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

Survivin expression pattern in the intestine of normoxic and ischemic rats

Abstract 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

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

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

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 and –3.3 mEq/l, 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

Pimonidazole staining within injured muscle area (representative figure).

<p>The animal was ventilated with 100% O₂. 450 minutes after trauma, the <i>Musculus gastrocnemius</i> of the traumatized right hind limb was harvested and section of the muscle specimen was analyzed for pimonidazole labelling within the injured area. Injured area is identified by e.g. necrotic muscle cells (arrow) and edema (arrowhead). Hypoxic muscle cells indicated by pimonidazole binding are stained brown (star). Scale bar: 500 µm.</p

Hypoxic fraction as indicated by pimonidazole staining.

<p>Animals were ventilated with 100% O₂. The traumatized <i>Musculus gastrocnemius</i> of the trauma group IIa (n = 4 for each time point) and the <i>Musculus gastrocnemius</i> of the control group IIc (n = 6) were harvested and sections of the muscle specimen were analyzed for pimonidazole binding. Hypoxic fraction, percentage area of pimonidazole-labelled muscle cells within an area; injured area, injured area of the traumatized muscle of the trauma group IIa; uninjured area, the adjacent uninjured area of the traumatized muscle of the trauma group IIa; control, tissue area of the non-traumatized muscle of the control group IIc. Values shown represent means ± SEM. *P<0.05 (versus control).</p

HIF-1α staining within injured muscle area (representative figure).

<p>The animal was ventilated with 100% O₂. 450 minutes after trauma, the <i>Musculus gastrocnemius</i> of the traumatized right hind limb was harvested and section of the muscle specimen was analyzed for HIF-1α expression within the injured area. No significant staining for HIF-1α of the muscle cells. Scale bar: 150 µm. Inlet: Positive staining for HIF-1α expression by myeloid cells (brown), invading to the traumatized muscle. Scale bar: 10 µm.</p

Effect of local blunt muscle trauma on microvascular blood flow, relative hemoglobin amount and hemoglobin O₂ saturation.

<p>Animals (n = 8) were ventilated with 100% O₂. Parameters were determined immediately before trauma (base values, base), 1–2 minutes after trauma (0) and every 40 minutes until the end of the experimental period. flow, microvascular blood flow (A); rHb, relative hemoglobin amount, representing blood filling of microvessels (B); SO₂, microvascular hemoglobin O₂ saturation (C); AU, arbitrary unit; trauma, injured area of traumatized muscle; contralateral, respective area of the contralateral non-traumatized muscle. Values shown represent means ± SEM. *P<0.05 (versus base). ^#P<0.05 (versus contralateral; significant to all post trauma time points of flow and rHb except for flow at time point 160 minutes; not significant for SO₂, but post-hoc t-tests reveal significant differences between the groups for the time points 200 and 240 minutes).</p

Probe positions at the injured area.

<p>Five different probe positions (A–E) were used for laser Doppler and white-light spectroscopic measurements with the spectrometer O2C (oxygen-to-see). The injured area of the dorsal compartment muscles of the shaved right lower hind limb is shown. A special glass fiber probe was used to collect data over 20 seconds at each position.</p