Antimicrobial effects of inhaled sphingosine against Pseudomonas aeruginosa in isolated ventilated and perfused pig lungs.

BACKGROUND: Ex-vivo lung perfusion (EVLP) is a save way to verify performance of donor lungs prior to implantation. A major problem of lung transplantation is a donor-to-recipient-transmission of bacterial cultures. Thus, a broadspectrum anti-infective treatment with sphingosine in EVLP might be a novel way to prevent such infections. Sphingosine inhalation might provide a reliable anti-infective treatment option in EVLP. Here, antimicrobial potency of inhalative sphingosine in an infection EVLP model was tested. METHODS: A 3-hour EVLP run using pig lungs was performed. Bacterial infection was initiated 1-hour before sphingosine inhalation. Biopsies were obtained 60 and 120 min after infection with Pseudomonas aeruginosa. Aliquots of broncho-alveolar lavage (BAL) before and after inhalation of sphingosine were plated and counted, tissue samples were fixed in paraformaldehyde, embedded in paraffin and sectioned. Immunostainings were performed. RESULTS: Sphingosine inhalation in the setting of EVLP rapidly resulted in a 6-fold decrease of P. aeruginosa CFU in the lung (p = 0.016). We did not observe any negative side effects of sphingosine. CONCLUSION: Inhalation of sphingosine induced a significant decrease of Pseudomonas aeruginosa at the epithelial layer of tracheal and bronchial cells. The inhalation has no local side effects in ex-vivo perfused and ventilated pig lungs.

Inhaled sphingosine has no adverse side effects in isolated ventilated and perfused pig lungs.

Ex-vivo lung perfusion (EVLP) systems like XVIVO are more and more common in the setting of lung transplantation, since marginal donor-lungs can easily be subjected to a performance test or be treated with corticosteroids or antibiotics in high dose regimes. Donor lungs are frequently positive in bronchoalveolar lavage (BAL) bacterial cultures (46-89%) which leads to a donor-to-recipient transmission and after a higher risk of lung infection with reduced posttransplant outcome. We have previously shown that sphingosine very efficiently kills a variety of pathogens, including Pseudomonas aeruginosa, Staphylococcus aureus and epidermidis, Escherichia coli or Haemophilus influenzae. Thus, sphingosine could be a new treatment option with broadspectrum antiinfective potential, which may improve outcome after lung transplantation when administered prior to lung re-implantation. Here, we tested whether sphingosine has any adverse effects in the respiratory tract when applied into isolated ventilated and perfused lungs. A 4-h EVLP run using minipig lungs was performed. Functional parameters as well as perfusate measurements where obtained. Biopsies were obtained 30 min and 150 min after inhalation of sphingosine. Tissue samples were fixed in paraformaldehyde, embedded in paraffin and sectioned. Hemalaun, TUNEL as well as stainings with Cy3-coupled anti-sphingosine or anti-ceramide antibodies were implemented. We demonstrate that tube-inhalation of sphingosine into ex-vivo perfused and ventilated minipig lungs results in increased levels of sphingosine in the luminal membrane of bronchi and the trachea without morphological side effects up to very high doses of sphingosine. Sphingosine also did not affect functional lung performance. In summary, the inhalation of sphingosine results in an increase of sphingosine concentrations in the luminal plasma membrane of tracheal and bronchial epithelial cells. The inhalation has no local side effects in ex-vivo perfused and ventilated minipig lungs.

Interferon regulatory factor 8 regulates expression of acid ceramidase and infection susceptibility in cystic fibrosis.

Most patients with cystic fibrosis (CF) suffer from acute and chronic pulmonary infections with bacterial pathogens, which often determine their life quality and expectancy. Previous studies have demonstrated a downregulation of the acid ceramidase in CF epithelial cells resulting in an increase of ceramide and a decrease of sphingosine. Sphingosine kills many bacterial pathogens, and the downregulation of sphingosine seems to determine the infection susceptibility of cystic fibrosis mice and patients. It is presently unknown how deficiency of the cystic fibrosis transmembrane conductance regulator (CFTR) connects to a marked downregulation of the acid ceramidase in human and murine CF epithelial cells. Here, we employed quantitative PCR, western blot analysis, and enzyme activity measurements to study the role of IRF8 for acid ceramidase regulation. We report that genetic deficiency or functional inhibition of CFTR/Cftr results in an upregulation of interferon regulatory factor 8 (IRF8) and a concomitant downregulation of acid ceramidase expression with CF and an increase of ceramide and a reduction of sphingosine levels in tracheal and bronchial epithelial cells from both human individuals or mice. CRISPR/Cas9- or siRNA-mediated downregulation of IRF8 prevented changes of acid ceramidase, ceramide, and sphingosine in CF epithelial cells and restored resistance to Pseudomonas aeruginosa infections, which is one of the most important and common pathogens in lung infection of patients with CF. These studies indicate that CFTR deficiency causes a downregulation of acid ceramidase via upregulation of IRF8, which is a central pathway to control infection susceptibility of CF cells.

Acid Ceramidase Rescues Cystic Fibrosis Mice from Pulmonary Infections.

Previous studies have shown that sphingosine kills a variety of pathogenic bacteria, including Pseudomonas aeruginosa and Staphylococcus aureus Sphingosine concentrations are decreased in airway epithelial cells of cystic fibrosis (CF) mice, and this defect has been linked to the infection susceptibility of these mice. Here, we tested whether the genetic overexpression of acid ceramidase rescues cystic fibrosis mice from pulmonary infections with P. aeruginosa We demonstrate that the transgenic overexpression of acid ceramidase in CF mice corresponds to the overexpression of acid ceramidase in bronchial and tracheal epithelial cells and normalizes ceramide and sphingosine levels in bronchial and tracheal epithelial cells. In addition, the expression of ß1-integrin, which is ectopically expressed on the luminal surface of airway epithelial cells in cystic fibrosis mice, an alteration that is very important for mediating pulmonary P. aeruginosa infections in cystic fibrosis, is normalized in cystic fibrosis airways upon the overexpression of acid ceramidase. Most importantly, the overexpression of acid ceramidase protects cystic fibrosis mice from pulmonary P. aeruginosa infections. Infection of CF mice or CF mice that inhaled sphingosine with P. aeruginosa or a P. aeruginosa mutant that is resistant to sphingosine indicates that sphingosine and not a metabolite kills P. aeruginosa upon pulmonary infection. These studies further support the use of acid ceramidase and its metabolite sphingosine as potential treatments of cystic fibrosis.

Sphingosine kills bacteria by binding to cardiolipin.

Sphingosine is a long-chain sphingoid base that has been shown to have bactericidal activity against many pathogens, including Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli We have previously demonstrated that sphingosine is present in nasal, tracheal, and bronchial epithelial cells and constitutes a central element of the defense of the airways against bacterial pathogens. Here, using assorted lipid-binding and cell biology assays, we demonstrate that exposing P. aeruginosa and S. aureus cells to sphingosine results in a very rapid, i.e. within minutes, permeabilization of the bacterial plasma membrane, resulting in leakiness of the bacterial cells, loss of ATP, and loss of bacterial metabolic activity. These alterations rapidly induced bacterial death. Mechanistically, we demonstrate that the presence of the protonated NH2 group in sphingosine, which is an amino-alcohol, is required for sphingosine's bactericidal activity. We also show that the protonated NH2 group of sphingosine binds to the highly negatively-charged lipid cardiolipin in bacterial plasma membranes. Of note, this binding was required for bacterial killing by sphingosine, as revealed by genetic experiments indicating that E. coli or P. aeruginosa strains that lack cardiolipin synthase are resistant to sphingosine, both in vitro and in vivo We propose that binding of sphingosine to cardiolipin clusters cardiolipin molecules in the plasma membrane of bacteria. This clustering results in the formation of gel-like or even crystal-like structures in the bacterial plasma membrane and thereby promotes rapid permeabilization of the plasma membrane and bacterial cell death.

Clinical Development of Sphingosine as Anti-Bacterial Drug: Inhalation of Sphingosine in Mini Pigs has no Adverse Side Effects.

BACKGROUND/AIMS: Pulmonary infections with Pseudomonas aeruginosa (P. aeruginosa) or Staphylococcus aureus (S. aureus) are of utmost clinical relevance in patients with cystic fibrosis, chronic obstructive pulmonary disease, after trauma and burn, upon ventilation or in immuno-compromised patients. Many P. aeruginosa and S. aureus strains are resistant to many known antibiotics and it is very difficult or often impossible to eradicate the pathogens in patient´s lungs. We have recently shown that the sphingoid base sphingosine very efficiently kills many pathogens, including for instance P. aeruginosa, S. aureus or Acinetobacter baumannii, in vitro. In vivo experiments of our group on cystic fibrosis mice indicated that inhalation of sphingosine prevents or eliminates existing acute or chronic pneumonia with P. aeruginosa or S. aureus in these mice. We also demonstrated that sphingosine is safe to use for inhalation up to high doses, at least in mice. To facilitate development of sphingosine to an anti-bactericidal drug that can be used in humans for inhalation, safety data on non-rodents, larger animals are absolutely required. METHODS: Here, we inhaled mini pigs with increasing doses of sphingosine for 10 days and analyzed the uptake of sphingosine into epithelial cells of bronchi as well as into the trachea and lung and the systemic circulation. Moreover, we measured the generation of ceramide and sphingosine 1-phosphate that potentially mediate inflammation, the influx of leukocytes, epithelial cell death and disruption of the epithelial cell barrier. RESULTS: We demonstrate that inhalation of sphingosine results in increased levels of sphingosine in the luminal membrane of bronchi and the trachea, but not in systemic accumulation. Inhaled sphingosine had no side effects up to very high doses. CONCLUSION: In summary, we demonstrate that inhalation of sphingosine results in an increase of sphingosine concentrations in the luminal plasma membrane of tracheal and bronchial epithelial cells. The inhalation has no systemic or local side effects.

Therapeutic effects of physostigmine during systemic inflammation.

INTRODUCTION: Usually, physostigmine is used as antidote for anticholinergic poisons in order to improve hemodynamics and cardiac output. In addition, it causes beneficial effects during sepsis when added timely. Here, we studied whether physostigmine improves hemodynamics when treatment during systemic inflammation was delayed. METHODS: Two series of randomized studies with overall 44 rats were conducted. Systemic inflammation was induced by lipopolysaccharide (LPS) infusion (0.5 mg LPS/kg×h). Physostigmine (PHY) was intravenously applied after an LPS infusion period of 90 minutes (50 µg PHY/kg within 10 minutes) with (series 1) and without (series 2) additional volume loading. Hemodynamic parameters, blood gases, and parameters for tissue damage were periodically determined for up to 180 minutes. RESULTS: Even though volume was additionally administered (series 1), LPS caused a reduction of peripheral blood flow. Treatment with PHY improved hemodynamics in macrocirculation (mean arterial blood pressure) and microcirculation (peripheral blood flow). PHY neither affected alterations in blood gases, electrolyte homeostasis, and glucose metabolism nor prevented intestinal damage induced by LPS. In series 2, without any additional volume loading, PHY likewise resulted in an improvement of the LPS-induced alterations in macro- and microcirculation, but finally worsened the LPS-mediated effects on plasma parameters for tissue damage such as creatine kinase, probably due to the lack of volume and a further damage to the heart. CONCLUSION: The present results demonstrated that hemodynamic responses to PHY may not only be visible in patients with anticholinergic drug overdose but also be visible in septic patients, provided that fluid intake of these patients is adequate.

The Liver and Small Intestine Can Partly Compensate Severe Normovolemic Hemodilution in a Rat Model.

BACKGROUND: Knowing the individual critical hematocrit for every organ is essential in operative scenarios in which extensive blood losses are expected. In the past, experimental settings were very heterogeneous resulting in the publication of widely differing values even for one organ in the same species. This study aimed to investigate the compensatory capacity of the liver and the small intestine in a rat model of severe normovolemic hemodilution. MATERIALS AND METHODS: Male rats were subjected to a stepwise hemodilution with a succinylated gelatin-containing solution to a final hematocrit of 10%, being observed for additional 150 min. During the course of the experiment, blood glucose and L-lactate, as well as D-lactate and intestinal fatty acid-binding protein-2 measurements, were performed eight times in total. The amino acids alanine and glutamine were measured during dilution and at the end of the experiment (four times in total). Hemodilutional effects on the blood and oxygen supply of the liver and the small intestine were measured in a minimally invasive manner. RESULTS: In the liver and the small intestine, there were no substantial changes in the blood flow of the microcirculation. Plasma glucose and lactate levels rose transiently, whereas lactate values did not exceed the upper threshold of aerobic metabolism. Plasma levels of the amino acids alanine and glutamine rose significantly and stayed elevated, whereas D-lactate and intestinal fatty acid-binding protein-2 were not significantly increased at any point during the whole experimental time compared to the initial value. CONCLUSIONS: Severe hemodilution with a succinylated gelatin-containing solution is tolerated at a profoundly low hematocrit value of 10% during the experimental phase of 150 min.

Impact of Acute Intestinal Ischemia and Reperfusion Injury on Hemodynamics and Remote Organs in a Rat Model.

BACKGROUND: Acute mesenteric ischemia following cardiovascular surgery is a rare but fatal complication. We established a new rat model for hemodynamic monitoring during mesenteric ischemia/reperfusion (I/R) and evaluated the impact of mesenteric I/R on hemodynamics and remote organ injury. METHODS: Mesenteric I/R was induced in male Wistar rats by superior mesenteric artery occlusion for 90 minutes, followed by 120 minutes of reperfusion. Before I/R, ventilation and hemodynamic monitoring including mean arterial blood pressure (MAP) and cardiac output (CO) were established. During reperfusion Geloplasma (I/R + Geloplasma, N = 6) and Ringer's solution (I/R + Ringer, N = 6) were titrated according to CO and compared with I/R without volume resuscitation (I/R only, N = 6) and a sham group (sham, N = 6). Blood samples were regularly taken for serum marker measurements. After reperfusion organs were harvested for histology studies. RESULTS: After acute mesenteric I/R, MAP and CO decreased (p < 0.01) while systemic and pulmonary vascular resistance increased (p < 0.01) continuously in the I/R group. Volume substitution according to CO initially stabilized hemodynamic parameters, but CO declined independently in the late stage. Compared with the I/R + Ringer group, the I/R + Geloplasma group required less volume for resuscitation (p < 0.01), experienced less metabolic acidosis. I/R groups had more organ injuries, more neutrophils sequestration, and higher creatine phosphokinase-MB levels than sham group. CONCLUSION: A new model for CO monitoring after mesenteric I/R injury demonstrated severe hypovolemic shock during reperfusion followed by remote myocardial and lung injury. Far less colloid volume is needed for hemodynamic stabilization after I/R compared with crystalloid volume.

Autodigestion by migrated trypsin is a major factor in small intestinal ischemia-reperfusion injury.

BACKGROUND: The pathophysiological role of pancreatic digestive hydrolases in intestinal ischemia-reperfusion (I/R) injury is still not clear. Here, we studied whether ischemia-induced injury to the small intestine can be explained by the autodigestion hypothesis. MATERIALS AND METHODS: Mesenteric I/R was induced in rats by superior mesenteric artery occlusion (90 min) and reopening (120 min). Thirty minutes before superior mesenteric artery occlusion, aprotinin (14.7 mg/kg), orlistat (5 mg/kg), and their combination or α1-proteinase inhibitor (60 mg/kg) were injected into the lumen of the small intestine. Systemic and vital parameters, intestinal microcirculation, and mucosal barrier function were monitored during the observation phase; markers of small intestinal injury, as well as trypsin-, chymotrypsin-, elastase-, and lipase-like activities in intestinal effluates were assessed at the end. RESULTS: The pattern of small intestinal injury correlated inversely with the local alterations in microvascular tissue perfusion and corresponded with the intestinal distribution of trypsin-like activity. Aprotinin almost completely inhibited trypsin-like activity (P < 0.05) and significantly reduced intestinal tissue injury. Combined with orlistat, it also increased the postischemic blood pressure (P < 0.05) but not the intestinal barrier function. Macroscopic as well as the histologic alterations were decreased by α1-proteinase inhibitor, which significantly improved postischemic blood pressure (P < 0.05). CONCLUSIONS: The I/R-induced pattern of small intestinal injury is likely to result from both local differences in tissue ischemia and the digestive activity of migrated pancreatic trypsin. Therefore, administration of aprotinin and orlistat into ischemic small intestines may be a therapeutic option in patients with a poor diagnosis.

Survivin expression pattern in the intestine of normoxic and ischemic rats.

BACKGROUND: Survivin, a member of the inhibitor of apoptosis protein (IAP) family, regulates mitosis and chromosome segregation. The expression of survivin proceeds during embryonic development and in addition has already been demonstrated in cancer cells. However, there is also evidence of survivin expression in differentiated tissues, including the gastro-intestinal tract of adult rats. A study with human colon specimens exhibited survivin in most basal crypt epithelial cells of normal mucosa. There is rather limited information on survivin expression in the small intestine. In order to paint a more detailed and thus complete picture of survivin expression patterns in the gastrointestinal tract, we used an immunohistochemical approach in normal adult rat small intestinal and ascending colonic tissue. Moreover, to get deeper insights in the regulation of survivin expression after tissue damage, we also studied its expression in mesenteric ischemia-reperfusion (I/R) injury. METHODS: Mesenteric ischemia-reperfusion injury was induced in male Wistar rats (six animals/group) by occlusion of the superior mesenteric artery for 90 min and subsequent reperfusion for 120 min. Paraffin sections of untreated or ischemically treated tissue were assessed immunohistochemically by survivin and Ki-67 staining. RESULTS: Survivin could be detected in the small intestine and ascending colon of the normoxia group. It was expressed mainly in the epithelial cells of the crypts and only marginally in the villi. The individual small intestinal segments studied revealed comparable staining intensities. Likewise, expression of survivin was detected in the ischemically damaged small intestine and ascending colon. The expression pattern corresponded to the normoxic animals, as far as verifiable due to the existing tissue damage. Comparison of the expression pattern of Ki-67, a protein that acts as a cellular marker for proliferation, and survivin demonstrated a coincidental localization of the two proteins in the small intestinal and ascending colonic tissue. CONCLUSIONS: Survivin was expressed strongly in epithelial cells of small intestinal as well as ascending colonic tissue. Its expression was located in cells with a high proliferation rate and regenerative capacity. This further supports the decisive role of survivin in cell division. Surprisingly, the ischemically damaged small intestinal and ascending colonic tissue showed a comparably high expression level. These results suggest that there is already a maximal survivin expression under normal conditions. However, the intestine is able to maintain the regenerative capacity even in spite of an ischemic injury. These findings reflect the important relevance of an intact intestinal barrier.

Transient dilutional acidosis but no lactic acidosis upon cardiopulmonary bypass in patients undergoing coronary artery bypass grafting.

INTRODUCTION: Dilutional acidosis may result from the introduction of a large fluid volume into the patients' systemic circulation, resulting in a considerable dilution of endogenous bicarbonate in the presence of a constant carbon dioxide partial pressure. Its significance or even existence, however, has been strongly questioned. Blood gas samples of patients operated on with standard cardiopulmonary bypass (CPB) were analyzed in order to provide further evidence for the existence of dilutional acidosis. MATERIAL AND METHODS: Between 07/2014 and 10/2014, a total of 25 consecutive patients scheduled for elective isolated coronary artery bypass grafting with CPB were enrolled in this prospective observational study. Blood gas samples taken regularly after CPB initiation were analyzed for dilutional effects and acid-base changes. RESULTS: After CPB initiation, hemoglobin concentration dropped from an average initial value of 12.8 g/dl to 8.8 g/dl. Before the beginning of CPB, the mean value of the patients' pH and base excess (BE) value averaged 7.41 and 0.5 mEq/l, respectively. After the onset of CPB, pH and BE values significantly dropped to a mean value of 7.33 (p < 0.0001) and -3.3 mEq/l (p < 0.0001), respectively, within the first 20 min. In the following period during CPB they recovered to 7.38 and -0.5 mEq/l, respectively, on average. Patients did not show overt lactic acidosis. CONCLUSIONS: The present data underline the general existence of dilutional acidosis, albeit very limited in its duration. In patients undergoing coronary artery bypass grafting it seems to be the only obvious disturbance in acid-base homeostasis during CPB.

Attenuation of intestinal ischemia-reperfusion-injury by ß-alanine: a potentially glycine-receptor mediated effect.

BACKGROUND: Acute mesenteric ischemia is often caused by embolization of the mesenteric arterial circulation. Coherent intestinal injury due to ischemia and following reperfusion get visible on macroscopic and histologic level. In previous studies, application of glycine caused an ameliorated intestinal damage after ischemia-reperfusion in rats. Because we speculated that glycine acted here as a signal molecule, we investigated whether the glycine-receptor agonist ß-alanine evokes the same beneficial effect in intestinal ischemia-reperfusion. MATERIALS AND METHODS: ß-alanine (10, 30, and 100 mg/kg) was administered intravenously. Ischemia/reperfusion of the small intestine was initiated by occluding and reopening the superior mesenteric artery in rats. After 90 min of ischemia and 120 min of reperfusion, the intestine was analyzed with regard to macroscopic and histologic tissue damage, the activity of the saccharase, and accumulation of macrophages. In addition, systemic parameters and metabolic ones (e.g., acid-base balance, electrolytes, and blood glucose) were measured at certain points in time. RESULTS: All three dosages of ß-alanine did not change systemic parameters but prevent from hyponatremia during the period of reperfusion. Most importantly, application of 100-mg ß-alanine clearly diminished intestinal tissue damage, getting visible on macroscopic and histologic level. In addition, I/R-mediated decrease of saccharase activity and accumulation of macrophages in the small intestine were ameliorated. CONCLUSIONS: The present study demonstrated that ß-alanine was a potent agent to ameliorate I/R-induced injury of the small intestine. Due to its diminishing effect on the accumulation of macrophages, ß-alanine is strongly expected to mediate its beneficial effect via glycine receptors.

Histidine and other amino acids in blood and urine after administration of Bretschneider solution (HTK) for cardioplegic arrest in patients: effects on N-metabolism.

Bretschneider (histidine-tryptophan-ketoglutarate, HTK) solution employed for induction of cardioplegic arrest possesses a high histidine concentration (198 mM). Due to the large volume administered, massive amounts of histidine are incorporated. The aim of the study was to evaluate alterations in amino acid and nitrogen metabolism originating from histidine degradation. Between 07/2014 and 10/2014, a total of 29 consecutive patients scheduled for elective isolated coronary artery bypass grafting with cardiopulmonary bypass (CPB) were enrolled in this prospective observational study. The patients received 1.6 L cardioplegic Bretschneider solution on average. Blood gas and urine samples obtained were analyzed for amino acid as well as urea and ammonium concentrations. After CPB initiation, plasma histidine concentration greatly increased to 21,000 µM to reach 8000 µM at the end. Within the operative period, plasma concentrations of aspartate, glutamate, asparagine, alanine, and glutamine increased variable in magnitude. During the same time, urinary analysis revealed histidine excretion of 19,500 µmol in total and marked elevations in glutamate and glutamine excretion. The absolute amounts of urea and ammonium excreted additionally were 3 mmol and 8 mmol, respectively. Already during CPB, distinct amounts of the histidine administered are metabolized, mainly to other amino acids, but only small amounts to urea and ammonia. Thus, the impact of the histidine incorporated on acid-base status in the intraoperative phase is minor. On the other hand, intraoperative provision of several amino acids arising from histidine metabolism might mitigate postaggression syndrome.

Excitation-contraction coupling in zebrafish ventricular myocardium is regulated by trans-sarcolemmal Ca2+ influx and sarcoplasmic reticulum Ca2+ release.

Zebrafish (Danio rerio) have become a popular model in cardiovascular research mainly due to identification of a large number of mutants with structural defects. In recent years, cardiomyopathies and other diseases influencing contractility of the heart have been studied in zebrafish mutants. However, little is known about the regulation of contractility of the zebrafish heart on a tissue level. The aim of the present study was to elucidate the role of trans-sarcolemmal Ca(2+)-flux and sarcoplasmic reticulum Ca(2+)-release in zebrafish myocardium. Using isometric force measurements of fresh heart slices, we characterised the effects of changes of the extracellular Ca(2+)-concentration, trans-sarcolemmal Ca(2+)-flux via L-type Ca(2+)-channels and Na(+)-Ca(2+)-exchanger, and Ca(2+)-release from the sarcoplasmic reticulum as well as beating frequency and ß-adrenergic stimulation on contractility of adult zebrafish myocardium. We found an overall negative force-frequency relationship (FFR). Inhibition of L-type Ca(2+)-channels by verapamil (1 µM) decreased force of contraction to 22 ± 7% compared to baseline (n=4, p<0.05). Ni(2+) was the only substance to prolong relaxation (5 mM, time after peak to 50% relaxation: 73 ± 3 ms vs. 101 ± 8 ms, n=5, p<0.05). Surprisingly though, inhibition of the sarcoplasmic Ca(2+)-release decreased force development to 54 ± 3% in ventricular (n=13, p<0.05) and to 52 ± 8% in atrial myocardium (n=5, p<0.05) suggesting a substantial role of SR Ca(2+)-release in force generation. In line with this finding, we observed significant post pause potentiation after pauses of 5 s (169 ± 7% force compared to baseline, n=8, p<0.05) and 10 s (198 ± 9% force compared to baseline, n=5, p<0.05) and mildly positive lusitropy after ß-adrenergic stimulation. In conclusion, force development in adult zebrafish ventricular myocardium requires not only trans-sarcolemmal Ca2+-flux, but also intact sarcoplasmic reticulum Ca(2+)-cycling. In contrast to mammals, FFR is strongly negative in the zebrafish heart. These aspects need to be considered when using zebrafish to model human diseases of myocardial contractility.

Attenuation of intestinal ischemic injury and shock by physostigmine.

BACKGROUND: Recently, protection in shock (hemorrhagic or septic) by physostigmine has been demonstrated. Here, we studied the protective effect of intravenous infusion of physostigmine in a rat model of severe intestinal ischemia-reperfusion (I/R) injury and shock. MATERIALS AND METHODS: Mesenteric I/R was induced in male Wistar rats by occlusion of the superior mesenteric artery (90 min) and subsequent reperfusion (120 min). Physostigmine (30 or 70 µg/kg) was administered as bolus injection before induction of I/R. One additional group received, subsequent to the bolus of 30-µg/kg physostigmine, a continuous infusion of 60-µg/kg physostigmine till the end of the experiment. RESULTS: Physostigmine at a dose of 70 µg/kg administered before I/R significantly decreased the macroscopically and microscopically visible intestinal damage. In addition to and presumably as a result of this local protective effect, physostigmine prevented shock induced by reperfusion of the ischemically injured intestine. Lower doses (30 µg/kg) or continuous application of physostigmine were less advantageous. CONCLUSIONS: Physostigmine is clearly protective in intestinal I/R injury and shock. However, for this purpose, physostigmine has to be applied at a dose (70 µg/kg), that is, approximately double the amount of the presently used clinical dose.

Severe blunt muscle trauma in rats: only marginal hypoxia in the injured area.

BACKGROUND: After severe muscle trauma, hypoxia due to microvascular perfusion failure is generally believed to further increase local injury and to impair healing. However, detailed analysis of hypoxia at the cellular level is missing. Therefore, in the present work, spectroscopic measurements of microvascular blood flow and O2 supply were combined with immunological detection of hypoxic cells to estimate O2 conditions within the injured muscle area. MATERIALS AND METHODS: Severe blunt muscle trauma was induced in the right Musculus gastrocnemius of male Wistar rats by a standardized "weight-drop" device. Microvascular blood flow, relative hemoglobin amount, and hemoglobin O2 saturation were determined by laser Doppler and white-light spectroscopy. Hypoxic cells were detected by histologic evaluation of covalent binding of pimonidazole and expression of HIF-1α. RESULTS: Directly after trauma and until the end of experiment (480 minutes), microvascular blood flow and relative hemoglobin amount were clearly increased. In contrast to blood flow and relative hemoglobin amount, there was no immediate but a delayed increase of microvascular hemoglobin O2 saturation. Pimonidazole immunostaining revealed a hypoxic fraction (percentage area of pimonidazole-labelled muscle cells within the injured area) between 8 to 3%. There was almost no HIF-1α expression detectable in the muscle cells under each condition studied. CONCLUSIONS: In the early phase (up to 8 hours) after severe blunt muscle trauma, the overall microvascular perfusion of the injured area and thus its O2 supply is clearly increased. This increased O2 supply is obviously sufficient to ensure normoxic (or even hyperoxic) conditions in the vast majority of the cells.

Glycine selectively reduces intestinal injury during endotoxemia.

BACKGROUND: Glycine is well known to protect the intestine against ischemia-reperfusion injury and during mechanical manipulation. Here, we studied whether glycine protects the small intestine during endotoxemia, even without being the site of the infection. MATERIALS AND METHODS: Lipopolysaccharide (LPS) was infused at a rate of 1 mg/kg × h over a period of 7 h (subacute endotoxemia) in male Wistar rats. Glycine (single dose: 50 mg/kg × 15 min) was applied intravenously at 180 and 270 min after the beginning of the LPS infusion. Systemic parameters were periodically determined. The small intestine was analyzed for macroscopic (hemorrhages) and histopathologic changes (hematoxylin and eosin staining), and markers of inflammation (myeloperoxidase activity). RESULTS: Glycine neither decreased mortality nor beneficially affected vital parameters (e.g., mean arterial blood pressure and breathing rate), electrolytes, blood gases including pH and base excess, and plasma parameters of tissue injury such as lactate concentration, hemolysis, and aminotransferases activities during experimental endotoxemia. It, however, specifically diminished the LPS-induced small intestinal injury, as indicated by less intestinal accumulation of blood, less intestinal hemorrhages, and reduced intestinal hemoglobin content. CONCLUSIONS: The present results demonstrate that glycine selectively protects the small intestine during subacute endotoxemia, even after manifestation of a severe systemic impairment. Because glycine is non-toxic at low doses, an administration of a moderate glycine dose (50-100 mg/kg) may be suitable to protect from intestinal damage during sepsis. Its true clinical potential, however, needs to be verified in further experimental studies and clinical trials.