Blood volume in patients likely to be preload responsive: a post hoc analysis of a randomized controlled trial.

BACKGROUND: Preload responsive postoperative patients with signs of inadequate organ perfusion are commonly assumed to be hypovolemic and therefore treated with fluids to increase preload. However, preload is influenced not only by blood volume, but also by venous vascular tone and the contribution of these factors to preload responsiveness in this setting is unknown. Based on this, the objective of this study was to investigate blood volume status in preload-responsive postoperative patients. METHODS: Data from a clinical trial including postoperative patients after major abdominal surgery were analyzed. Patients with signs of inadequate organ perfusion and with data from a passive leg raising test (PLR) were included. An increase in pulse pressure by ≥ 9% was used to identify patients likely to be preload responsive. Blood volume was calculated from plasma volume measured using radiolabelled albumin and hematocrit. Patients with a blood volume of at least 10% above or below estimated normal volume were considered hyper- and hypovolemic, respectively. RESULTS: A total of 63 patients were included in the study. Median (IQR) blood volume in the total was 57 (50-65) ml/kg, and change in pulse pressure after PLR was 14 (7-24)%. A total of 43 patients were preload responsive. Of these patients, 44% were hypovolemic, 28% euvolemic and 28% hypervolemic. CONCLUSIONS: A large fraction of postoperative patients with signs of hypoperfusion that are likely to be preload responsive, are hypervolemic. In these patients, treatments other than fluid administration may be a more rational approach to increase cardiac output. Trial registration EudraCT 2013-004446-42.

Albumin infusion rate and plasma volume expansion: a randomized clinical trial in postoperative patients after major surgery.

BACKGROUND: Optimal infusion rate of colloids in patients with suspected hypovolemia is unknown, and the primary objective of the present study was to test if plasma volume expansion by 5% albumin is greater if fluid is administered slowly rather than rapidly. METHODS: Patients with signs of hypoperfusion after major abdominal surgery were randomized to intravenous infusion of 5% albumin at a dose of 10 ml/kg (ideal body weight) either rapidly (30 min) or slowly (180 min). Plasma volume was measured using radiolabeled albumin at baseline, at 30 min, and at 180 min after the start of infusion. Primary outcome was change in plasma volume from the start of infusion to 180 min after the start of infusion. Secondary outcomes included the change in the area under the plasma volume curve and transcapillary escape rate (TER) for albumin from 180 to 240 min after the start of albumin infusion. RESULTS: A total of 33 and 31 patients were included in the analysis in the slow and rapid groups, respectively. The change in plasma volume from the start of infusion to 180 min did not differ between the slow and rapid infusion groups (7.4 ± 2.6 vs. 6.5 ± 4.1 ml/kg; absolute difference, 0.9 ml/kg [95%CI, - 0.8 to 2.6], P = 0.301). Change in the area under the plasma volume curve was smaller in the slow than in the rapid infusion group and was 866 ± 341 and 1226 ± 419 min ml/kg, respectively, P < 0.001. TER for albumin did not differ and was 5.3 ± 3.1%/h and 5.4 ± 3%/h in the slow and in the rapid infusion groups, respectively, P = 0.931. CONCLUSIONS: This study does not support our hypothesis that a slow infusion of colloid results in a greater plasma volume expansion than a rapid infusion. Instead, our result of a smaller change in the area under the plasma volume curve indicates that a slow infusion results in a less efficient plasma volume expansion, but further studies are required to confirm this finding. A rapid infusion has no effect on vascular leak as measured after completion of the infusion. TRIAL REGISTRATION: EudraCT2013-004446-42 registered December 23, 2014.

Neutrophil extracellular traps in the central nervous system hinder bacterial clearance during pneumococcal meningitis.

Neutrophils are crucial mediators of host defense that are recruited to the central nervous system (CNS) in large numbers during acute bacterial meningitis caused by Streptococcus pneumoniae. Neutrophils release neutrophil extracellular traps (NETs) during infections to trap and kill bacteria. Intact NETs are fibrous structures composed of decondensed DNA and neutrophil-derived antimicrobial proteins. Here we show NETs in the cerebrospinal fluid (CSF) of patients with pneumococcal meningitis, and their absence in other forms of meningitis with neutrophil influx into the CSF caused by viruses, Borrelia and subarachnoid hemorrhage. In a rat model of meningitis, a clinical strain of pneumococci induced NET formation in the CSF. Disrupting NETs using DNase I significantly reduces bacterial load, demonstrating that NETs contribute to pneumococcal meningitis pathogenesis in vivo. We conclude that NETs in the CNS reduce bacterial clearance and degrading NETs using DNase I may have significant therapeutic implications.

The importance of albumin infusion rate for plasma volume expansion following major abdominal surgery - AIR: study protocol for a randomised controlled trial.

BACKGROUND: Administration of fluids to restore normovolaemia is one of the most common therapeutic interventions performed peri-operatively and in the critically ill, but no study has evaluated the importance of infusion rate for the plasma volume-expanding effect of a resuscitation fluid. The present study is designed to test the hypothesis that a slow infusion of resuscitation fluid results in better plasma volume expansion than a rapid infusion. METHODS/DESIGN: The study is a single-centre, assessor-blinded, parallel-group, randomised prospective study. Patients over 40 years of age admitted to the post-operative care unit after a Whipple procedure or major gynaecological surgery and presenting with signs of hypovolaemia are eligible for inclusion. Patients are randomised in a 1:1 fashion with no stratification to either rapid (30 minutes) or slow (180 minutes) infusion of 5% albumin at a dose of 10 ml/kg ideal body weight. Plasma volume is measured using 125I human serum albumin at baseline (prior to albumin infusion) as well as at 30 minutes and 180 minutes after infusion start. The primary endpoint is change in plasma volume from baseline to 180 minutes after the start of 5% albumin infusion. Secondary endpoints include the integral of plasma volume over time from baseline to 180 minutes after the start of the infusion and transcapillary escape rate of albumin (%/h) from 180 minutes to 240 minutes after the start of albumin infusion. In addition, diuresis, change in central venous oxygen saturation, lactate and blood pressure will be evaluated. A total of 70 patients will be included in the study, and the study has 80% power to detect a difference of 4 ml/kg in plasma volume expansion between the two groups. DISCUSSION: The present study is the first clinical investigation of the importance of infusion rate for the plasma volume-expanding effect of a resuscitation fluid. TRIAL REGISTRATION: EudraCT identifier: 2013-004446-42 . Registration date: 20 December 2013. ClinicalTrials.gov identifier: NCT02728921 . Registration date: 31 March 2016.

Increased Rho activation and PKC-mediated smooth muscle contractility in the absence of caveolin-1.

Caveolae are omega-shaped membrane invaginations that are abundant in smooth muscle cells. Since many receptors and signaling proteins co-localize with caveolae, these have been proposed to integrate important signaling pathways. The aim of this study was to test whether RhoA/Rho-kinase and protein kinase C (PKC)-mediated Ca(2+) sensitization depends on caveolae using caveolin (Cav)-1-deficient (KO) and wild-type (WT) mice. In WT smooth muscle, caveolae were detected and Cav-1, -2 and -3 proteins were expressed. Relative mRNA expression levels were approximately 15:1:1 for Cav-1, -2, and -3, respectively. Caveolae were absent in KO and reduced levels of Cav-2 and Cav-3 proteins were seen. In intact ileum longitudinal muscle, no differences in the responses to 5-HT or the muscarinic agonist carbachol were found, whereas contraction elicited by endothelin-1 was reduced. Rho activation by GTPgammaS was increased in KO compared with WT as shown using a pull-down assay. Following alpha-toxin permeabilization, no difference in Ca(2+) sensitivity or in Ca(2+) sensitization was detected. In KO femoral arteries, phorbol 12,13-dibutyrate (PDBu)-induced and PKC-mediated contraction was increased. This was associated with increased alpha(1)-adrenergic contraction. Following inhibition of PKC, alpha(1)-adrenergic contraction was normalized. PDBu-induced Ca(2+) sensitization was not increased in permeabilized femoral arteries. In conclusion, Rho activation, but not Ca(2+) sensitization, depends on caveolae in the ileum. Moreover, PKC driven arterial contraction is increased in the absence of caveolin-1. This depends on an intact plasma membrane and is not associated with altered Ca(2+) sensitivity.

Actions downstream of cyclic GMP/protein kinase G can reverse protein kinase C-mediated phosphorylation of CPI-17 and Ca²âº sensitization in smooth muscle.

Ca(2+) sensitivity of smooth muscle contraction is modulated by several systems converging on myosin light chain phosphatase (MLCP). Rho-Rho kinase is considered to inhibit MLCP via phosphorylation, whereas protein kinase C (PKC) induced sensitization has been shown to be dependent on phosphorylation of the inhibitory protein CPI-17. We have explored the interaction of cGMP-dependent protein kinase (PKG) with Ca(2+) sensitization pathways using permeabilized mouse smooth muscle. Three conditions giving approximately 50% of maximal active force were compared in small intestinal preparations: 1). Ca(2+)-activated unsensitized muscle (pCa 5.9 with Rho kinase inhibitor Y27632); 2). Rho-Rho kinase-sensitized muscle (pCa 6.1 with guanosine 5'-3-O-(thio)triphosphate); and 3). PKC-sensitized muscle (pCa 6.0 with Y27632 and PKC activator phorbol 12,13-dibutyrate). 8-Br-cGMP relaxed the sensitized muscles but had marginal effects on unsensitized preparations, showing that PKG reverses both PKC and Rho-mediated Ca(2+) sensitization. CPI-17 was present in permeabilized intestinal tissue. In PKC-sensitized preparations, CPI-17 phosphorylation decreased in response to 8-Br-cGMP. The rate of PKC-mediated phosphorylation in the presence of the MLCP inhibitor microcystin-LR was not influenced by 8-Br-cGMP. PKC-induced Ca(2+) sensitization also was reversed in vascular smooth muscle tissues (portal vein and femoral artery). We conclude that actions downstream of cGMP/PKG can reverse PKC-mediated phosphorylation of CPI-17 and Ca(2+) sensitization in smooth muscle.

Modulation of Ca2+ sensitivity by cyclic nucleotides in smooth muscle from protein kinase G-deficient mice.

The cGMP-dependent protein kinase (PKG) is the main mediator of nitric oxide-induced relaxation of smooth muscle. Although this pathway is well established, the cellular action of PKG, nitric oxide, and cGMP is complex and not fully understood. A cross-talk between the cGMP-PKG and other pathways (e.g. cAMP-protein kinase A) seems to exist. We have explored cGMP- and cAMP-dependent relaxation of smooth muscle using PKG-deficient mice (cGKI-/-). In intact ileum strips of wild type mice (cGKI+/+), 8-Br-cGMP inhibited the sustained phase of carbachol contractions by approximately 80%. The initial peak was less inhibited (approximately 30%). This relaxation was associated with a reduction in intracellular [Ca2+] and decreased Ca2+ sensitivity. Contractions of cGKI-/- ileum were not influenced by 8-Br-cGMP. EC50 for 8-Br-cGMP for PKG was estimated to be 10 nm. PKG-independent relaxation by 8-Br-cGMP had an EC50 of 10 microm. Relaxation by cAMP (approximately 50% at 100 microm), Ca2+ sensitivity of force, and force potentiation by GTPgammaS were similar in cGKI+/+ and cGKI-/- tissues. The results show that PKG is the main target for cGMP-induced relaxation in intestinal smooth muscle. cGMP desensitize the contractile system to Ca2+ via PKG. PKG-independent pathways are activated at 1000-fold higher cGMP concentrations. Relaxation by cAMP can occur independently of PKG. Long term deficiency of PKG does not lead to an apparent up-regulation of the cAMP-dependent pathways or changes in Ca2+ sensitivity.

Sustained norepinephrine contraction in the rat portal vein is lost when Ca(2+) is replaced with Sr(2+).

Agonist-induced activation of smooth muscle involves a rise in intracellular Ca(2+) concentration and sensitization of myosin light chain phosphorylation to Ca(2+). Sr(2+) can enter through Ca(2+) channels, be sequestered and released from sarcoplasmic reticulum, and replace Ca(2+) in activation of myosin light chain phosphorylation. Sr(2+) cannot replace Ca(2+) in facilitation of agonist-activated Ca(2+)-dependent nonselective cation channels. It is not known whether Sr(2+) can replace Ca(2+) in small G protein-mediated sensitization of phosphorylation. To explore mechanisms involved in alpha-receptor-activated contractions in smooth muscle, effects of replacing Ca(2+) with Sr(2+) were examined in rat portal vein. Norepinephrine (NE) at >3.0 x 10(-7) M in the presence of Ca(2+) resulted in a strong sustained contraction, whereas this sustained component was absent in the presence of Sr(2+); only the amplitude of phasic contractions increased. Pretreatment with low (approximately 0.05 mM) free Ca(2+) followed by 2.5 mM Sr(2+) resulted in a sustained component of the NE response. In beta-escin-permeabilized preparations, phenylephrine in the presence of GTP or guanosine 5'-O-(3-thiotriphosphate) alone induced sensitization to Sr(2+). In conclusion, a Ca(2+)-regulated membrane/channel process is required for the sustained component of NE responses in rat portal vein. Sensitization alone is not responsible for the sustained phase of the NE contraction.