Hypertonic saccharide solution delays pyroptosis in murine macrophages regardless of the membrane binding of gasdermin D N-terminal.

Pyroptosis is a type of programmed lytic cell death that could be activated by either the canonical or noncanonical inflammasome pathway. In this study, we aimed to examine the effect of hypertonic solution on noncanonical pyroptosis in macrophage. We found that although hypertonic solution had a general inhibitory effect on noncanonical pyroptosis, the underlying mechanism varied by the solute causing hypertonicity. Specifically, hypertonic NaCl or KCl solution inhibited the cleavage of gasdermin D, the pore-forming protein in pyroptosis, whereas hypertonic saccharide solution did not affect the cleavage or membrane binding of gasdermin D. In this case, nevertheless, pyroptosis was still inhibited as evidenced by the preserved mitochondria activity and cell membrane permeability.

A rapid and efficient method for the extraction and identification of menaquinones from Actinomycetes in wet biomass.

BACKGROUND: Menaquinones are constituents of prokaryote cell membranes where they play important functions during electron transport. Menaquinone profiles are strongly recommended for species classification when proposing a new Actinomycetes taxon. Presently, the most widely used methods to determine menaquinones are based on freeze-dried cells. Taxonomic research in our lab has revealed that menaquinone concentrations are low for some species of the genus Microbacterium, leading to difficulties in identifying menaquinones. RESULTS: Menaquinones extracted using the novel lysozyme-chloroform-methanol (LCM) method were comparable in quality to those obtained using the Collins method, the most widely used method. All tested strains extracted via the LCM method showed higher concentrations of menaquinones than those extracted via the Collins method. For some Microbacterium strains, the LCM method exhibited higher sensitivity than the Collins method, and more trace menaquinones were detected with the LCM method than the Collins method. In addition, LCM method is faster than the Collins method because it uses wet cells. CONCLUSION: The LCM method is a simple, rapid and efficient technique for the extraction and identification of menaquinones from Actinomycetes.

Hypertonic Mannitol for the Prevention of Intradialytic Hypotension: A Randomized Controlled Trial.

RATIONALE & OBJECTIVE: Intradialytic hypotension (IDH) is a common complication at the initiation of hemodialysis (HD) therapy, is associated with greater mortality, and may be related to relatively rapid shifts in plasma osmolality. This study sought to evaluate the effect of an intervention to minimize intradialytic changes in plasma osmolality on the occurrence of IDH. STUDY DESIGN: Double-blind, single-center, randomized, controlled trial. SETTING & PARTICIPANTS: Individuals requiring initiation of HD for acute or chronic kidney disease. INTERVENTION: Mannitol, 0.25g/kg/h, versus a similar volume of 0.9% saline solution during the first 3 HD sessions. OUTCOMES: The primary end point was average decline in systolic blood pressure (SBP). The secondary end point was the proportion of total sessions complicated by IDH (defined as a decrease ≥ 20mm Hg from the pre-HD SBP). Exploratory end points included biomarkers of cardiac and kidney injury. RESULTS: 52 patients were randomly assigned and contributed to 156 study visits. There were no significant differences in average SBP decline between the mannitol and placebo groups (15±11 vs 19±16mm Hg; P = 0.3). The proportion of total sessions complicated by IDH was lower in the mannitol group compared to placebo (25% vs 43%), with a nominally lower risk for developing an episode of IDH (OR, 0.38; 95% CI, 0.14-1.00), though this finding was of borderline statistical significance (P = 0.05). There were no consistent differences in cardiac and kidney injury biomarker levels between treatment groups. LIMITATIONS: Modest sample size and number of events. CONCLUSIONS: In this pilot randomized controlled trial studying patients requiring initiation of HD, we found no difference in absolute SBP decline between those who received mannitol and those who received saline solution. However, there were fewer overall IDH events and a nominally lower risk for dialysis sessions being complicated by IDH in the mannitol group. A larger multicenter randomized controlled trial is warranted. FUNDING: Government funding to an author (Dr Mc Causland is supported by National Institute of Diabetes and Digestive and Kidney Diseases grant K23DK102511). TRIAL REGISTRATION: Registered at ClinicalTrials.gov with study number NCT01520207.

Protective effects of hydrogen-rich saline against N-methyl-N-nitrosourea-induced photoreceptor degeneration.

The N-methyl-N-nitrosourea (MNU)-treated rat is typically used as an animal model of chemically-induced retinitis pigmentosa (RP). Reactive oxygen species (ROS) have been recognized as the crucial contributor to the retinal photoreceptor apoptosis seen in MNU-treated rats. In the present study, we explored the therapeutic effects of hydrogen-rich saline (HRS), a selective ROS scavenger, on MNU-induced photoreceptor degeneration. Intraperitoneal (IP) administration of HRS ameliorated MNU-induced photoreceptor degeneration in terms of morphology and function: Sharply decreased thickness of the retinal outer nuclear layer (ONL) and flattened photopic and scotopic electroretinogram (ERG) waveforms, typically seen in response to MNU treatment, were substantially rescued in rats cotreated with MNU and HRS (MNU + HRS). Moreover, the terminal deoxyuridine triphosphate nick-end labeling (TUNEL) assay revealed a smaller number of apoptotic photoreceptors in the MNU + HRS group compared that in the MNU group. Compared to MNU-treated rats, retinal malondialdehyde (MDA) content in MNU + HRS rats significantly decreased while superoxide dismutase (SOD) activity significantly increased. Morphological and multi-electrode array (MEA) analyses revealed more efficient preservation of the architecture and field potential waveforms in particularly the peripheral regions of the retinas within the MNU + HRS group, compared to that in the MNU group. However, this enhanced protection of structure and function in the peripheral retina is unlikely the result of site-dependent variation in the efficacy of HRS; rather, it is most likely due to reduced susceptibility of peripheral photoreceptors to MNU-induced degeneration. Inner retinal neuron function in the MNU + HRS rats was better preserved, with fewer apoptotic photoreceptors in the ONL. Collectively, these results support the rationale for future clinical evaluation of HRS as a therapeutic agent for human RP.

Optimizing Irreversible Electroporation Ablation with a Bipolar Electrode.

PURPOSE: To optimize single-insertion bipolar irreversible electroporation (IRE) by characterizing effects of electric parameters and controlling tissue electric properties in a porcine model. MATERIALS AND METHODS: Single-insertion electrode bipolar IRE was performed in 28 in vivo pig livers (78 ablations). First, effects of voltage (2,700-3,000 V), number of pulses, repeated cycles (1-6 cycles), and pulse width (70-100 µs) were studied. Next, electric conductivity was altered by instillation of hypertonic and hypotonic fluids. Finally, effects of thermal stabilization were assessed using internal electrode cooling. Treatment effect was evaluated 2-3 hours after IRE. Dimensions were compared and subjected to statistical analysis. RESULTS: Delivering 3,000 V at 70 µs for a single 90-pulse cycle yielded 3.8 cm ± 0.4 × 2.0 cm ± 0.3 of ablation. Applying 6 cycles of energy increased ablation to 4.5 cm ± 0.4 × 2.6 cm ± 0.3 (P < .001). Further increasing pulse lengths to 100 µs (6 cycles) increased ablation to 5.0 cm ± 0.4 × 2.9 cm ± 0.3 (P < .001) but resulted in electric spikes and system crashes in 40%-50% of cases. Increasing tissue electric conductivity via hypertonic solution instillation in surrounding tissues increased frequency of generator crashes, whereas continuous instillation of distilled water eliminated this arcing phenomenon but reduced ablation to 2.3 cm ± 0.1. Controlled instillation of distilled water when electric arcing was suspected from audible popping produced ablations of 5.3 cm ± 0.6 × 3.1 cm ±0.3 without crashes. Finally, 3.1 cm ± 0.1 short-axis ablation was achieved without system crashes with internal electrode perfusion at 37°C versus 2.3 cm ± 0.1 with 4°C-10°C perfusion (P < .001). CONCLUSIONS: Bipolar IRE ablation zones can be increased with repetitive high voltage and greater pulse widths accompanied by either judicious instillation of hypotonic fluids or internal electrode perfusion to minimize unwanted electric arcing.

Meta-analysis of high- versus low-chloride content in perioperative and critical care fluid resuscitation.

BACKGROUND: The objective of this systematic review and meta-analysis was to assess the relationship between the chloride content of intravenous resuscitation fluids and patient outcomes in the perioperative or intensive care setting. METHODS: Systematic searches were performed of PubMed/MEDLINE, Embase and Cochrane Library (CENTRAL) databases in accordance with PRISMA guidelines. Randomized clinical trials, controlled clinical trials and observational studies were included if they compared outcomes in acutely ill or surgical patients receiving either high-chloride (ion concentration greater than 111 mmol/l up to and including 154 mmol/l) or lower-chloride (concentration 111 mmol/l or less) crystalloids for resuscitation. Endpoints examined were mortality, measures of kidney function, serum chloride, hyperchloraemia/metabolic acidosis, blood transfusion volume, mechanical ventilation time, and length of hospital and intensive care unit stay. Risk ratios (RRs), mean differences (MDs) or standardized mean differences (SMDs) and confidence intervals were calculated using fixed-effect modelling. RESULTS: The search identified 21 studies involving 6253 patients. High-chloride fluids did not affect mortality but were associated with a significantly higher risk of acute kidney injury (RR 1.64, 95 per cent c.i. 1.27 to 2.13; P < 0.001) and hyperchloraemia/metabolic acidosis (RR 2.87, 1.95 to 4.21; P < 0.001). High-chloride fluids were also associated with greater serum chloride (MD 3.70 (95 per cent c.i. 3.36 to 4.04) mmol/l; P < 0.001), blood transfusion volume (SMD 0.35, 0.07 to 0.63; P = 0.014) and mechanical ventilation time (SMD 0.15, 0.08 to 0.23; P < 0.001). Sensitivity analyses excluding heavily weighted studies resulted in non-statistically significant effects for acute kidney injury and mechanical ventilation time. CONCLUSION: A weak but significant association between higher chloride content fluids and unfavourable outcomes was found, but mortality was unaffected by chloride content.

Allogeneic human tissue-engineered blood vessel.

BACKGROUND: Arterial bypass graft implantation remains the primary therapy for patients with advanced cardiovascular disease; however, there is no available synthetic small-diameter vascular graft. METHODS: Tissue-engineered vessels were grown from human smooth muscle cells that were seeded on a biodegradable scaffold using a biomimetic perfusion system. The human tissue-engineered vessels (hTEV) were decellularized by a two-step process using a combination of detergents and hypertonic solutions. The mechanical characteristics were assessed by suture retention strength and burst pressure. The decellularized hTEV were implanted as aortic interpositional grafts in nude rats to evaluate in vivo performance as an arterial graft over a 6-week period. RESULTS: The human tissue-engineered structure formed a vessel composed of smooth muscle cells and the extracellular matrix proteins, including collagen. After decellularization, the collagen matrix remained intact while the cellular components were removed. The mechanical strength of the hTEV after decellularization was similar to human vein in vitro, with a burst pressure of 1,567 ± 384 mm Hg (n = 3) versus 1,680 ± 307 mm Hg for human saphenous vein. The hTEVs had a high patency rate (four of five grafts) without evidence of rupture or aneurysm over a 6-week period as an aortic interpositional graft in a nude rat model. Histologic analysis showed a thin neointima with a confluent endothelium and a subendothelial layer of smooth muscle cells on the explanted tissue-engineered vessels. Transmission electron microscopy on the explanted tissue demonstrated elastin formation in the neointima and intact residual collagen fibers from the tissue-engineered vessel. CONCLUSIONS: The hTEV had a high patency rate and remained mechanically stable as an aortic interpositional graft in a nude rat. The vessel supported the growth of a neointima with endothelial cells and smooth muscle cells. The host remodeling suggested the engineered matrix had a positive effect to create a regenerated vascular graft.

Cryobiological properties of immature zebrafish oocytes assessed by their ability to be fertilized and develop into hatching embryos.

As a step to develop a cryopreservation method for zebrafish oocytes, we investigated the cryobiological properties of immature oocytes at stage III by examining their ability to mature and to develop into hatching embryos after fertilization. When oocytes were chilled at -5°C for 30min, the maturation rate decreased, but the rates of fertilization and hatching were not significantly different from those of controls. When oocytes were exposed to hypotonic solutions for 60min at 25°C, the rates of maturation, fertilization, and hatching decreased in a solution with 0.16Osm/kg or below. When oocytes were exposed to hypertonic solutions (containing sucrose) at 25°C for 30min, the maturation rate decreased in solution with 0.51Osm/kg, whereas the hatching rate decreased with lower osmolality (0.40Osm/kg). In an experiment on the toxicity of cryoprotectants (∼10%, at 25°C), it was found that glycerol and ethylene glycol were toxic both by the assessment of maturation and hatching. Propylene glycol, DMSO and methanol were less toxic by the assessment of maturation, but were found to be toxic by the assessment of hatching. Methanol was the least toxic, but it was less effective to make a solution vitrify than propylene glycol. Therefore, a portion of methanol was replaced with propylene glycol. The replacement increased the toxicity, but could be effective to reduce chilling injury at -5°C. These results clarified the sensitivity of immature oocytes to various cryobiological properties accurately, which will be useful for realizing cryopreservation of zebrafish oocytes.

Measurement of the membrane elasticity of red blood cell with osmotic pressure by optical tweezers.

Cells have to undergo many changes in osmotic pressure during their long-term preservation, and will have injuries before they return to their normal states. Mechanics of a cell with deformation, either small or large, is usually used to describe the change of the cell quantitatively. However, there are few reports on the deformation of cells subjected to the change of osmotic pressures during preservation. Here, we report our study of the elasticity of the human red blood cells under osmotic pressures using optical tweezers. We find that the deformation characteristics of erythrocytes are strongly dependent on the osmotic pressure. We also find the RBCs will become stiff with increasing osmotic pressure, suggesting a potential reason for membrane injury during preservation.

[Correction of intracranial hypertension syndrome using hyperosmolar solutions in patients with severe brain damage (multicenter randomized clinical study)].

The paper presents the results of a muticenter study of the effect of 3 hyperosmolar solutions (15% mannitol solution, 10% sodium chloride solution, and the combined solution HyperHAES containing 7.2% sodium chloride and hydroxyethyl starch 200/0.5) on the value of intracranial pressure (ICP) (invasive ICP monitoring) and systemic hemodynamic parameters (PiCCOplus) in 94 clinical cases of intracranial hypertension (ICP more than 20 mm Hg) in 25 patients with acute cerebral pathology (severe brain injury, aneurysmatic subarachnoid hemorrhage). Intravenous infusion of the solutions was found to induce a reduction in ICP; however, this was most pronounced (by 30-40%) and longer (up to 4 hours) when HyperHAES solution was used. This solution produced not only an osmotic, but also hemodynamic effect.

Erythrocyte storage in hypertonic (SAGM) or isotonic (PAGGSM) conservation medium: influence on cell properties.

BACKGROUND AND OBJECTIVES: Prolonged red blood cell (RBC) storage may be associated with increased post-transfusion morbidity and mortality. A contributing factor is RBC storage lesions. We analysed the role of additive conservation solutions, either hypertonic or isotonic, on such cell properties. MATERIALS AND METHODS: After blood donation in citrate-phosphate-dextrose as an anticoagulant, 10 RBC units were stored with saline-adenine-glucose-mannitol (SAGM; 376 mOsm/l) and 10 units with phosphate-adenine-glucose-guanosin-saline-mannitol (PAGGSM; 285 mOsm/l). Measurements were made on days 1 and 42 of storage. RESULTS: The mean cellular volume measured by centrifuged microhaematocrit increased from 87.6 +/- 3.1 fl to 100.7 +/- 4.3 fl in PAGGSM and to 92.2 +/- 2.5 fl in SAGM (P < 0.001) on day 1, after 42 days it was 95.8 +/- 4.0 fl and 93.8 +/- 3.9 fl, respectively. Spontaneous haemolysis and osmotic fragility were lower after storage in PAGGSM. Both additives showed a similar degree of echinocytosis, decreased RBC aggregability and deformability, and increased RBC suspension viscosity after storage. CONCLUSIONS: The isotonic PAGGSM prevented the initial RBC swelling caused by citrate-phosphate-dextrose less than hypertonic SAGM, but reduced the spontaneous haemolysis rate and osmotic fragility after 42 days of storage. All other parameters, such as echinocytosis, decreased RBC deformability and aggregability, and increased blood viscosity was similar for both additive solutions and remained a major problem of blood banking.

Low osmolality and shear stress during liposuction impair cell viability in autologous fat grafting.

BACKGROUND: Liposuction and subsequent autologous fat grafting have become essential techniques for fat augmentation in plastic surgery. However, standard harvesting techniques that ensure the survival of adipocytes and stromal vascular fraction (SVF) cells and thus preserve the transplanted fat volume are lacking. In particular, the effect of different parameters of the tumescent solution has not been studied in this context. We hypothesized that the osmolality of the tumescent solution could have a significant effect on the survival of adipocytes and SVF cells. METHODS: We developed two distinct in vitro models based on freshly harvested excision fat from patients undergoing surgical treatment. First, we investigated the effect of osmolality by incubating excision fat in different tumescent solutions and analyzed the total cell survival and the differentiation potential of SVF cells. Vital whole-mount staining, isolation yield of SVF cells, clonogenicity, and osteogenic and adipogenic differentiation capacities were analyzed. Second, we addressed the additional effect of mechanical stress by simulating a liposuction on pieces of excision fat after incubation with the tumescent solutions. RESULTS: Osmolality of the tumescent solution by itself did not have a significant effect on adipocyte and SVF viability or SVF differentiation. However, when osmolality was combined with liposuction, a significant trend toward lower viability and more lipid droplets with lower osmolality was observed. Especially, SVF viability was significantly lower after liposuction with a hypotonic (150 mOsm/kg) solution. CONCLUSION: This study demonstrates the considerable effect of osmolality during liposuction and may lead to the development of "cell-protective" tumescent solutions.

Improvement of endothelial cell viability at 4 degrees C by addition of lazaroid U74500A to preservation solutions.

Lazaroids are potent inhibitors of lipid peroxidation. Endothelial cell damage has been shown to occur during cold storage preservation of lung and liver. This study examines the effects on endothelial cell viability of the addition of four lazaroids, U74006F, U78518F, U74500A, or U75412E to preservation solutions. Human umbilical vein endothelial cell cultures were stored at 4 degrees C for 48 and 96 hr in EuroCollins or 5% polyethylene glycol in buffered saline (PEG). U78518F, U74500A, U74006F, U75412E, or dexamethasone (each 50 microM) was added to EC (n = 32) or PEG (n = 32) and compared with control solutions of EC or PEG alone. Endothelial cell viability was determined by measuring cellular reduction of 3-[4,5-dimethylthiazol-2-yl]-2,3-diphenyltetrazolium bromide to a purple formazan dye. The reduction occurs only in viable cells and requires mitochondrial dehydrogenase activity. Results were quantified by measuring dye absorbance (Ab) with a micro-ELISA spectrophotometer. Absorbance values were compared by ANOVA and reported as mean values +/- standard deviation. Addition of U74500A to EC (Ab = 0.474 +/- 0.055) and PEG (Ab = 0.462 +/- .005) improved viability at 48 hr when compared with EC (Ab = 0.289 +/- 0.069) and PEG (Ab = 0.287 +/- 0.052) alone (P less than 0.05). At 96 hr, addition of U74500A resulted in improved viability in both EC (Ab = 0.377 +/- 0.068) and PEG (Ab = 0.195 +/- 0.09) or PEG alone (Ab = 0.212 +/- 0.1) (P less than 0.05). Other lazaroids tested were also effective in improving cellular viability, but to a lesser degree than U74500A. This study demonstrates that the addition of lazaroids to organ preservation solutions improves endothelial cell viability.

The role of osmolality in the absorption of a nutrient solution.

BACKGROUND: Loss of water during enteral nutrition following massive intestinal resection may be severe. Low osmolality of oral rehydration solutions has recently been shown to mediate an increase in water absorption. AIM: To evaluate the effect of osmolality of a nutrient solution on the intraluminal duodenojejunal water flow, and the net absorption rates of total nitrogen and carbohydrate. METHODS: Eight healthy volunteers with a mean age of 27 (range 25-29) years participated in the study. Enteral nutrition (17% protein, 59% carbohydrate, 24% lipid plus 5 g/L PEG 4000) was infused (5 mL/min 2.64 kcal/min) into the descending duodenum either as a hypotonic (160 mOsmol/kg) or as an isotonic solution in a random order. Intestinal samples were aspirated 20 and 45 cm distally to the infusion point. RESULTS: Intraluminal water flow rates were significantly lower with the hypotonic solution than with the isotonic solution, both in the duodenum (4.9 +/- 0.3 vs. 6.7 +/- 0.5 mL/min; P < 0.02) and the upper jejunum (3.0 +/- 0.1 vs. 3.9 +/- 0.2 mL/min; P < 0.005). The net absorption rates of total nitrogen and carbohydrate were similar with both solutions. CONCLUSION: Low osmolality of a nutrient solution decreases intraluminal water flow rates in the upper intestine without affecting the absorption rates of total nitrogen and carbohydrate. Compared with an isotonic solution, the use of a hypotonic solution might lower the water loss in patients with extensive short bowel intestinal resection.

Impact of initial flush potassium concentration on the adequacy of lung preservation.

The effects of initial lung flushing with intracellular and extracellular fluid type solutions were studied in lungs stored with the University of Wisconsin solution. Excised Sprague-Dawley rat lungs (n = 39) were flushed first with one of the following solutions: (1) the University of Wisconsin solution (K+ = 140 mmol/L), (2) modified (low potassium) University of Wisconsin solution (K+ = 20 mmol/L), (3) phosphate buffered saline solution (K+ = 3.9 mmol/L), (4) modified low-potassium phosphate-buffered saline solution (K+ = 20 mmol/L), (5) modified high-potassium phosphate-buffered saline solution (K+ = 40 mmol/L), and (6) Euro-Collins solution (K+ = 115 mmol/L) followed by secondary flush with storage solution and cold (4 degrees C) storage in University of Wisconsin solution for 24 hours. The lungs were then reperfused in the isolated, pulsatile, blood-perfused working lung system for 2 hours or until lung failure. Blood gas analysis and shunt fraction, aerodynamic parameters (airway resistance, lung compliance, elastic work, and flow resistive work), and total pulmonary vascular resistance were measured throughout the perfusion period. The mean oxygen tensions (in millimeters of mercury) at 30 minutes after the onset of reperfusion for University of Wisconsin solution, modified University of Wisconsin solution, phosphate-buffered saline solution, modified phosphate-buffered saline solutions (20 and 40 mmol/L), and Euro-Collins solution were 56.1 +/- 4.2, 72.7 +/- 9.1, 87.7 +/- 6.9 (p < 0.01 versus University of Wisconsin solution; p < 0.01 versus Euro-Collins solution), 86.0 +/- 9.6 (p < 0.01 versus University of Wisconsin solution; p < 0.01 versus Euro-Collins solution), 87.9 +/- 7.7 (p < 0.01 versus University of Wisconsin solution; p < 0.01 versus Euro-Collins solution), and 53.5 +/- 6.0, respectively. All aerodynamic parameters in the lungs flushed with extracellular fluid type solutions were superior to those flushed with intracellular fluid type solutions. We conclude that the efficacy of initial flushing was essential for successful lung preservation and that extracellular fluid type solutions were superior to intracellular fluid type solutions, at least for flushing the lung before storage with University of Wisconsin solution. Potassium concentration in flushing solution should be 20 mmol/L or less to obtain appropriate flushing and subsequent adequate distribution of the storage solution.

Superior protection in orthotopic rat lung transplantation with cyclic adenosine monophosphate and nitroglycerin-containing preservation solution.

BACKGROUND: Primary lung graft failure is common, and current lung preservation strategies are suboptimal. Because the decline in lung levels of cyclic adenosine monophosphate and cyclic guanosine monophosphate during preservation could enhance adhesiveness of endothelial cells for leukocytes as well as increase vascular permeability and vasoconstriction, we hypothesized that buttressing these levels by means of a preservation solution would significantly improve lung preservation. METHODS: An orthotopic rat left lung transplantation model was used. Lungs were harvested from male Lewis rats and preserved for 6 hours at 4 degrees C with (1) Euro-Collins solution (n = 8); (2) University of Wisconsin solution (n = 8); (3) low-potassium dextran glucose solution (n = 8); (4) Columbia University solution (n = 8), which contains a cyclic adenosine monophosphate analog (dibutyryl cyclic adenosine monophosphate) and a nitric oxide donor (nitroglycerin) to buttress cyclic guanosine monophosphate levels; or (5) Columbia University solution without cyclic adenosine monophosphate or nitroglycerin (n = 8). PaO2, pulmonary vascular resistance, and recipient survival were evaluated 30 minutes after left lung transplantation and removal of the nontransplanted right lung from the pulmonary circulation. RESULTS: Among all groups studied, grafts stored with Columbia University solution demonstrated the highest Pa O2 (355 +/- 25 mm Hg for Columbia University solution versus 95 +/- 22 mm Hg for Euro-Collins solution, P <.01, 172 +/- 55 mm Hg for University of Wisconsin solution, P <.05, 76 +/- 15 mm Hg for low-potassium dextran glucose solution, P <.01, and 82 +/- 25 mm Hg for Columbia University solution without cyclic adenosine monophosphate or nitroglycerin, P <.01) and the lowest pulmonary vascular resistances (1 +/- 0.2 mm Hg * mL-1 * min-1 for Columbia University solution versus 12 +/- 4 mm Hg * mL-1 * min-1 for Euro-Collins solution, P <.01, 9 +/- 2 mm Hg * mL-1 * min-1 for University of Wisconsin solution, 14 +/- 6 mm Hg * mL-1 * min-1 for low-potassium dextran glucose solution, P <.01, and 8 +/- 2 mm Hg * mL-1 * min-1 for Columbia University solution without cyclic adenosine monophosphate and nitroglycerin). These functional and hemodynamic improvements provided by Columbia University solution were accompanied by decreased graft leukostasis and decreased recipient tumor necrosis factor alpha and interleukin 1alpha levels compared with the other groups. In toto, these improvements translated into superior survival among recipients of Columbia University solution-preserved grafts (100% for Columbia University solution, 37% for Euro-Collins solution, P <.01, 50% for University of Wisconsin solution, P <.05, 50% for low-potassium dextran glucose solution, P <.05, and 13% for Columbia University solution without cyclic adenosine monophosphate and nitroglycerin, P <.01). CONCLUSION: Nitroglycerin and cyclic adenosine monophosphate confer beneficial vascular effects that make Columbia University solution a superior lung preservation solution in a stringent rat lung transplantation model.

Influence of the potassium concentration on functional and structural preservation of the lung: where is the optimum?

BACKGROUND: Low-potassium solutions have been shown to improve lung preservation. The optimal potassium concentration, however, has not been investigated systematically. The purpose of this study was to evaluate the effect of solutions with different potassium concentrations on functional and structural preservation after flush-perfusion and ischemia. We used our established extracorporeal working heart-lung model and a modification of this model with isolated pulmonary perfusion at defined flow rates. METHODS: In two sets of experiments 42 rat heart-lung blocks (experiment I and II: n=7/group) were used. Lungs were flush-preserved with 20 ml Euro-Collins solution (EC115; K+ 115 mmol/L), potassium-reduced Euro-Collins solution (EC40; K+ 40 mmol/L), or low-potassium Euro-Collins solution (EC10; K+ 10 mmol/L) and stored for 2 hours at 10 degrees C. Reperfusion was performed for 40 minutes with Krebs-Henseleit solution containing washed bovine red blood cells (38%) while the lungs were ventilated with room air. In experiment I pulsatile perfusion of the lungs was achieved by the working right side of the heart. In experiment II lungs were perfused at defined flow rates by a roller pump. Postischemic function was assessed by means of oxygenation capacity and pulmonary vascular resistance. The degree of structural damage to the air-blood barrier was assessed by quantitative stereologic light and electron microscopic evaluation. RESULTS: In both experiments after 40 minutes reperfusion oxygenation capacity was significantly higher in EC40 than in EC115 and EC10, whereas pulmonary vascular resistance was significantly higher in EC115 than in EC40 and EC10. Quantitative histologic examination showed surprisingly modest damage to the endothelial side of the air-blood barrier but a considerable degree of damage to the epithelium in both experiments. The alterations in the pump-perfused isolated lung experiments exceeded those of the pulsatile perfused heart-lung experiments. The comparative analysis of the study groups revealed a minor degree of epithelial swelling and fragmentation in EC40 than in EC115 and EC10, respectively. CONCLUSIONS: The results obtained with two modifications of an extracorporeal model indicate that flush perfusion of the lung with a potassium-reduced solution results in better functional and structural preservation than flush perfusion with either high- or low-potassium solutions. The optimum may lie in the vicinity of 40 mmol/L. Further studies are necessary to verify these initial findings.

Studies of rat lung viability and adenine nucleotide metabolism after death.

BACKGROUND: Prior studies from our laboratory have supported the use of cadaveric lungs for transplantation. In this study we investigated different preservation strategies for lungs retrieved from cadavers 4 hours after circulatory arrest. METHODS: Seventy-two Sprague-Dawley rats were sacrificed and then ventilated with 100% oxygen for 4 hours. The lungs were then flushed with modified Euro-Collins, University of Wisconsin, or Carolina rinse solution, either alone, with prostaglandin E1, or with prostaglandin E1 plus the free radical scavenger dimethylthiourea. After an additional 4-hour cold storage, the left lung was flushed with trypan blue solution to quantify cell viability, whereas the right lung was used to determine wet-to-dry weight ratios and to measure the levels of the adenine nucleotides adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate by high-performance liquid chromatography. RESULTS: Viability was consistently better in the lungs flushed with Carolina rinse solution; these differences were statistically significant compared with those in the corresponding modified Euro-Collins subgroups (p < 0.005). The addition of prostaglandin E1 to all three preservation solutions improved the total adenine nucleotide levels; this increase was statistically significant for the modified Euro-Collins subgroup (p < 0.005). The total adenine nucleotide levels for the University of Wisconsin subgroups were higher than those for the corresponding modified Euro-Collins subgroups. The highest total adenine nucleotide levels were obtained in lungs flushed with Carolina rinse plus prostaglandin E1. Wet-to-dry weight ratios were always significantly lower in the lungs preserved with University of Wisconsin solution (p < 0.05), with a value similar to that of fresh tissue. CONCLUSIONS: The characteristics of the solution used to flush and to store rat cadaveric lungs have an impact on lung viability and adenine nucleotide metabolism. The ideal preservation strategy may allow for lung retrieval from cadavers for safe transplantation.

Hypertonic resuscitation and blood coagulation: in vitro comparison of several hypertonic solutions for their action on platelets and plasma coagulation.

Resuscitation with hypertonic saline (HS) appears to aggravate bleeding in a model of uncontrolled hemorrhage [J. Trauma 28 (1988) 751; J. Trauma 29 (1989) 79; Arch. Surg. 127 (1992) 93]. This property may be related to the anticoagulant effects of HS on plasma clotting factors and platelets [J. Trauma 31 (1991) 8]. The hypothesis in this study is that a hypertonic solution can be developed that would not disturb the blood coagulation mechanism and could be used as an alternative to hypertonic saline.HS and four different 2400 mosM solutions containing monosaccharides and/or glycine were screened for their in vitro effects on plasma clotting times and platelets. Significant prolongations falling outside the normal range were detected in prothrombin time (PT) and thrombin rime (TT) when only 5% of the volume of normal plasma is HS. Platelet function as measured by extent of shape change (ESC) induced by ADP and aggregation induced by thrombin were also critically impaired by HS at a 5% dilution. All alternative solutions-hypertonic glucose, sorbitol, glycine, glucose/glycine, glucose/mannitol/glycine, sorbitol/glycine-caused a significantly reduced impairment in platelet function and the plasma coagulation system. Hypertonic glycine showed a unique ability to fully preserve the function and integrity of the plasma coagulation system. Considering the pre-deposition of the trauma patient to coagulopathy, administration of HS which clearly is a potent anticoagulant and anti-platelet risks further aggravating the coagulopathy. In contrast, hypertonic glycine preserves the blood coagulation mechanism and exhibits the potential for numerous therapeutic applications. Therefore, prompt evaluation of hypertonic glycine as a resuscitative fluid is highly desirable.

Digestion of maize and sunflower pollen by the spotted maize beetle Astylus atromaculatus (Melyridae): is there a role for osmotic shock?

We investigated the mechanism and efficiency of digestion of two types of pollen, maize, Zea mays, and sunflower, Helianthus annuus, by the spotted maize beetle, Astylus atromaculatus (Melyridae). We found similar and high extraction efficiencies, but different mechanisms of digestion. Osmotic shock was apparently involved in digestion of the large and thin-walled maize pollen grains. In the anterior midgut most maize pollen grains were already ruptured, in contrast with the intact exines of sunflower pollen, which suggests another mechanism of digestion for the latter, such as enzymatic action. We investigated the effect of osmotic shock on maize pollen in vitro by looking at the behavior of pollen grains in varying osmotic concentrations. Maize pollen grains burst in both distilled water and sugar solutions of various concentrations, and the amount of rupturing decreased with an increase in sugar concentration. Digestion of maize pollen was much slower in honeybees than in spotted maize beetles. Maize pollen bursts early in the midgut of maize beetles, but remains intact in honeybees: this suggests that osmotic shock may not be as important for honeybees as previously suggested.