Neurohistological abnormalities during early porcine endotoxemia.

BACKGROUND Brain dysfunction is common in sepsis. We aimed to assess whether cerebral perfusion, oxygenation, and/or metabolism are abnormal during early endotoxemia, and how they may relate to potential neurohistological changes. METHODS In this prospective animal study, we included 12 pigs (weight: 42 ± 4 kg; mean ± SD) that were exposed to Escherichia coli lipopolysaccharide (E. coli LPS B0111 : B4, 0.4 μg/kg/h) or saline infusion (n = 6, each) for 10 h. Systemic hemodynamics, cerebral blood flow, intracranial pressure, and brain tissue oxygen tension were continuously measured. At the end of the experiment, formalin-fixed brains were cut in coronal sections and embedded in paraffin. Afterwards, the sections were cut at 5 microns and stained with hematoxylin and eosin. RESULTS Stable systemic hemodynamics in both groups were associated with higher carotid arterial blood flow after 10 h of endotoxemia (9.0 ± 2.2 ml/kg/min) compared to controls (6.6 ± 1.2 ml/kg/min; time-group interaction: P = 0.014). Intracranial pressure, cerebral perfusion pressure, brain oxygen consumption, and brain tissue oxygen tension were similar in both groups. In four of the six endotoxemic animals but in none of the controls, cerebral tissue lesions were found (encephalomalacia with spongy degeneration of white matter, axonal swelling, and ischemic neuronal thalamic necrosis), including significant venous vascular alterations, predominantly in the brainstem, in three of the four animals. CONCLUSIONS Early endotoxemia seems to be associated with histological signs of brain damage unrelated to systemic or cerebral hemodynamics or oxygenation.

Oxygen supply and oxygen-dependent gene expression in differentiating embryonic stem cells.

Blastocyst-derived pluripotent mouse embryonic stem cells can differentiate in vitro to form so-called embryoid bodies (EBs), which recapitulate several aspects of murine embryogenesis. We used this in vitro model to study oxygen supply and consumption as well as the response to reduced oxygenation during the earliest stages of development. EBs were found to grow equally well when cultured at 20% (normoxia) or 1% (hypoxia) oxygen during the first 5 days of differentiation. Microelectrode measurements of pericellular oxygen tension within 13- to 14-day-old EBs (diameter 510-890 micron) done at 20% oxygen revealed efficient oxygenation of the EBs' core region. Confocal laser scanning microscopy analysis of EBs incubated with fluorescent dyes that specifically stain living cells confirmed that the cells within an EB were viable. To determine the EBs' capability to sense low oxygen tension and to specifically respond to low ambient oxygen by modulating gene expression we quantified aldolase A and vascular endothelial growth factor (VEGF) mRNAs, since expression of these genes is upregulated by hypoxia in a variety of cells. Compared with the normoxic controls, we found increased aldolase A and VEGF mRNA levels after exposing 8- to 9-day-old EBs to 1% oxygen. We propose that EBs represent a powerful tool to study oxygen-regulated gene expression during the early steps of embryogenesis, where the preimplantation conceptus resides in a fluid environment with low oxygen tension until implantation and vascularization allow efficient oxygenation.

Possibility for a full optical determination of photodynamic therapy outcome

The efficacy of photodynamic therapy (PDT) depends on a variety of parameters: concentration of the photosensitizer at the time of treatment, light wavelength, fluence, fluence rate, availability of oxygen within the illuminated volume, and light distribution in the tissue. Dosimetry in PDT requires the congregation of adequate amounts of light, drug, and tissue oxygen. The adequate dosimetry should be able to predict the extension of the tissue damage. Photosensitizer photobleaching rate depends on the availability of molecular oxygen in the tissue. Based on photosensitizers photobleaching models, high photobleaching has to be associated with high production of singlet oxygen and therefore with higher photodynamic action, resulting in a greater depth of necrosis. The purpose of this work is to show a possible correlation between depth of necrosis and the in vivo photosensitizer (in this case, Photogem (R)) photodegradation during PDT. Such correlation allows possibilities for the development of a real time evaluation of the photodynamic action during PDT application. Experiments were performed in a range of fluence (0-450 J/cm(2)) at a constant fluence rate of 250 mW/cm(2) and applying different illumination times (0-1800 s) to achieve the desired fluence. A quantity was defined (psi) as the product of fluorescence ratio (related to the photosensitizer degradation at the surface) and the observed depth of necrosis. The correlation between depth of necrosis and surface fluorescence signal is expressed in psi and could allow, in principle, a noninvasive monitoring of PDT effects during treatment. High degree of correlation is observed and a simple mathematical model to justify the results is presented.

Retinal and lenticular ultrastructure in the aestivating salamanderfish, Lepidogalaxias salamandroides (Galaxiidae, Teleostei) with special reference to a new type of photoreceptor mosaic

The salamanderfish, Lepidogalaxias salamandroides (Galaxiidae, Teleostei) is endemic to southwestern Australia and inhabits shallow, freshwater pools which evaporate during the hot summer months. Burrowing into the substrate in response to falling water levels allows these fish to aestivate for extended periods of time while encapsulated in a mucous cocoon even when the pools contain no water. Only a few minutes after a major rainfall, these fish emerge into relatively clear water which subsequently becomes laden with tannin, turning the water black and reducing the pH to approximately 4.3. As part of a large study of the visual adaptations of this unique species, the retinal and lenticular morphology of the aestivating salamanderfish is examined at the level of the light and electron microscopes. The inner retina is highly vascularised by a complex system of vitreal blood vessels, while the outer retina receives a blood supply by diffusion from a choriocapillaris. This increased retinal blood supply may be an adaptation for reducing the oxygen tension during critical periods of aestivation. Large numbers of Muller cells traverse the thickness of the retina from the inner to the outer limiting membranes. The ganglion cells are arranged in two ill-defined layers, separated from a thick inner nuclear layer containing two layers of horizontal cells by a soma-free inner plexiform layer. The photoreceptors can be divided into three types typical of many early actinopterygian representatives; equal double cones, small single cones and large rods (2:1:1). These photoreceptors are arranged into a unique regular square mosaic comprising a large rod bordered by four equal double cones with a small single cone located at the corner of each repeating unit. The double cones may optimise perception of mobile prey which it tracks by flexion of its head and neck and the large rods may increase sensitivity in the dark tannin-rich waters in which it lives. Each single cone also possesses a dense collection of polysomes and glycogen (a paraboloid) beneath its ellipsoid, the first such finding in teleosts. The retinal pigment epithelium possesses melanosomes, pha,oocytes and a large number of mitochondria. The anatomy of the retina and the photoreceptor mosaic is discussed in relation to the primitive phylogeny of this species and its unique life history.

HEMODYNAMIC AND PERFUSION END POINTS FOR VOLEMIC RESUSCITATION IN SEPSIS

Sepsis is the systemic inflammatory response syndrome secondary to a local infection, and severe sepsis and septic shock are the more devastating scenarios of this disease. In the last decade, considerable achievements were obtained in sepsis knowledge, and an international campaign was developed to improve the treatment of this condition. However, sepsis is still one of the most important causes of death in intensive care units. The early stages of sepsis are characterized by a variety of hemodynamic derangements that induce a systemic imbalance between tissue oxygen supply and demand, leading to global tissue hypoxia. This dysfunction, which may occur in patients presenting normal vital signs, can be accompanied by a significant increase in both morbidity and mortality. The early identification of high-risk sepsis patients through tissue perfusion markers such as lactate and venous oxygen saturation is crucial for prompt initiation of therapeutic support, which includes early goal-directed therapy as necessary. The purpose of this article was to review the most commonly used hemodynamic and perfusion parameters for hemodynamic optimization in sepsis, emphasizing the physiological background for their use and the studies that demonstrated their effectiveness as goals of volemic resuscitation.

Hypoxia modulates phenotype, inflammatory response, and leishmanial infection of human dendritic cells

Development of hypoxic areas occurs during infectious and inflammatory processes and dendritic cells (DCs) are involved in both innate and adaptive immunity in diseased tissues. Our group previously reported that macrophages exposed to hypoxia were infected with the intracellular parasite Leishmania amazonensis, but showed reduced susceptibility to the parasite. This study shows that although hypoxia did not alter human DC viability, it significantly altered phenotypic and functional characteristics. The expression of CD1a, CD80, and CD86 was significantly reduced in DCs exposed to hypoxia, whereas CD11c, CD14, CD123, CD49 and HLA-DR expression remained unaltered in DCs cultured in hypoxia or normoxia. DC secretion of IL-12p70, the bioactive interleukin-12 (IL-12), a cytokine produced in response to inflammatory mediators, was enhanced under hypoxia. In addition, phagocytic activity (Leishmania uptake) was not impaired under hypoxia, although this microenviroment induced infected DCs to reduce parasite survival, consequently controlling the infection rate. All these data support the notion that a hypoxic microenvironment promotes selective pressure on DCs to assume a phenotype characterized by pro-inflammatory and microbial activities in injured or inflamed tissues and contribute to the innate immune response.

PrrC from Rhodobacter sphaeroides, a homologue of eukaryotic Sco proteins, is a copper-binding protein and may have a thiol-disulfide oxidoreductase activity

PrrC from Rhodobacter sphaeroides provides the signal input to a two-component signal transduction system that senses changes in oxygen tension and regulates expression of genes involved in photosynthesis (Eraso, J.M. and Kaplan, S. (2000) Biochemistry, 39, 2052-2062; Oh, J.-I. and Kaplan, S. (2000) EMBO J. 19, 42374247). It is also a homologue of eukaryotic Sco proteins and each has a C-x-x-x-C-P sequence. In mitochondrial Sco proteins these cysteines appear to be essential for the biogenesis Of the Cu-A centre of respiratory cytochrome oxidase. Overexpression and purification of a water-soluble and monomeric form of PrrC has provided sufficient material for a chemical and spectroscopic study of the properties of the four cysteine residues of PrrC, and its ability to bind divalent cations, including copper. PrrC expressed in the cytoplasm of Escherichia coli binds Ni2+ tightly and the data are consistent with a mononuclear metal site. Following removal of Ni2+ and formation of renatured metal-free rPrrC (apo-PrrC), Cu2+ could be loaded into the reduced form of PrrC to generate a protein with a distinctive UV-visible spectrum, having absorbance with a lambda(max) of 360 nm. The copper:PrrC ratio is consistent with the presence of a mononuclear metal centre. The cysteines of metal-free PrrC oxidise in the presence of air to form two intramolecular disulfide bonds, with one pair being extremely reactive. The cysteine thiols with extreme O-2 sensitivity are involved in copper binding in reduced PrrC since the same copper-loaded protein could not be generated using oxidised PrrC. Thus, it appears that PrrC, and probably Sco proteins in general, could have both a thiol-disulfide oxidoreductase function and a copper-binding role. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Identification and in vivo characterization of PpaA, a regulator of photosystem formation in Rhodobacter sphaeroides

A regulatory protein, PpaA, involved in photosystem formation in the anoxygenic phototrophic proteobacterium Rhodobacter sphaeroides has been identified and characterized in vivo. Based on the phenotypes of cells expressing the ppaA gene in extra copy and on the phenotype of the ppaA null mutant, it was concluded that PpaA activates photopigment production and puc operon expression under aerobic conditions. This is in contrast to the function of the PpaA homologue from Rhodobacter capsulatus, AerR, which acts as a repressor under aerobic conditions [Dong, C., Elsen, S., Swem, L. R. & Bauer, C. E. (2002). J Bacteriol 184, 2805-2814]. The expression of the ppaA gene increases several-fold in response to a decrease in oxygen tension, suggesting that the PpaA protein is active under conditions of low or no oxygen. However, no discernible phenotype of a ppaA null mutant was observed under anaerobic conditions tested thus far. The photosystem gene repressor PpsR mediates repression of ppaA gene expression under aerobic conditions. Sequence analysis of PpaA homologues from several anoxygenic phototrophic bacteria revealed a putative corrinoid-binding domain. It is suggested that PpaA binds a corrinoid cofactor and the availability or structure of this cofactor affects PpaA activity.

Pseudomonas aeruginosa diversification during infection development in cystic fibrosis lungs

Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Engenharia Clínica)

Options chirurgicates pour l'insuffisance cardiaque terminale [Surgical options for terminal heart failure]

Terminal heart failure can be the cause or the result of major dysfunctions of the organisms. Although, the outcome of the natural history is the same in both situations, it is of prime importance to differentiate the two, as only heart failure as the primary cause allows for successful mechanical circulatory support as bridge to transplantation or towards recovery. Various objective parameters allow for the establishment of the diagnosis of terminal heart failure despite optimal medical treatment. A cardiac index <2.0 l/min, and a mixed venous oxygen saturation <60%, in combination with progressive renal failure, should trigger a diagnostic work-up in order to identify cardiac defects that can be corrected or to list the patient for transplantation with/without mechanical circulatory support.

Use of levosimendan as rescue therapy in children with low cardiac output syndrome.

Objective: Aim of post operative treatments after cardiac surgery is to avoid low cardiac output syndrome (LCOS). Levosimendan, a new inotrope agent, has been demonstrated in adult patient to be an effective treatment for this purpose when classical therapy is not effective. It shows a positive effect on cardiac output, with fewer adverse effects and lower mortality than with dopamine. There is very few data on its benefit in the paediatric population. The aim of this study is to evaluate the effect of levosimendan in cardiac children with LCOS.Methods: Retrospective analysis of 25 children hospitalised in our PICU after cardiac surgery that demonstrated LCOS not responding to classical catecholamine therapy and who received levosimendan as rescue. LCOS parameters like urine output, mixed venous oxygen saturation (SvO2), arterio-venous differences in CO2 (AVCO2) and plasmatic lactate were compared before therapy and at 12, 24, 48 and 72 hours after the beginning of the levosimendan infusion. We also analyzed the effect on the utilisation of amines (amine score), adverse events and mortality.Results: After the beginning of levosimendan infusion, urine output (3.1 vs 5.3ml/kg/h, p=0.003) and SVO2 (56 vs 64mmHg, p=0.001) increase significantly during first 72 hours and at the same time plasmatic lactate (2.6 vs 1.4 mmole/l, p<0.001), AVCO2 (11 vs 8 mmHg, p=0.002) and amine score (63 vs 39, p=0.007) decrease significantly. No side effects were noted during administration of levosimendan. In this group of patients, mortality was 0%.Conclusion: Levosimendan is an effective treatment in children after congenital heart surgery. Our study, with a greater sample of patient than other studies, confirms the improvement of cardiac output already shown in other paediatric studies.

Hyperoxemia in newborn infants: detection by pulse oximetry.

Pulse oximetry has been proposed as a noninvasive continuous method for transcutaneous monitoring of arterial oxygen saturation of hemoglobin (tcSO2) in the newborn infant. The reliability of this technique in detecting hyperoxemia is controversial, because small changes in saturation greater than 90% are associated with relatively large changes in arterial oxygen tension (PaO2). The purpose of this study was to assess the reliability of pulse oximetry using an alarm limit of 95% tcSO2 in detecting hyperoxemia (defined as PaO2 greater than 90 mm Hg) and to examine the effect of varying the alarm limit on reliability. Two types of pulse oximeter were studied alternately in 50 newborn infants who were mechanically ventilated with indwelling arterial lines. Three arterial blood samples were drawn from every infant during routine increase of inspired oxygen before intratracheal suction, and PaO2 was compared with tcSO2. The Nellcor N-100 pulse oximeter identified all 26 hyperoxemic instances correctly (sensitivity 100%) and alarmed falsely in 25 of 49 nonhyperoxemic instances (specificity 49%). The Ohmeda Biox 3700 pulse oximeter detected 13 of 35 hyperoxemic instances (sensitivity 37%) and alarmed falsely in 7 of 40 nonhyperoxemic instances (specificity 83%). The optimal alarm limit, defined as a sensitivity of 95% or more associated with maximal specificity, was determined for Nellcor N-100 at 96% tcSO2 (specificity 38%) and for Ohmeda Biox 3700 at 89% tcSO2 (specificity 52%). It was concluded that pulse oximeters can be highly sensitive in detecting hyperoxemia provided that type-specific alarm limits are set and a low specificity is accepted.

Transcriptional control of the hydrogen cyanide biosynthetic genes hcnABC by the anaerobic regulator ANR and the quorum-sensing regulators LasR and RhlR in Pseudomonas aeruginosa.

Virulence factors of Pseudomonas aeruginosa include hydrogen cyanide (HCN). This secondary metabolite is maximally produced at low oxygen tension and high cell densities during the transition from exponential to stationary growth phase. The hcnABC genes encoding HCN synthase were identified on a genomic fragment complementing an HCN-deficient mutant of P. aeruginosa PAO1. The hcnA promoter was found to be controlled by the FNR-like anaerobic regulator ANR and by the quorum-sensing regulators LasR and RhlR. Primer extension analysis revealed two transcription starts, T1 and T2, separated by 29 bp. Their function was confirmed by transcriptional lacZ fusions. The promoter sequence displayed an FNR/ANR box at -42.5 bp upstream of T2 and a lux box centered around -42.5 bp upstream of T1. Expression of the hcn genes was completely abolished when this lux box was deleted or inactivated by two point mutations in conserved nucleotides. The lux box was recognized by both LasR [activated by N-(oxododecanoyl)-homoserine lactone] and RhlR (activated by N-butanoyl-homoserine lactone), as shown by expression experiments performed in quorum-sensing-defective P. aeruginosa mutants and in the N-acyl-homoserine lactone-negative heterologous host P. fluorescens CHA0. A second, less conserved lux box lying 160 bp upstream of T1 seems to account for enhanced quorum-sensing-dependent expression. Without LasR and RhlR, ANR could not activate the hcn promoter. Together, these data indicate that expression of the hcn promoter from T1 can occur under quorum-sensing control alone. Enhanced expression from T2 appears to rely on a synergistic action between LasR, RhlR, and ANR.

Cerebral extracellular lactate increase is predominantly nonischemic in patients with severe traumatic brain injury.

Growing evidence suggests that endogenous lactate is an important substrate for neurons. This study aimed to examine cerebral lactate metabolism and its relationship with brain perfusion in patients with severe traumatic brain injury (TBI). A prospective cohort of 24 patients with severe TBI monitored with cerebral microdialysis (CMD) and brain tissue oxygen tension (PbtO2) was studied. Brain lactate metabolism was assessed by quantification of elevated CMD lactate samples (>4 mmol/L); these were matched to CMD pyruvate and PbtO2 values and dichotomized as glycolytic (CMD pyruvate >119 μmol/L vs. low pyruvate) and hypoxic (PbtO2 <20 mm Hg vs. nonhypoxic). Using perfusion computed tomography (CT), brain perfusion was categorized as oligemic, normal, or hyperemic, and was compared with CMD and PbtO2 data. Samples with elevated CMD lactate were frequently observed (41±8%), and we found that brain lactate elevations were predominantly associated with glycolysis and normal PbtO2 (73±8%) rather than brain hypoxia (14±6%). Furthermore, glycolytic lactate was always associated with normal or hyperemic brain perfusion, whereas all episodes with hypoxic lactate were associated with diffuse oligemia. Our findings suggest predominant nonischemic cerebral extracellular lactate release after TBI and support the concept that lactate may be used as an energy substrate by the injured human brain.

In Vivo Modulation of Mitochondrial Activity Determines HSC Engraftment and Post-Transplant Survival in Mice

Cellular metabolism is emerging as a potential fate determinant in cancer and stem cell biology, constituting a crucial regulator of the hematopoietic stem cell (HSC) pool [1-4]. The extremely low oxygen tension in the HSC microenvironment of the adult bone marrow forces HSCs into a low metabolic profile that is thought to enable their maintenance by protecting them from reactive oxygen species (ROS). Although HSC quiescence has for long been associated with low mitochondrial activity, as testified by the low rhodamine stain that marks primitive HSCs, we hypothesized that mitochondrial activation could be an HSC fate determinant in its own right. We thus set to investigate the implications of pharmacologically modulating mitochondrial activity during bone marrow transplantation, and have found that forcing mitochondrial activation in the post-transplant period dramatically increases survival. Specifically, we examined the mitochondrial content and activation profile of each murine hematopoietic stem and progenitor compartment. Long-term-HSCs (LT-HSC, Lin-cKit+Sca1+ (LKS) CD150+CD34-), short-term-HSCs (ST-HSC, LKS+150+34+), multipotent progenitors (MPPs, LKS+150-) and committed progenitors (PROG, Lin-cKit+Sca1-) display distinct mitochondrial profiles, with both mitochondrial content and activity increasing with differentiation. Indeed, we found that overall function of the hematopoietic progenitor and stem cell compartment can be resolved by mitochondrial activity alone, as illustrated by the fact that low mitochondrial activity LKS cells (TMRM low) can provide efficient long-term engraftment, while high mitochondrial activity LKS cells (TMRM high) cannot engraft in lethally irradiated mice. Moreover, low mitochondrial activity can equally predict efficiency of engraftment within the LT-HSC and ST-HSC compartments, opening the field to a novel method of discriminating a population of transitioning ST-HSCs that retain long-term engraftment capacity. Based on previous experience that a high-fat bone marrow microenvironment depletes short-term hematopoietic progenitors while conserving their long-term counterparts [5], we set to measure HSC mitochondrial activation in high-fat diet fed mice, known to decrease metabolic rate on a per cell basis through excess insulin/IGF-1 production. Congruently, we found lower mitochondrial activation as assessed by flow cytometry and RT-PCR analysis as well as a depletion of the short-term progenitor compartment in high fat versus control chow diet fed mice. We then tested the effects of a mitochondrial activator known to counteract the negative effects of high fat diet. We first analyzed the in vitro effect on HSC cell cycle kinetics, where no significant change in proliferation or division time was found. However, HSCs responded to the mitochondrial activator by increasing asynchrony, a behavior that is thought to directly correlate with asymmetric division [6]. As opposed to high-fat diet fed mice, mice fed with the mitochondrial activator showed an increase in ST-HSCs, while all the other hematopoietic compartments were comparable to mice fed on control diet. Given the dependency on short-term progenitors to rapidly reconstitute hematopoiesis following bone marrow transplantation, we tested the effect of pharmacological mitochondrial activation on the recovery of mice transplanted with a limiting HSC dose. Survival 3 weeks post-transplant was 80% in the treated group compared to 0% in the control group, as predicted by faster recovery of platelet and neutrophil counts. In conclusion, we have found that mitochondrial activation regulates the long-term to short-term HSC transition, unraveling mitochondrial modulation as a valuable drug target for post-transplant therapy. Identification of molecular pathways accountable for the metabolically mediated fate switch is currently ongoing.