Carbachol and bradykinin elevate cyclic AMP and rapidly deplete ATP in cultured rat sympathetic neurons

The agonists carbachol (CCh) and bradykinin (BK) and 54 mM KCl (high K+) were among the most potent stimulants of cyclic AMP (cAMP) production in cultured rat sympathetic neurons, measured with the use of a high-fidelity assay developed for small samples. The rise in cAMP evoked by CCh (through muscarinic receptors), BK, and high K+ was inhibited in Ca2(+)-depleted medium (1.3 mM Ca2+ and 2 mM BAPTA or EGTA), which also prevented the sustained rise in [Ca2+]i evoked by each of these stimuli, showing that elevation of cAMP requires extracellular Ca2+ and, possibly, Ca2+ influx. Preliminary results obtained with the novel calmodulin inhibitor CGS 9343B, which blocked the elevation of cAMP, and with the cyclogenase inhibitor indomethacin, which partially blocked the actions of the agonists but not those of high K+, suggest that calmodulin and arachidonate metabolites may be two components of the signaling pathway. In addition to their effects on cAMP metabolism, CCh, muscarine, and BK, but not nicotine, caused a 30-40% decrease in ATP levels. This effect was much greater than that evoked by high K+ and was largely inhibited by CGS 9343B but slightly enhanced in the Ca(+)-depleted medium, showing that agonists are still active in the absence of [Ca2+]o. Thus, agonists that activate phosphoinositide metabolism can also increase cAMP production and substantially deplete cells of ATP. These novel actions may have to be taken into account when the mechanisms by which such agonists regulate cell function are being considered

Ca2+ transients are not required as signals for long-term neurite outgrowth from cultured sympathetic neurons

A method for clamping cytosolic free Ca2+ ([Ca2+]i) in cultures of rat sympathetic neurons at or below resting levels for several days was devised to determine whether Ca2+ signals are required for neurite outgrowth from neurons that depend on Nerve Growth Factor (NGF) for their growth and survival. To control [Ca2+]i, normal Ca2+ influx was eliminated by titration of extracellular Ca2+ with EGTA and reinstated through voltage-sensitive Ca2+ channels. The rate of neurite outgrowth and the number of neurites thus became dependent on the extent of depolarization by KCl, and withdrawal of KCl caused an immediate cessation of growth. Neurite outgrowth was completely blocked by the L type Ca2+ channel antagonists nifedipine, nitrendipine, D600, or diltiazem at sub- or micromolar concentrations. Measurement of [Ca2+]i in cell bodies using the fluorescent Ca2+ indicator fura-2 established that optimal growth, similar to that seen in normal medium, was obtained when [Ca2+]i was clamped at resting levels. These levels of [Ca2+]i were set by serum, which elevated [Ca2+]i by integral of 30 nM, whereas the addition of NGF had no effect on [Ca2+]i. The reduction of [Ca2+]o prevented neurite fasciculation but this had no effect on the rate of neurite elongation or on the number of extending neurites. These results show that neurite outgrowth from NGF-dependent neurons occurs over long periods in the complete absence of Ca2+ signals, suggesting that Ca2+ signals are not necessary for operating the basic machinery of neurite outgrowth

Lack of Tryptophan Hydroxylase-1 in Mice Results in Gait Abnormalities

The role of peripheral serotonin in nervous system development is poorly understood. Tryptophan hydroxylase-1 (TPH1) is expressed by non-neuronal cells including enterochromaffin cells of the gut, mast cells and the pineal gland and is the rate-limiting enzyme involved in the biosynthesis of peripheral serotonin. Serotonin released into circulation is taken up by platelets via the serotonin transporter and stored in dense granules. It has been previously reported that mouse embryos removed from Tph1-deficient mothers present abnormal nervous system morphology. The goal of this study was to assess whether Tph1-deficiency results in behavioral abnormalities. We did not find any differences between Tph1-deficient and wild-type mice in general motor behavior as tested by rotarod, grip-strength test, open field and beam walk. However, here we report that Tph1 (−/−) mice display altered gait dynamics and deficits in rearing behavior compared to wild-type (WT) suggesting that tryptophan hydroxylase-1 expression has an impact on the nervous system

A fatigue damage model for seismic response of RC structures

Numerous damage models have been developed in order to analyze seismic behavior. Among the different possibilities existing in the literature, it is very clear that models developed along the lines of continuum damage mechanics are more consistent with the definition of damage as a phenomenon with mechanical consequences because they include explicitly the coupling between damage and mechanical behavior. On the other hand, for seismic processes, phenomena such as low cycle fatigue may have a pronounced effect on the overall behavior of the frames and, therefore, its consideration turns out to be very important. However, most of existing models evaluate the damage only as a function of the maximum amplitude of cyclic deformation without considering the number of cycles. In this paper, a generalization of the simplified model proposed by Cipollina et al. [Cipollina A, López-Hinojosa A, Flórez-López J. Comput Struct 1995;54:1113–26] is made in order to include the low cycle fatigue. Such a model employs in its formulation irreversible thermodynamics and internal state variable theory

Electronic patient-reported outcomes from home in patients recovering from major gynecologic cancer surgery: A prospective study measuring symptoms and health-related quality of life

We previously reported on the feasibility of a Web-based system to capture patient-reported outcomes (PROs) in the immediate postoperative period. The purpose of this study was to update the experience of these patients and assess patient and provider satisfaction and feedback regarding the system

A hematopoietic contribution to microhemorrhage formation during antiviral CD8 T cell-initiated blood-brain barrier disruption

<p>Abstract</p> <p>Background</p> <p>The extent to which susceptibility to brain hemorrhage is derived from blood-derived factors or stromal tissue remains largely unknown. We have developed an inducible model of CD8 T cell-initiated blood-brain barrier (BBB) disruption using a variation of the Theiler's murine encephalomyelitis virus (TMEV) model of multiple sclerosis. This peptide-induced fatal syndrome (PIFS) model results in severe central nervous system (CNS) vascular permeability and death in the C57BL/6 mouse strain, but not in the 129 SvIm mouse strain, despite the two strains' having indistinguishable CD8 T-cell responses. Therefore, we hypothesize that hematopoietic factors contribute to susceptibility to brain hemorrhage, CNS vascular permeability and death following induction of PIFS.</p> <p>Methods</p> <p>PIFS was induced by intravenous injection of VP2_121-130peptide at 7 days post-TMEV infection. We then investigated brain inflammation, astrocyte activation, vascular permeability, functional deficit and microhemorrhage formation using T2*-weighted magnetic resonance imaging (MRI) in C57BL/6 and 129 SvIm mice. To investigate the contribution of hematopoietic cells in this model, hemorrhage-resistant 129 SvIm mice were reconstituted with C57BL/6 or autologous 129 SvIm bone marrow. Gadolinium-enhanced, T1-weighted MRI was used to visualize the extent of CNS vascular permeability after bone marrow transfer.</p> <p>Results</p> <p>C57BL/6 and 129 SvIm mice had similar inflammation in the CNS during acute infection. After administration of VP2_121-130peptide, however, C57BL/6 mice had increased astrocyte activation, CNS vascular permeability, microhemorrhage formation and functional deficits compared to 129 SvIm mice. The 129 SvIm mice reconstituted with C57BL/6 but not autologous bone marrow had increased microhemorrhage formation as measured by T2*-weighted MRI, exhibited a profound increase in CNS vascular permeability as measured by three-dimensional volumetric analysis of gadolinium-enhanced, T1-weighted MRI, and became moribund in this model system.</p> <p>Conclusion</p> <p>C57BL/6 mice are highly susceptible to microhemorrhage formation, severe CNS vascular permeability and morbidity compared to the 129 SvIm mouse. This susceptibility is transferable with the bone marrow compartment, demonstrating that hematopoietic factors are responsible for the onset of brain microhemorrhage and vascular permeability in immune-mediated fatal BBB disruption.</p

Induction of Blood Brain Barrier Tight Junction Protein Alterations by CD8 T Cells

Disruption of the blood brain barrier (BBB) is a hallmark feature of immune-mediated neurological disorders as diverse as viral hemorrhagic fevers, cerebral malaria and acute hemorrhagic leukoencephalitis. Although current models hypothesize that immune cells promote vascular permeability in human disease, the role CD8 T cells play in BBB breakdown remains poorly defined. Our laboratory has developed a novel murine model of CD8 T cell mediated central nervous system (CNS) vascular permeability using a variation of the Theiler's virus model of multiple sclerosis. In previous studies, we observed that MHC class II−/− (CD4 T cell deficient), IFN-γR−/−, TNF-α−/−, TNFR1−/−, TNFR2−/−, and TNFR1/TNFR2 double knockout mice as well as those with inhibition of IL-1 and LTβ activity were susceptible to CNS vascular permeability. Therefore, the objective of this study was to determine the extent immune effector proteins utilized by CD8 T cells, perforin and FasL, contributed to CNS vascular permeability. Using techniques such as fluorescent activated cell sorting (FACS), T1 gadolinium-enhanced magnetic resonance imaging (MRI), FITC-albumin leakage assays, microvessel isolation, western blotting and immunofluorescent microscopy, we show that in vivo stimulation of CNS infiltrating antigen-specific CD8 T cells initiates astrocyte activation, alteration of BBB tight junction proteins and increased CNS vascular permeability in a non-apoptotic manner. Using the aforementioned techniques, we found that despite having similar expansion of CD8 T cells in the brain as wildtype and Fas Ligand deficient animals, perforin deficient mice were resistant to tight junction alterations and CNS vascular permeability. To our knowledge, this study is the first to demonstrate that CNS infiltrating antigen-specific CD8 T cells have the capacity to initiate BBB tight junction disruption through a non-apoptotic perforin dependent mechanism and our model is one of few that are useful for studies in this field. These novel findings are highly relevant to the development of therapies designed to control immune mediated CNS vascular permeability

Comparison of risk-scoring systems in the prediction of outcome after liver resection

Background: Risk prediction techniques commonly used in liver surgery include the American Society of Anesthesiologists (ASA) grading, Charlson Comorbidity Index (CCI) and cardiopulmonary exercise tests (CPET). This study compares the utility of these techniques along with the number of segments resected as predictive tools in liver surgery. Methods: A review of a unit database of patients undergoing liver resection between February 2008 and January 2015 was undertaken. Patient demographics, ASA, CCI and CPET variables were recorded along with resection size. Clavien-Dindo grade III–V complications were used as a composite outcome in analyses. Association between predictive variables and outcome was assessed by univariate and multivariate techniques. Results: One hundred and seventy-two resections in 168 patients were identified. Grade III–V complications occurred after 42 (24.4%) liver resections. In univariate analysis of CPET variables, ventilatory equivalents for CO2 (VEqCO2) was associated with outcome. CCI score, but not ASA grade, was also associated with outcome. In multivariate analysis, the odds ratio of developing grade III–V complications for incremental increases in VEqCO2, CCI and number of liver segments resected were 1.09, 1.49 and 2.94, respectively. Conclusions: Of the techniques evaluated, resection size provides the simplest and most discriminating predictor of significant complications following liver surgery

Mast cell tryptase stimulates myoblast proliferation; a mechanism relying on protease-activated receptor-2 and cyclooxygenase-2

<p>Abstract</p> <p>Background</p> <p>Mast cells contribute to tissue repair in fibrous tissues by stimulating proliferation of fibroblasts through the release of tryptase which activates protease-activated receptor-2 (PAR-2). The possibility that a tryptase/PAR-2 signaling pathway exists in skeletal muscle cell has never been investigated. The aim of this study was to evaluate whether tryptase can stimulate myoblast proliferation and determine the downstream cascade.</p> <p>Methods</p> <p>Proliferation of L6 rat skeletal myoblasts stimulated with PAR-2 agonists (tryptase, trypsin and SLIGKV) was assessed. The specificity of the tryptase effect was evaluated with a specific inhibitor, APC-366. Western blot analyses were used to evaluate the expression and functionality of PAR-2 receptor and to assess the expression of COX-2. COX-2 activity was evaluated with a commercial activity assay kit and by measurement of PGF₂α production. Proliferation assays were also performed in presence of different prostaglandins (PGs).</p> <p>Results</p> <p>Tryptase increased L6 myoblast proliferation by 35% above control group and this effect was completely inhibited by APC-366. We confirmed the expression of PAR-2 receptor <it>in vivo </it>in skeletal muscle cells and in satellite cells and <it>in vitro </it>in L6 cells, where PAR-2 was found to be functional. Trypsin and SLIGKV increased L6 cells proliferation by 76% and 26% above control, respectively. COX-2 activity was increased following stimulation with PAR-2 agonist but its expression remained unchanged. Inhibition of COX-2 activity by NS-398 abolished the stimulation of cell proliferation induced by tryptase and trypsin. Finally, 15-deoxy-Δ-^12,14-prostaglandin J₂(15Δ-PGJ₂), a product of COX-2-derived prostaglandin D₂, stimulated myoblast proliferation, but not PGE₂and PGF₂α.</p> <p>Conclusions</p> <p>Taken together, our data show that tryptase can stimulate myoblast proliferation and this effect is part of a signaling cascade dependent on PAR-2 activation and on the downstream activation of COX-2.</p