Role of Rho-kinase in mediating contraction of chicken embryo femoral arteries

Rho-kinase-dependent Ca(2+) sensitization is an essential process for contraction of mammalian vascular smooth muscle but the information about its effects in non-mammalian vessels is scarce. We aimed to investigate, using the Rho-kinase inhibitor hydroxyfasudil, the potential role of the Rho-kinase pathway of Ca(2+) sensitization in depolarization- and agonist-mediated contraction of chicken embryo (at day 19 of the 21 days of incubation) femoral arteries. Contraction elicited by KCl (125 mM) comprised two phases (phasic and tonic contraction), both of which were abolished in the absence of extracellular Ca(2+). Hydroxyfasudil (10 μM) left the initial phasic component nearly intact but abolished the tonic component. Hydroxyfasudil also induced a marked impairment of the contractions elicited by phenylephrine (PE), the thromboxane A(2) mimetic U46619, and endothelin-1. In contrast, inhibition of protein kinase C (PKC) by chelerythrine did not affect KCl- or PE-induced contractions, indicating lack of participation of PKC-mediated Ca(2+) sensitization. Incubation under chronic hypoxia (15% O(2) from day 0) impaired embryonic growth but did not significantly affect hydroxyfasudil-mediated relaxation. In summary, our findings are indicative of a role for Rho-kinase activity in depolarization- and agonist-induced force generation in chicken embryo femoral arteries

Endotoxin Induced Chorioamnionitis Prevents Intestinal Development during Gestation in Fetal Sheep

Chorioamnionitis is the most significant source of prenatal inflammation and preterm delivery. Prematurity and prenatal inflammation are associated with compromised postnatal developmental outcomes, of the intestinal immune defence, gut barrier function and the vascular system. We developed a sheep model to study how the antenatal development of the gut was affected by gestation and/or by endotoxin induced chorioamnionitis

Hypoxia sensing in the fetal chicken femoral artery is mediated by the mitochondrial electron transport chain

Zoer B, Cogolludo AL, Perez-Vizcaino F, De Mey JG, Blanco CE, Villamor E. Hypoxia sensing in the fetal chicken femoral artery is mediated by the mitochondrial electron transport chain. Am J Physiol Regul Integr Comp Physiol 298: R1026-R1034, 2010. First published January 20, 2010; doi: 10.1152/ajpregu.00500.2009.Vascular hypoxia sensing is transduced into vasoconstriction in the pulmonary circulation, whereas systemic arteries dilate. Mitochondrial electron transport chain (mETC), reactive O-2 species (ROS), and K+ channels have been implicated in the sensing/signaling mechanisms of hypoxic relaxation in mammalian systemic arteries. We aimed to investigate their putative roles in hypoxia-induced relaxation in fetal chicken (19 days of incubation) femoral arteries mounted in a wire myograph. Acute hypoxia (PO2 similar to 2.5 kPa) relaxed the contraction induced by norepinephrine (1 mu M). Hypoxia-induced relaxation was abolished or significantly reduced by the mETC inhibitors rotenone (complex I), myxothiazol and antimycin A (complex III), and NaN3 (complex IV). The complex II inhibitor 3-nitroproprionic acid enhanced the hypoxic relaxation. In contrast, the relaxations mediated by acetylcholine, sodium nitroprusside, or forskolin were not affected by the mETC blockers. Hypoxia induced a slight increase in ROS production (as measured by 2,7-dichlorofluorescein-fluorescence), but hypoxia-induced relaxation was not affected by scavenging of superoxide (polyethylene glycol-superoxide dismutase) or H2O2 (polyethylene glycol-catalase) or by NADPH-oxidase inhibition (apocynin). Also, the K+ channel inhibitors tetraethylammonium (nonselective), diphenyl phosphine oxide-1 (voltage-gated K+ channel 1.5), glibenclamide (ATP-sensitive K+ channel), iberiotoxin (large-conductance Ca2+-activated K+ channel), and BaCl2 (inward-rectifying K+ channel), as well as ouabain (Na+-K+-ATPase inhibitor) did not affect hypoxia-induced relaxation. The relaxation was enhanced in the presence of the voltage-gated K+ channel blocker 4-aminopyridine. In conclusion, our experiments suggest that the mETC plays a critical role in O-2 sensing in fetal chicken femoral arteries. In contrast, hypoxia-induced relaxation appears not to be mediated by ROS or K+ channels

Mitochondrial DNA damage analysis in bronchoalveolar lavage cells of preterm infants

In mechanically ventilated preterm infants, the combination of immaturity, volutrauma, oxidative stress, and inflammatory processes can lead to chronic lung injury. Mitochondrial DNA (mtDNA) is more susceptible to oxidative damage than nuclear DNA. We aimed to investigate the level of mtDNA damage (deletions, mutations and changes in copy number) in bronchoalveolar lavage (BAL) cells from 10 preterm infants (27-30 weeks). A first BAL (BAL1) was done within 24 h of endotracheal intubation and BAL2 was performed 30-103 h thereafter. Deletions were analyzed by long range PCR, point mutations by heteroduplex analysis of the D-loop region, and copy number changes by real-time PCR. Using these methods, no deletions were found in any of the BAL samples. When BAL1 and BAL2 samples were compared no new mutations were found. In contrast, a marked decrease in mtDNA copy number was observed in 5 patients. In conclusion, we found that exposure of preterm infants to short term mechanical ventilation did not lead to detrimental consequences for the mtDNA in the form of mutations or deletions

Pulmonary Vascular Endothelial Growth Factor Expression and Disaturated Phospholipid Content in a Chicken Model of Hypoxia-Induced Fetal Growth Restriction

Background: Prenatal hypoxia is an important cause of intrauterine growth retardation that affects fetal lung maturation, although previous studies have rendered conflicting results. The fetal chicken model allows the study of the isolated effects of hypoxia during development. Objectives: We hypothesized that prenatal hypoxia would differentially affect surfactant synthesis, depending on timing and duration of hypoxia. Pulmonary vascular endothelial growth factor (VEGF) expression was analyzed as a possible link between oxygen sensing and surfactant production. Methods: Fertilized White Leghorn eggs were incubated in normoxia, hyperoxia (60% O-2) from day 15 or hypoxia (15% O-2) from either day 6 or day 15 of incubation. Whole lung disaturated phospholipids (DSPL) and mRNA expression of VEGF isoforms were quantified at day 16 and 19. Results: Lung DSPL content increased approximately threefold between day 16 and 19 in control animals. Both hypoxia and hyperoxia from day 15 significantly increased DSPL content at day 19 versus control (103 +/- 22 and 116 +/- 18 vs. 81 +/- 15 mu g/mg protein, p <0.01 and p <0.001, respectively), while long-term hypoxia tended to decrease DSPL content (65 +/- 17 mu g/mg protein, p = 0.056). No differences in DSPL content were observed at day 16. Short-term hypoxia transiently up-regulated VEGF146 1.5-fold at day 16 (p <0.05). A similar trend was observed for VEGF122 (p = 0.058) and VEGF190 (p = 0.08), while no differences were present at day 19. Conclusions: Both prenatal hypoxia and hyperoxia induced during critical windows of lung development differentially modulate surfactant synthesis. Our data support the concept that fetal oxygen tension is a key signal in the regulation of the surfactant system

Brain apoptosis and carotid artery reactivity in fetal asphyctic preconditioning

We aimed to develop a model of fetal hypoxia-ischemia (HI) preconditioning that reflects the pathophysiological conditions of perinatal asphyxia more closely than the existing neonatal stroke models. Fetal asphyxia (FA) was induced by clamping the uterine vasculature on embryonic day E17. At birth (P0), severe perinatal asphyxia (SPA) was induced during cesarean section. At P4, carotid arteries were studied in a wire myograph and at P8 brains were analyzed for apoptotic cell death in the prefrontal cortex and striatum. The contraction induced by K+ was significantly reduced in the carotid arteries from the SPA group and endothelium-dependent relaxation (mediated by acetylcholine) was augmented in the FA group. These changes in vascular responsiveness were not present in the animals exposed to both insults (FA + SPA). Additionally, FA+SPA animals showed lower numbers of apoptotic cells compared to SPA animals in both the prefrontal cortex and striatum. Exposure to a global fetal asphyctic insult seems to protect against the vascular alterations and the increase of apoptosis in striatum and prefrontal cortex induced by severe asphyxia at birth

A Systematic Search and Mapping Review of Studies on Intracerebral Microdialysis of Amino Acids, and Systematized Review of Studies on Circadian Rhythms

Background: Microdialysis can be used to measure amino acids in the extracellular space in vivo, based on the principle of diffusion. Variations in experimental set-up result in variations in baseline levels of the compounds measured. Variations may also be due to circadian rhythms. Method: We systematically searched and mapped the literature on all studies reporting baseline microdialysis measurements of histamine and the amino acids asparagine, aspartate, GABA, glutamate, glutamine, glycine, proline and taurine. We fully reviewed the studies describing circadian rhythms for histamine and the selected amino acids. Results: We retrieved 2331 papers describing baseline measurements of one or more of the compounds of interest. We provide a numerical summary and lists of the publications by compound. We retrieved 11 references describing studies on the circadian rhythms of the compounds of interest. Aspartate, glutamate and histamine are generally higher during the dark than during the light phase in nocturnal rodents. For glutamine, no rhythmicity was observed. For GABA, the results were too inconsistent to generalise. For asparagine, glycine, proline and taurine, insufficient data are available. Conclusion: The literature on intracerebral microdialysis measurements of the amino acids is vast, but certain primary studies are still warranted. Future systematic reviews on the individual compounds can shed light on the effects of experimental variations on baseline concentrations