Functional and developmental studies of the peripheral arterial chemoreceptors in rat: effects of nicotine and possible relation to sudden infant death syndrome.

The drive on respiration mediated by the peripheral arterial chemoreceptors was assessed by the hyperoxic test in 3-day-old rat pups. They accounted for 22.5 +/- 8.8% during control conditions, but only for 6.9 +/- 10.0% after nicotine exposure, an effect counteracted by blockade of peripheral dopamine type 2 receptors (DA2Rs). Furthermore, nicotine reduced dopamine (DA) content and increased the expression of tyrosine hydroxylase (TH) in the carotid bodies, further suggesting that DA mediates the acute effect of nicotine on arterial chemoreceptor function. During postnatal development TH and DA2R mRNA levels in the carotid bodies decreased. Thus, nicotine from smoking may also interfere with the postnatal resetting of the oxygen sensitivity of the peripheral arterial chemoreceptors by increasing carotid body TH mRNA, as well as DA release in this period. Collectively these effects of nicotine on the peripheral arterial chemoreceptors may increase the vulnerability to hypoxic episodes and attenuate the protective chemoreflex response. These mechanisms may underlie the well-known relation between maternal smoking and sudden infant death syndrome.

Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension.

Hypoxia-inducible factor 1 (HIF-1) is found in mammalian cells cultured under reduced O2 tension and is necessary for transcriptional activation mediated by the erythropoietin gene enhancer in hypoxic cells. We show that both HIF-1 subunits are basic-helix-loop-helix proteins containing a PAS domain, defined by its presence in the Drosophila Per and Sim proteins and in the mammalian ARNT and AHR proteins. HIF-1 alpha is most closely related to Sim. HIF-1 beta is a series of ARNT gene products, which can thus heterodimerize with either HIF-1 alpha or AHR. HIF-1 alpha and HIF-1 beta (ARNT) RNA and protein levels were induced in cells exposed to 1% O2 and decayed rapidly upon return of the cells to 20% O2, consistent with the role of HIF-1 as a mediator of transcriptional responses to hypoxia.

Localization of specific erythropoietin binding sites in defined areas of the mouse brain.

The main physiological regulator of erythropoiesis is the hematopoietic growth factor erythropoietin (EPO), which is induced in response to hypoxia. Binding of EPO to the EPO receptor (EPO-R), a member of the cytokine receptor superfamily, controls the terminal maturation of red blood cells. So far, EPO has been reported to act mainly on erythroid precursor cells. However, we have detected mRNA encoding both EPO and EPO-R in mouse brain by reverse transcription-PCR. Exposure to 0.1% carbon monoxide, a procedure that causes functional anemia, resulted in a 20-fold increase of EPO mRNA in mouse brain as quantified by competitive reverse transcription-PCR, whereas the EPO-R mRNA level was not influenced by hypoxia. Binding studies on mouse brain sections revealed defined binding sites for radioiodinated EPO in distinct brain areas. The specificity of EPO binding was assessed by homologous competition with an excess of unlabeled EPO and by using two monoclonal antibodies against human EPO, one inhibitory and the other noninhibitory for binding of EPO to EPO-R. Major EPO binding sites were observed in the hippocampus, capsula interna, cortex, and midbrain areas. Functional expression of the EPO-R and hypoxic upregulation of EPO suggest a role of EPO in the brain.

Mecânica ventilatória, padrão ventilatório e custo metabólico da ventilação emTrachemys scripta e Chelonoidis carbonarius (Testudines: Cryptodira)

Dentro da classe Reptilia, a ordem Testudines possui algumas características exclusivas, tais como a fusão das costelas e da coluna vertebral e a perda dos músculos intercostais, inviabilizando a ventilação costal. Além disso, as posições naturais que os Testudines exibem podem influenciar a mecânica ventilatória. O presente estudo teve como objetivo testar a influência do posicionamento do corpo sobre a mecânica ventilatória através da complacência estática e dinâmica e analisar através da respirometria aberta o padrão ventilatório e o custo metabólico da ventilação através da exposição em normóxia, hipóxia e hipercarbia em Trachemys scripta e Chelonoidis carbonarius. Os volumes pulmonares, complacência estática e dinâmica em C. carbonarius foram inferiores aos de T. scripta e outras espécies de Testudines já estudadas. Verificou-se também influência das posições sobre a mecânica ventilatória nas duas espécies, sendo a posição de membros e cabeça retraídos na carapaça apresentando os menores valores (p<0,05). Hipóxia e hipercarbia estimularam o aumento da ventilação nas duas espécies estudadas (p<0,05), sendo observadas maiores alterações da frequência ventilatória e volume corrente em C. carbonarius. Os valores de custo metabólico da ventilação foram baixos devido à uma diminuição no consumo de oxigênio em hipóxia e hipercarbia, indicando depressão metabólica em ambas as espécies ou então o método para calcular esse custo não ser ideal. Ao relacionar os dados de consumo de oxigênio com os de ventilação, verificou-se a possibilidade de shunt cardíaco esquerdo-direito. Será necessário calcular o trabalho mecânico da ventilação a fim de entender melhor a mecânica ventilatória nas duas espécies e posteriormente relacionar os dados de ventilação e custo metabólico da ventilação com os de trabalho mecânico.

BDNF impairs recovery of synaptic transmission after hypoxia even with blockage of adenosine A2A receptors

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Angiopoietin-1 receptor Tie2 distinguishes multipotent differentiation capability in bovine coccygeal nucleus pulposus cells.

BACKGROUND The intervertebral disc (IVD) has limited self-healing potential and disc repair strategies require an appropriate cell source such as progenitor cells that could regenerate the damaged cells and tissues. The objective of this study was to identify nucleus pulposus-derived progenitor cells (NPPC) and examine their potential in regenerative medicine in vitro. METHODS Nucleus pulposus cells (NPC) were obtained from 1-year-old bovine coccygeal discs by enzymatic digestion and were sorted for the angiopoietin-1 receptor Tie2. The obtained Tie2- and Tie2+ fractions of cells were differentiated into osteogenic, adipogenic, and chondrogenic lineages in vitro. Colony-forming units were prepared from both cell populations and the colonies formed were analyzed and quantified after 8 days of culture. In order to improve the preservation of the Tie2+ phenotype of NPPC in monolayer cultures, we tested a selection of growth factors known to have stimulating effects, cocultured NPPC with IVD tissue, and exposed them to hypoxic conditions (2 % O2). RESULTS After 3 weeks of differentiation culture, only the NPC that were positive for Tie2 were able to differentiate into osteocytes, adipocytes, and chondrocytes as characterized by calcium deposition (p < 0.0001), fat droplet formation (p < 0.0001), and glycosaminoglycan content (p = 0.0095 vs. Tie2- NPC), respectively. Sorted Tie2- and Tie2+ subpopulations of cells both formed colonies; however, the colonies formed from Tie2+ cells were spheroid in shape, whereas those from Tie2- cells were spread and fibroblastic. In addition, Tie2+ cells formed more colonies in 3D culture (p = 0.011) than Tie2- cells. During expansion, a fast decline in the fraction of Tie2+ cells was observed (p < 0.0001), which was partially reversed by low oxygen concentration (p = 0.0068) and supplementation of the culture with fibroblast growth factor 2 (FGF2) (p < 0.0001). CONCLUSIONS Our results showed that the bovine nucleus pulposus contains NPPC that are Tie2+. These cells fulfilled formally progenitor criteria that were maintained in subsequent monolayer culture for up to 7 days by addition of FGF2 or hypoxic conditions. We propose that the nucleus pulposus represents a niche of precursor cells for regeneration of the IVD.

Porewater and particulate geochemistry during Maria S. Merian cruise MSM17/4

We present sedimentary geochemical data and in situ benthic flux measurements of dissolved inorganic nitrogen (DIN: NO3-, NO2-, NH4+) and oxygen (O2) from 7 sites with variable sand content along 18°N offshore Mauritania (NW Africa). Bottom water O2 concentrations at the shallowest station were hypoxic (42 µM) and increased to 125 µM at the deepest site (1113 m). Total oxygen uptake rates were highest on the shelf (-10.3 mmol O2 /m2 d) and decreased quasi-exponentially with water depth to -3.2 mmol O2 /m2 d. Average denitrification rates estimated from a flux balance decreased with water depth from 2.2 to 0.2 mmol N /m2 d. Overall, the sediments acted as net sink for DIN. Observed increases in delta 15NNO3 and delta 18ONO3 in the benthic chamber deployed on the shelf, characterized by muddy sand, were used to calculate apparent benthic nitrate fractionation factors of 8.0 pro mille (15epsilon app) and 14.1 pro mille (18epsilon app). Measurements of delta 15NNO2 further demonstrated that the sediments acted as a source of 15N depleted NO2-. These observations were analyzed using an isotope box model that considered denitrification and nitrification of NH4+ and NO2-. The principal findings were that (i) net benthic 14N/15N fractionation (epsilon DEN) was 12.9 ± 1.7pro mille, (ii) inverse fractionation during nitrite oxidation leads to an efflux of isotopically light NO2- (-22 ± 1.9 pro mille), and (iii) direct coupling between nitrification and denitrification in the sediment is negligible. Previously reported epsilon DEN for fine-grained sediments are much lower (4-8 pro mille). We speculate that high benthic nitrate fractionation is driven by a combination of enhanced porewater-seawater exchange in permeable sediments and the hypoxic, high productivity environment. Although not without uncertainties, the results presented could have important implications for understanding the current state of the marine N cycle.