 levels in water, although it does so in air. The observed ventilatory responses to hypercarbia may indicate that this species is similar to other anurans in possessing central chemoreceptors. (C) 2012 Elsevier Inc. All rights reserved.

Central leptin replacement enhances chemorespiratory responses in leptin-deficient mice independent of changes in body weight

Previous studies showed that leptin-deficient (ob/ob) mice develop obesity and impaired ventilatory responses to CO2 . In this study, we examined if leptin replacement improves chemorespiratory responses to hypercapnia (7 % CO2) in ob/ob mice and if these effects were due to changes in body weight or to the direct effects of leptin in the central nervous system (CNS). was measured via plethysmography in obese leptin-deficient- (ob/ob) and wild-type- (WT) mice before and after leptin (10 mu g/2 mu l day) or vehicle (phosphate buffer solution) were microinjected into the fourth ventricle for four consecutive days. Although baseline was similar between groups, obese ob/ob mice exhibited attenuated compared to WT mice (134 +/- 9 versus 196 +/- 10 ml min(-1)). Fourth ventricle leptin treatment in obese ob/ob mice significantly improved (from 131 +/- 15 to 197 +/- 10 ml min(-1)) by increasing tidal volume (from 0.38 +/- 0.03 to 0.55 +/- 0.02 ml, vehicle and leptin, respectively). Subcutaneous leptin administration at the same dose administered centrally did not change in ob/ob mice. Central leptin treatment in WT had no effect on . Since the fourth ventricle leptin treatment decreased body weight in ob/ob mice, we also examined in lean pair-weighted ob/ob mice and found it to be impaired compared to WT mice. Thus, leptin deficiency, rather than obesity, is the main cause of impaired in ob/ob mice and leptin appears to play an important role in regulating chemorespiratory response by its direct actions on the CNS.

Periaqueductal gray matter modulates the hypercapnic ventilatory response

The periaqueductal gray (PAG) is a midbrain structure directly involved in the modulation of defensive behaviors. It has direct projections to several central nuclei that are involved in cardiorespiratory control. Although PAG stimulation is known to elicit respiratory responses, the role of the PAG in the CO2-drive to breathe is still unknown. The present study assessed the effect of chemical lesion of the dorsolateral and dorsomedial and ventrolateral/lateral PAG (dlPAG, dmPAG, and vPAG, respectively) on cardiorespiratory and thermal responses to hypercapnia. Ibotenic acid (IBO) or vehicle (PBS, Sham group) was injected into the dlPAG, dmPAG, or vPAG of male Wistar rats. Rats with lesions outside the dlPAG, dmPAG, or vPAG were considered as negative controls (NC). Pulmonary ventilation (Ve), mean arterial pressure (MAP), heart rate (HR), and body temperature (Tb) were measured in unanesthetized rats during normocapnia and hypercapnic exposure (5, 15, 30 min, 7 % CO2). IBO lesioning of the dlPAG/dmPAG caused 31 % and 26.5 % reductions of the respiratory response to CO2 (1,094.3 +/- 115 mL/kg/min) compared with Sham (1,589.5 +/- 88.1 mL/kg/min) and NC groups (1,488.2 +/- 47.7 mL/kg/min), respectively. IBO lesioning of the vPAG caused 26.6 % and 21 % reductions of CO2 hyperpnea (1,215.3 +/- 108.6 mL/kg/min) compared with Sham (1,657.3 +/- 173.9 mL/kg/min) and NC groups (1,537.6 +/- 59.3). Basal Ve, MAP, HR, and Tb were not affected by dlPAG, dmPAG, or vPAG lesioning. The results suggest that dlPAG, dmPAG, and vPAG modulate hypercapnic ventilatory responses in rats but do not affect MAP, HR, or Tb regulation in resting conditions or during hypercapnia.

Distinct mechanisms underlie adaptation of proximal tubule Na+/H+ exchanger isoform 3 in response to chronic metabolic and respiratory acidosis

The Na+/H+ exchanger isoform 3 (NHE3) is essential for HCO3- reabsorption in renal proximal tubules. The expression and function of NHE3 must adapt to acid-base conditions. The goal of this study was to elucidate the mechanisms responsible for higher proton secretion in proximal tubules during acidosis and to evaluate whether there are differences between metabolic and respiratory acidosis with regard to NHE3 modulation and, if so, to identify the relevant parameters that may trigger these distinct adaptive responses. We achieved metabolic acidosis by lowering HCO3- concentration in the cell culture medium and respiratory acidosis by increasing CO2 tension in the incubator chamber. We found that cell-surface NHE3 expression was increased in response to both forms of acidosis. Mild (pH 7.21 +/- 0.02) and severe (6.95 +/- 0.07) metabolic acidosis increased mRNA levels, at least in part due to up-regulation of transcription, whilst mild (7.11 +/- 0.03) and severe (6.86 +/- 0.01) respiratory acidosis did not up-regulate NHE3 expression. Analyses of the Nhe3 promoter region suggested that the regulatory elements sensitive to metabolic acidosis are located between -466 and -153 bp, where two consensus binding sites for SP1, a transcription factor up-regulated in metabolic acidosis, were localised. We conclude that metabolic acidosis induces Nhe3 promoter activation, which results in higher mRNA and total protein level. At the plasma membrane surface, NHE3 expression was increased in metabolic and respiratory acidosis alike, suggesting that low pH is responsible for NHE3 displacement to the cell surface.

Low Apgar scores at 5 minutes in a low risk population: maternal and obstetrical factors and postnatal outcome

Objective: To evaluate the association between Apgar scores of less than seven at five minutes (AS(5min) < 7) and antenatal factors and postnatal outcomes. Methods: A retrospective cohort and case-control study of 27,252 consecutive term newborns in a low risk obstetrical population between January 2003 and December 2010. Maternal and infant databases were reviewed from all cases with AS(5min) < 7 (n = 121; 0.4%) and 363 cases with AS(5min) >= 7 at 5 minutes who were randomly selected by a computer program. The main outcomes were neonatal death, newborn respiratory distress, need for orotracheal intubation and neonatal intensive care unit (NICU), and hypoxic-ischemic-encephalopathy. Results: After multiple regression analysis, repeated late decelerations on cardiotocography (OR: 2.4; 95% CI: 1.4-4.1) and prolonged second stage of labor (OR: 3.3; 95% CI: 1.3-8.3) were associated with AS(5min) < 7, as well as neonatal respiratory distress (OR: 3.0; 95% CI: 1.3-6.9), orotracheal intubation (OR: 2.5; 95% CI: 1.2-4.8), need for NICU (OR: 9.5; 95% CI: 6.7-16.8), and hypoxic-ischemic-encephalopathy (OR: 14.1; 95% CI: 3.6-54.7). No other antenatal factors were associated with AS(5min) < 7 (p > 0.05). Conclusion: Repeated late decelerations and prolonged second stage of labor in the low-risk population are predictors of AS(5min) < 7, a situation associated with increased risk of neonatal respiratory distress, need for mechanical ventilatory support and NICU, and hypoxic-ischemic-encephalopathy.

Cerebral White Matter Oxidation and Nitrosylation in Young Rodents With Kaolin-Induced Hydrocephalus

Hydrocephalus is associated with reduced blood flow in periventricular white matter. To investigate hypoxic and oxidative damage in the brains of rats with hydrocephalus, kaolin was injected into the cisterna magna of newborn 7- and 21-day-old Sprague-Dawley rats, and ventricle size was assessed by magnetic resonance imaging at 7, 21, and 42 days of age. In-situ evidence of hypoxia in periventricular capillaries and glial cells was shown by pimonidazole hydrochloride binding. Biochemical assay of thiobarbituric acid reaction and immunohistochemical detection of malondialdehyde and 4-hydroxy-2-nonenal indicated the presence of lipid peroxidation in white matter. Biochemical assay of nitrite indicated increased nitric oxide production. Nitrotyrosine immunohistochemistry showed nitrosylated proteins in white matter reactive microglia and astrocytes. Activities of the antioxidant enzymes catalase and glutathione peroxidase were not increased, and altered hypoxia-inducible factor 1 alpha was not detected by quantitative reverse transcription-polymerase chain reaction. Cerebral vascular endothelial growth factor expression determined by quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay was not changed, but vascular endothelial growth factor immunoreactivity was increased in reactive astrocytes of hydrocephalic white matter. To determine if nitric oxide synthase is involved in the pathogenesis, we induced hydrocephalus in 7-day-old wild-type and neuronal nitric oxide synthase-deficient mice. At 7 days, the wild-type and mutant mice exhibited equally severe ventriculomegaly and no behavioral differences, although increased glial fibrillary acidic protein was less in the mutant mice. We conclude that hypoxia, via peroxidation and nitrosylation, contributes to brain changes in young rodents with hydrocephalus and that compensatory mechanisms are negligible.

Near-Term Fetal Hypoxia-Ischemia in Rabbits MRI Can Predict Muscle Tone Abnormalities and Deep Brain Injury

Background and Purpose-The pattern of antenatal brain injury varies with gestational age at the time of insult. Deep brain nuclei are often injured at older gestational ages. Having previously shown postnatal hypertonia after preterm fetal rabbit hypoxia-ischemia, the objective of this study was to investigate the causal relationship between the dynamic regional pattern of brain injury on MRI and the evolution of muscle tone in the near-term rabbit fetus. Methods-Serial MRI was performed on New Zealand white rabbit fetuses to determine equipotency of fetal hypoxia-ischemia during uterine ischemia comparing 29 days gestation (E29, 92% gestation) with E22 and E25. E29 postnatal kits at 4, 24, and 72 hours after hypoxia-ischemia underwent T2- and diffusion-weighted imaging. Quantitative assessments of tone were made serially using a torque apparatus in addition to clinical assessments. Results-Based on the brain apparent diffusion coefficient, 32 minutes of uterine ischemia was selected for E29 fetuses. At E30, 58% of the survivors manifested hind limb hypotonia. By E32, 71% of the hypotonic kits developed dystonic hypertonia. Marked and persistent apparent diffusion coefficient reduction in the basal ganglia, thalamus, and brain stem was predictive of these motor deficits. Conclusions-MRI observation of deep brain injury 6 to 24 hours after near-term hypoxia-ischemia predicts dystonic hypertonia postnatally. Torque-displacement measurements indicate that motor deficits in rabbits progressed from initial hypotonia to hypertonia, similar to human cerebral palsy, but in a compressed timeframe. The presence of deep brain injury and quantitative shift from hypo-to hypertonia may identify patients at risk for developing cerebral palsy. (Stroke. 2012;43:2757-2763.)

Dichotomous effects of VEGF-A on adipose tissue dysfunction

Obese fat pads are frequently undervascularized and hypoxic, leading to increased fibrosis, inflammation, and ultimately insulin resistance. We hypothesized that VEGF-A-induced stimulation of angiogenesis enables sustained and sufficient oxygen and nutrient exchange during fat mass expansion, thereby improving adipose tissue function. Using a doxycycline (Dox)-inducible adipocyte-specific VEGF-A overexpression model, we demonstrate that the local up-regulation of VEGF-A in adipocytes improves vascularization and causes a "browning" of white adipose tissue (AT), with massive up-regulation of UCP1 and PGC1 alpha. This is associated with an increase in energy expenditure and resistance to high fat diet-mediated metabolic insults. Similarly, inhibition of VEGF-A-induced activation of VEGFR2 during the early phase of high fat diet-induced weight gain, causes aggravated systemic insulin resistance. However, the same VEGF-A-VEGFR2 blockade in ob/ob mice leads to a reduced body-weight gain, an improvement in insulin sensitivity, a decrease in inflammatory factors, and increased incidence of adipocyte death. The consequences of modulation of angiogenic activity are therefore context dependent. Proangiogenic activity during adipose tissue expansion is beneficial, associated with potent protective effects on metabolism, whereas antiangiogenic action in the context of preexisting adipose tissue dysfunction leads to improvements in metabolism, an effect likely mediated by the ablation of dysfunctional proinflammatory adipocytes.

Aspergillus fumigatus mitochondrial electron transport chain mediates oxidative stress homeostasis, hypoxia responses and fungal pathogenesis

We previously observed that hypoxia is an important component of host microenvironments during pulmonary fungal infections. However, mechanisms of fungal growth in these in vivo hypoxic conditions are poorly understood. Here, we report that mitochondrial respiration is active in hypoxia (1% oxygen) and critical for fungal pathogenesis. We generated Aspergillus fumigatus alternative oxidase (aoxA) and cytochrome C (cycA) null mutants and assessed their ability to tolerate hypoxia, macrophage killing and virulence. In contrast to ?aoxA, ?cycA was found to be significantly impaired in conidia germination, growth in normoxia and hypoxia, and displayed attenuated virulence. Intriguingly, loss of cycA results in increased levels of AoxA activity, which results in increased resistance to oxidative stress, macrophage killing and long-term persistence in murine lungs. Thus, our results demonstrate a previously unidentified role for fungal mitochondrial respiration in the pathogenesis of aspergillosis, and lay the foundation for future research into its role in hypoxia signalling and adaptation.

Suppression of Hypoxia-Inducible Factor-1 alpha Contributes to the Antiangiogenic Activity of Red Propolis Polyphenols in Human Endothelial Cells

Polyphenol-enriched fractions from natural sources have been proposed to interfere with angiogenesis in pathological conditions. We recently reported that red propolis polyphenols (RPP) exert antiangiogenic activity. However, molecular mechanisms of this activity remain unclear. Here, we aimed at characterizing molecular mechanisms to explain the impact of RPP on endothelial cells' (EC) physiology. We used in vitro and ex and in vivo models to test the hypothesis that RPP inhibit angiogenesis by affecting hypoxia-inducible factor-1 alpha (HIF1 alpha) stabilization in EC. RPP (10 mg/L) affected angiogenesis by reducing migration and sprouting of EC, attenuated the formation of new blood vessels, and decreased the differentiation of embryonic stem cells into CD31-positive cells. Moreover, RPP (10 mg/L) inhibited hypoxia- or dimethyloxallylglycine-induced mRNA and protein expression of the crucial angiogenesis promoter vascular endothelial growth factor (VEGF) in a time-dependent mariner. Under hypoxic conditions, RPP at 10 mg/L, supplied for 1-4 h, decreased HIF1 alpha protein accumulation, which in turn attenuated VEGF gene expression. In addition, RPP reduced the HIF1 alpha protein half-life from similar to 58 min to 38 min under hypoxic conditions. The reduced HIF1 alpha protein half-life was associated with an increase in the von Hippel-Lindau (pVHL)-dependent proteasomal degradation of HIF1 alpha. RPP (10 mg/L, 4 h) downregulated Cdc42 protein expression. This caused a corresponding increase in pVHL protein levels and a subsequent degradation of HIF1 alpha. In summary, we have elucidated the underlying mechanism for the antiangiogenic action of RPP, which attenuates HIF1 alpha protein accumulation and signaling. J. Nutr. 142: 441-447, 2012.

Mechanisms of blunted muscle vasodilation during peripheral chemoreceptor stimulation in heart failure patients

We described recently that systemic hypoxia provokes vasoconstriction in heart failure (HF) patients. We hypothesized that either the exaggerated muscle sympathetic nerve activity and/or endothelial dysfunction mediate the blunted vasodilatation during hypoxia in HF patients. Twenty-seven HF patients and 23 age-matched controls were studied. Muscle sympathetic nerve activity was assessed by microneurography and forearm blood flow (FBF) by venous occlusion plethysmography. Peripheral chemoreflex control was evaluated through the inhaling of a hypoxic gas mixture (10% O-2 and 90% N-2). Basal muscle sympathetic nerve activity was greater and basal FBF was lower in HF patients versus controls. During hypoxia, muscle sympathetic nerve activity responses were greater in HF patients, and forearm vasodilatation in HF was blunted versus controls. Phentolamine increased FBF responses in both groups, but the increase was lower in HF patients. Phentolamine and N-G-monomethyl-L-arginine infusion did not change FBF responses in HF but markedly blunted the vasodilatation in controls. FBF responses to hypoxia in the presence of vitamin C were unchanged and remained lower in HF patients versus controls. In conclusion, muscle vasoconstriction in response to hypoxia in HF patients is attributed to exaggerated reflex sympathetic nerve activation and blunted endothelial function (NO activity). We were unable to identify a role for oxidative stress in these studies. (Hypertension. 2012; 60: 669-676.) . Online Data Supplement

Differential expression of HIF-1α in CD44+CD24-/low breast ductal carcinomas

Abstract Background Cancer stem cell (CSC) hypothesis postulates that tumors are maintained by a self-renewing CSC population that is also capable of differentiating into non-self-renewing cell populations that constitute the bulk of tumor. Stem cells renewal and differentiation can be directly influenced by the oxygen levels of determined tissues, probably by the reduction of oxidative DNA damage in hypoxic regions, thus leading to a friendlier microenvironment, regarding to clonal expansion and for resistance to chemotherapeutic regimens. Furthermore, there have been strong data indicating a pivotal role of hypoxic niche in cancer stem cells development. There are evidence that hypoxia could drive the maintenance of CSC, via HIF-1α expression, but it still to be determined whether hypoxia markers are expressed in breast tumors presenting CD44+CD24-/low immunophenotype. Methods Immunohistochemical analysis of CD44+CD24-/low expression and its relationship with hypoxia markers and clinical outcome were evaluated in 253 samples of breast ductal carcinomas. Double-immunolabeling was performed using EnVision Doublestain System (Dako, Carpinteria, CA, USA). Slides were then scanned into high-resolution images using Aperio ScanScope XT and then, visualized in the software Image Scope (Aperio, Vista, CA, USA). Results In univariate analysis, CD44+CD24-/low expression showed association with death due to breast cancer (p = 0.035). Breast tumors expressing CD44+CD24-/low immunophenotype showed relationship with HIF-1α (p = 0.039) and negativity for HER-2 (p = 0.013). Conclusion Considering that there are strong evidences that the fraction of a tumour considered to be cancer stem cells is plastic depending upon microenvironmental signals, our findings provide further evidence that hypoxia might be related to the worse prognosis found in CD44+CD24-/low positive breast tumors.