Peritoneal, systemic, and distant organ inflammatory responses are reduced by a laparoscopic approach and carbon dioxide vs air

BACKGROUND: Advantages of laparoscopic surgery have, among other factors, been attributed to a shorter length of abdominal incision and the use of CO2 versus air. An analysis of these factors taking pressure-induced alterations into account is lacking. The objective of the study was to determine the impact of laparoscopy and laparotomy with exposure to CO2 and room air under a similar pressure on local, systemic, and distant organ immune responses. METHODS: Twenty piglets were randomized into four groups: CO2 laparoscopy, air laparoscopy, CO2 laparotomy, and air laparotomy. Laparotomy was performed in a sterile balloon pressurized similar to laparoscopy. Peritoneal interleukin-1, interleukin-6, tumor necrosis factor-a, and counts of polymorphnuclear cells (PMNs), and macrophages (MFs) were determined in abdominal lavage fluids at 0, 2, and 48 h. Macrophages were assessed for reactive oxygen species (ROS) production. Systemic responses were gauged by white blood cell count (WBC) and cytokines. Alveolar lavage was performed at 48 h to determine cytokine levels, cell counts, and MF ROS production. Blood, lavage fluids, and mesenteric lymph nodes were tested for bacterial translocation. RESULTS: Regarding the peritoneal response, laparotomy versus laparoscopy when performed with CO2 significantly increased PMN and decreased the percentage of macrophages (%MF) up to 48 h. There was a significant increase in interleukin-6, and there was a fourfold increase in MF ROS production. Similar differences between the procedures were found with exposure to air. The use of air versus CO2 in laparoscopy, but not in laparotomy, resulted in an increase of peritoneal PMN and a decrease of the %MF up to 48 h. Air increased the local interleukin-6 release in both procedures and increased MF ROS production fourfold. Regarding the systemic response, laparotomy produced a significant increase in WBC, which was significantly more pronounced with exposure to air. No alteration of other systemic cytokines was seen. Regarding the pulmonary response, the number of MFs and MF ROS production were significantly increased after air versus CO2 laparoscopy. There were no such differences between the laparotomy groups. Regarding bacterial translocation, no bacteria were cultured from peritoneal fluids, lymph nodes, or blood. CONCLUSIONS: Inflammatory responses were reduced by a laparoscopic approach and by exposure to CO2 versus air. Peritoneal responses were affected to a larger degree than systemic parameters. Laparotomy overruled the effects of CO2 on chemotaxis and distant organ injury but not on peritoneal cytokine release.     

Improved cytosolic free calcium mobilization and superoxide production in bicarbonate-based peritoneal dialysis solution

Background. Intraperitoneal phagocytes play an important role in local host defence to prevent CAPD peritonitis. The intracellular calcium [Ca²⁺]i is thought to be involved in the regulation of various cell functions. This study therefore investigates the effect of lactate-based dialysis solution (LBDS) and bicarbonate-based dialysis solution (BBDS) on cytosolic free calcium mobilization and superoxide production (SP) as important steps in signal transduction and bacterial killing. Methods. We studied changes in [Ca²⁺]i and SP following stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP) in polymorphonuclear neutrophils (PMNs) incubated in either LBDS-pH 5.2, LBDS adjusted to pH 7.4, 1:10 diluted spent and fresh LBDS or BBDS-pH 7.4 with different glucose concentrations, comparing the data with cells treated with Hanks buffer (HBSS) pH 7.4 as control. To elucidate the effect of glucose and lactate PMNs were additionally incubated in HBSS-pH 7.4, containing glucose (HBSS-Glu-pH 7.4) or lactate (HBSS-Lact-pH 7.4) in the same concentrations as contained in CAPD solutions and tested as above. PMNs were isolated from healthy blood donors and incubated with dialysis solution 10 min prior to stimulation with fMLP. Results. [Ca²⁺]i mobilization and SP were completely inhibited in PMNs incubated in LBDS pH 5.2. pH adjustment of LBDS to 7.4 and 1:10 dilution of spent and fresh LBDS corrected some of the suppression of the calcium influx and superoxide production. BBDS pH 7.4, however, preserved physiological cell function significantly better at low (1.5 and 2.3%) glucose concentrations. Conclusion. In comparison to conventional lactate-based dialysis solution, pH adjusted and 1:10 diluted LBDS, bicarbonate-based dialysis solution is more biocompatible since it preserves significantly better neutrophil cell functions.     

Growth factor regulation of intracellular pH homeostasis under hypoxic conditions in isolated equine articular chondrocytes

Hypoxia and acidosis are recognized features of inflammatory arthroses. This study describes the effects of IGF‐1 and TGF‐β₁ on pH regulatory mechanisms in articular cartilage under hypoxic conditions. Acid efflux, reactive oxygen species (ROS), and mitochondrial membrane potential were measured in equine articular chondrocytes isolated in the presence of serum (10% fetal calf serum), IGF‐1 (1, 10, 50, 100 ng/ml) or TGF‐β₁ (0.1, 1, 10 ng/ml) and then exposed to a short‐term (3 h) hypoxic insult (1% O₂). Serum and 100 ng/ml IGF‐1 but not TGF‐β₁ attenuated hypoxic regulation of pH homeostasis. IGF‐1 appeared to act through mitochondrial membrane potential stabilization and maintenance of intracellular ROS levels in very low levels of oxygen. Using protein phosphorylation inhibitors PD98059 (25 µM) and wortmannin (200 nM) and Western blotting, ERK1/2 and PI‐3 kinase pathways are important for the effect of IGF‐1 downstream to ROS generation in normoxia but only PI‐3 kinase is implicated in hypoxia. These results show that oxygen and growth factors interact to regulate pH recovery in articular chondrocytes by modulating intracellular oxygen metabolites. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 197–203, 2013     

Inhibition of cyclic-3',5'-nucleotide phosphodiesterase abrogates the synergism of hypoxia with lipopolysaccharide in the induction of macrophage TNF-alpha production.

BACKGROUND: Local tumor necrosis factor (TNF)-alpha production by resident macrophages (M phi) contributes to posttraumatic tissue injury. Hypoxia decreases cellular cyclic adenosine monophosphate (cAMP) levels and enhances M phi secretion of TNF-alpha following lipopolysaccharide (LPS) stimulation. Thus, tissue hypoxia associated with trauma likely synergizes with proinflammatory mediators in the induction of M phi TNF-alpha production through an influence on cAMP generation or degradation. It is unclear whether elevation of cellular cAMP inhibits LPS-stimulated TNF-alpha production by hypoxic M phi. Moreover, it is unknown whether the synergism of hypoxia with LPS can be abrogated by promotion of cAMP generation or inhibition of cAMP degradation. METHODS: Rat peritoneal M phi were stimulated with Escherichia coli LPS (20 ng/ml) in a normoxic (room air with 5% CO(2)) or hypoxic (95% N(2) with 5% CO(2)) condition. TNF-alpha levels in cell-free supernatants were measured by enzyme-linked immunoassay. The beta-adrenoceptor agonist isoproterenol (ISP; 5.0 microM) and the adenylate cyclase activator forskolin (FSK; 50 microM) were applied to promote cAMP generation. The nonselective cyclic-3',5'-nucleotide phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX; 1.0 mM) and the PDE III-specific inhibitor milrinone (200 microM) were used to inhibit cAMP degradation. The nondegradable cAMP analogue dibutyryl cAMP (dbcAMP; 100 microM) was applied to further determine the role of PDE. RESULTS. Although hypoxia alone had a minimal effect on TNF-alpha production, it dramatically enhanced LPS-stimulated TNF-alpha production (4.08 +/- 0.28 ng/10(6) cells in hypoxia plus LPS vs 1.63 +/- 0.26 ng/10(6) cells in LPS, 2.5-fold, P < 0.01). Promotion of cAMP generation by either ISP or FSK reduced TNF-alpha production by hypoxic cells. However, neither of these two agents abolished the synergism of hypoxia with LPS (1.68 +/- 0.13 ng/10(6) cells in ISP plus hypoxia plus LPS vs 0.55 +/- 0.04 ng/10(6) cells in ISP plus LPS, threefold; 1.17 +/- 0.03 ng/10(6) cells in FSK plus hypoxia plus LPS vs 0.33 +/- 0.02 ng/10(6) cells in FSK plus LPS, 3.5-fold; both P < 0.01). Inhibition of cAMP degradation with IBMX reduced TNF-alpha production in hypoxic cells and abrogated the synergism (0.31 +/- 0.11 ng/10(6) cells in IBMX plus hypoxia plus LPS vs 0.27 +/- 0.04 ng/10(6) cells in IBMX plus LPS, P > 0.05), and the PDE III inhibitor milrinone had a comparable effect. Moreover, dbcAMP also attenuated TNF-alpha production with abrogation of the synergistic effect of hypoxia (0.56 +/- 0.08 ng/10(6) cells in dbcAMP plus hypoxia plus LPS vs 0.46 +/- 0.04 ng/10(6) cells in dbcAMP plus LPS, P > 0.05). CONCLUSIONS: The results show that elevation of cellular cAMP, either by promotion of generation or by inhibition of degradation, suppresses LPS-stimulated TNF-alpha production in hypoxic M phi. It appears that hypoxia synergizes with LPS in the induction of M phi TNF-alpha production through PDE-mediated cAMP degradation. Inhibition of PDE may be a therapeutic approach for suppression of synergistic induction of M phi TNF-alpha production by hypoxia and LPS in posttraumatic tissue.     

Carbon monoxide paradoxically protects against hypoxia

Background: Tissue hypoxia is fundamental to the development of cellular injury caused by multiple processes, including hemorrhagic shock. Others and we have previously demonstrated that endogenous production of carbon monoxide (CO) by heme oxygenase or exogenous administration of CO can protect against organ injury induced by hemorrhage. The purpose of these experiments was to test the hypotheses that CO abrogates the development of tissue hypoxia in vivo and diminishes effects of hypoxia in vitro.Methods: Male C57/BL6 mice were hemorrhaged to a mean arterial pressure of 25 mmHg. Control animals underwent the same procedure but were not hemorrhaged (sham). Inhalational CO exposure (250 ppm) was initiated concurrently with hemorrhage Mice were administered EF5 (10 μl/g; ip), which is an agent that irreversibly binds to intracellular proteins under hypoxic conditions and can then be detected by immunohistochemistry. Mice were sacrificed after 75 minutes of shock and livers were harvested. For in vitro experiments, primary mouse hepatocytes were exposed to normoxia (21% O₂) or hypoxia 1% O₂) with and without CO (250 ppm). Cells were harvested and assayed for viability, ATP content, or VEGF production. Results: Untreated hemorrhaged mice had a 17-fold increase in EF5 staining compared to shams. Hepatic hypoxia was most pronounced around the central veins. CO-treated hemorrhaged mice had a 79±13% reduction in EF5 staining compared to untreated hemorrhaged mice (P < 0.05). Additionally, CO-treated sham mice demonstrated no significant liver hypoxia. In vitro, CO significantly protected against hypoxia-induced cell death (P < 0.05). Hepatocytes exposed to hypoxia demonstrated a 54 ± 4% reduction in intracellular ATP levels after 6 hours, whereas CO treatment of hypoxic cells resulted in only a 9 ± 3% reduction in ATP (P < 0.01). Likewise, CO significantly inhibited VEGF elaboration by hypoxic hepatocytes. Conclusions: Inhaled low dose CO can paradoxically abrogate liver hypoxia during hemorrhage. Additionally, CO diminishes the consequences of hypoxia in vitro. CO may exert these effects by decreasing cellular metabolic activity and oxygen requirements, though further investigation is required. CO may prove to be a useful clinical adjunct in the treatment of hemorrhagic shock.     

Hypoxic pulmonary vasoconstriction is not potentiated by repeated intermittent hypoxia in closed chest dogs

Hypoxic pulmonary vasoconstrictor (HPV) responses were measured with repeated intermittent hypoxic challenges in eight non-traumatized closed chest dogs anesthetized with pentobarbital. The right lung was ventilated continuously with 100% O2 while the left lung was either ventilated with 100% O2 (control) or ventilated with a gas mixture containing 3-4% O2 (hypoxia). Mean per cent left lung blood flow for all four normoxic periods was 43.1 +/- 1.5% (mean +/- SE) of the total blood flow by the SF6 excretion method and 40.8 +/- 1.1% by the differential CO2 excretion method, corrected for the Haldane effect. With hypoxic ventilation, flow diversion from the hypoxic lung was maximal with the first exposure and did not change subsequently with a total of four alternating exposures to normoxia and hypoxia. Flow diversion during hypoxia was approximately 50.5 +/- 2.4% by the SF6 method and 50.3 +/- 3.5% by the VCO2 method. This result contrasts with the increasing flow diversion response with intermittant hypoxic exposure that has been reported in animals exposed first to thoracotomy and surgical dissection. It is concluded that in the absence of surgical trauma the initial response to hypoxia is maximal and is not potentiated by repeated hypoxic stimulation.     

Cell viability,reactive oxygen species,apoptosis, and necrosis in myoblast cultures exposed to low-level infrared laser

Low-level infrared laser is considered safe and effective for treatment of muscle injuries. However, the mechanism involved on beneficial effects of laser therapy are not understood. The aim was to evaluate cell viability, reactive oxygen species, apoptosis, and necrosis in myoblast cultures exposed to low-level infrared laser at therapeutic fluences. C2C12 myoblast cultures at different (2 and 10 %) fetal bovine serum (FBS) concentrations were exposed to low-level infrared laser (808 nm, 100 mW) at different fluences (10, 35, and 70 J/cm²) and evaluated after 24, 48, and 72 h. Cell viability was evaluated by WST-1 assay; reactive oxygen species (ROS), apoptosis, and necrosis were evaluated by flow cytometry. Cell viability was decreased atthe lowest FBS concentration. Laser exposure increased the cell viability in myoblast cultures at 2 % FBS after 48 and 72 h, but no significant increase in ROS was observed. Apoptosis was decreased at the higher fluence and necrosis was increased at lower fluence in myoblast cultures after 24 h of laser exposure at 2 % FBS. No laser-induced alterations were obtained at 10 % FBS. Results show that level of reactive oxygen species is not altered, at least to those evaluated in this study, but low-level infrared laser exposure affects cell viability, apoptosis, and necrosis in myoblast cultures depending on laser fluence and physiologic conditions of cells.     

The influence of lung volume and body position on pulmonary blood flow under hypoxic exposure]

We examined the changes in the electromagnetically measured left lower lobe blood flow (QLLL) when the left lower lobe (LLL) was exposed to alveolar hypoxia selectively under two different positions (supine and right lateral position in 18 dogs. QLLL/CO, Qs/Qt and other cardiopulmonary parameters were obtained during the following experimental sequence; 1) the whole lung ventilated with 100% O2 (control), 2) LLL collapsed and the remainder ventilated with 100% O2, 3) N2 expansion of LLL (N2 CPAP) and the remainder ventilated with 100% O2, 4) O2 expansion of LLL (O2 CPAP) and the remainder ventilated with 100% O2, and 5) The whole lung ventilated with 100% O2. In the supine position group (n = 9), both selective collapse and N2 CPAP of LLL caused QLLL/CO to decrease significantly (P less than 0.02) from control values (14.9 +/- 1.7%) to 10.6 +/- 0.9% and 11.9 +/- 1.6%, respectively. But there was no difference in QLLL/CO between collapse and N2 CPAP of LLL. In the right lateral position group (n = 9), hypoxic exposure of LLL caused no decrease in QLLL/CO from control values. But QLLL/CO during N2 CPAP (8.5 +/- 1.1%) decreased significantly than that during collapse (10.8 +/- 1.5%) (P less than 0.02). Qs/Qt during N2 CPAP (15.6 +/- 1.4%) also decreased significantly (P less than 0.05) from that during collapse (18.5 +/- 2.2%). We conclude that the difference of the changes in QLLL/CO under hypoxic exposure between supine and right lateral position was caused by hydrostatic pressure which influenced more during lateral position than during supine position.     

Acidification enhances peritoneal macrophage phagocytic activity

BACKGROUND: Laparoscopic surgery is currently used in an array of diverse clinical situations, including cases with potential bacterial contamination. Previous studies have shown that CO2 insufflation during laparoscopic procedures modulates the immune response due to the acidification of the peritoneum. In the present study, we investigated whether exposure of macrophages to an acidic environment, such as that produced by CO2 insufflation, could affect phagocytosis, which is the fundamental process for bacterial clearance. MATERIALS AND METHODS: A murine peritoneal macrophage line (J774) was pre-incubated at pH levels of 6.0 or 7.4 for 3 h at 37 degrees C and returned to neutral pH (7.4). Phagocytosis was evaluated by incubation with fluorescein isothiocyanate-conjugated IgG-opsonized bacterial particles, IgG-opsonized fluorescent latex beads, and non-opsonized fluorescent latex beads at 37 degrees C, pH 7.4. The intensity of the internalized signal was measured by using a fluorometer. RESULTS: Pre-incubation of macrophages at a pH of 6.0 resulted in a significant increase of phagocytic activity of opsonized particles. However, it did not change the uptake of non-opsonized particles. This effect was due to the internalization process since there were no differences in foreign particle binding of cells exposed to acidic or neutral pH levels. CONCLUSIONS: This study demonstrates that environmental acidification increases the phagocytosis of opsonized particles by macrophages. These results suggest that CO2 insufflation during laparoscopic surgery may be beneficial for the clearance of pathogens, particularly in cases where there is a high risk of potential intra-abdominal infections.     

Carbon dioxide differentially affects the cytokine release of macrophage subpopulations exclusively via alteration of extracellular pH

BACKGROUND: The improved outcome after endoscopic surgery has been attributed to less surgical trauma. However, the underlying mechanisms are not fully understood, and direct effects of CO2 used for pneumoperitoneum, cellular acidification, and/or the lack of air contamination have been postulated to additionally modulate immune functions during endoscopic surgery. We investigated the effects of CO2 incubation, extracellular acidification, and air contamination on the inflammatory response of two distinct macrophage populations. METHODS: R2 and NR 8383 rat macrophage cell lines were used. Interleukin-6 (IL-6) and nitric oxide after lipopolysaccharide (LPS) stimulation were determined in these sets of experiments: incubation in 100% CO2, 5% CO2, and room air for 2h; incubation at pH 7.4, 6.5, and 5.5 for 2 h in 5% CO2; and incubation in 100% CO2, 5% CO2 and room air in fixed pH 6.3. The extracellular pH was monitored during incubation. We determined the alteration of intracellular pH in cells subjected to extracellular acidification by fluorescence microscopy. RESULTS: Extracellular pH decreased to 6.3 during 100% CO2 incubation. IL-6 release was reduced after CO2 incubation in NR 8383 cells and increased in R2 cells (p < 0.05). It was not altered by air incubation. Decreasing the extracellular pH to 6.5 mimicked the effects of CO2 and a decrease to 5.5 suppressed IL-6 release in both cell lines. In fixed pH at 6.3, CO2 and air incubation had no effect. CO2 and pH had no impact on nitric oxide release and vitality. Intracellular pH decreased with extracellular acidification without significant difference between the two cell lines. CONCLUSIONS: A decrease in extracellular pH during incubation in CO2 differentially affects IL-6 release in macrophage subpopulations. This may explain contradictory results in the literature. Moreover, we demonstrated that air contamination does not affect macrophage cytokine release. The decrease in extracellular pH is the primary underlying mechanism of the alteration of macrophage cytokine release after CO2 incubation, and it appears that the ability to maintain intracellular pH is not determined by the effects of CO2 or extracellular acidification.     

Different effects of thiopental in severe hypoxia, total ischemia, and low-flow ischemia in rat heart muscle

The effect of thiopental (100 mg X 1(-1] during total ischemia, low-flow ischemia, and severe hypoxia with maintained flow was investigated in the isolated perfused rat heart. During total ischemia the rate of decline of tissue creatine phosphate and adenosine triphosphate was no different in thiopental-treated and untreated hearts. The development of ultrastructural damage during total ischemia, the release of creatine kinase on reperfusion, and the exacerbation of ultrastructural damage after reperfusion were unaffected by thiopental. When thiopental was added to the perfusate during hypoxia and during low-flow ischemia at a normal pH(7.4), creatine kinase release during reoxygenation and during reperfusion was significantly less (P less than 0.005 and P less than 0.05, respectively) than in the untreated groups. After low-flow ischemia at a low pH (6.5), creatine kinase release was no different in thiopental-treated and untreated hearts. Thus, thiopental afforded protection of the myocardium in hypoxia and low-flow ischemia at pH 7.4 but not in total ischemia and low-flow ischemia at pH 6.5. The data are consistent with the hypothesis that during total ischemia and low-flow ischemia at pH 6.5, acidosis favors the entry of thiopental into the cell, causing inhibition of mitochondrial function and reduction of ATP production. During hypoxic perfusion and low-flow ischemia at pH 7.4, when the decrease in pH is less, the cardiodepressant effect of thiopental may offset any deleterious effect of the drug on intracellular organelles such as mitochondria.     

Carbon dioxide reactivity, pressure autoregulation, and metabolic suppression reactivity after head injury: a transcranial Doppler study.

OBJECT: Contemporary management of head-injured patients is based on assumptions about CO2 reactivity, pressure autoregulation (PA), and vascular reactivity to pharmacological metabolic suppression. In this study, serial assessments of vasoreactivity of the middle cerebral artery (MCA) were performed using bilateral transcranial Doppler (TCD) ultrasonography. METHODS: Twenty-eight patients (mean age 33 +/- 13 years, median Glasgow Coma Scale score of 7) underwent a total of 61 testing sessions during postinjury Days 0 to 13. The CO2 reactivity (58 studies in 28 patients), PA (51 studies in 23 patients), and metabolic suppression reactivity (35 studies in 16 patients) were quantified for each cerebral hemisphere by measuring changes in MCA velocity in response to transient hyperventilation, arterial blood pressure elevation, or propofol-induced burst suppression, respectively. One or both hemispheres registered below normal vasoreactivity scores in 40%, 69%, and 97% of study sessions for CO2 reactivity, PA, and metabolic suppression reactivity (p < 0.0001), respectively. Intracranial hypertension, classified as intracranial pressure (ICP) greater than 20 mm Hg at the time of testing, was associated with global impairment of CO2 reactivity, PA, and metabolic suppression reactivity (p < 0.05). A low baseline cerebral perfusion pressure (CPP) was also predictive of impaired CO2 reactivity and PA (p < 0.01). Early postinjury hypotension or hypoxia was also associated with impaired CO2 reactivity (p < 0.05), and hemorrhagic brain lesions in or overlying the MCA territory were predictive of impaired metabolic suppression reactivity (p < 0.01). The 6-month Glasgow Outcome Scale score correlated with the overall degree of impaired vasoreactivity (p < 0.05). CONCLUSIONS: During the first 2 weeks after moderate or severe head injury, CO2 reactivity remains relatively intact, PA is variably impaired, and metabolic suppression reactivity remains severely impaired. Elevated ICP appears to affect all three components of vasoreactivity that were tested, whereas other clinical factors such as CPP, hypotensive and hypoxic insults, and hemorrhagic brain lesions have distinctly different impacts on the state of vasoreactivity. Incorporation of TCD ultrasonography-derived vasoreactivity data may facilitate more injury- and time-specific therapies for head-injured patients.     

Influence of isoflurane on hypoxic pulmonary vasoconstriction in dogs

The authors studied the influence of locally administered isoflurane anesthesia on the pulmonary vascular response to regional alveolar hypoxia (hypoxic pulmonary vasoconstriction [HPV]) over a range of cardiac outputs (COs) in seven mechanically ventilated, closed-chest dogs. The right lung was ventilated with 100% O2 throughout the study. The left lung was ventilated with either 100% O2 (normoxia) or an hypoxic gas mixture (hypoxia). Different alveolar concentrations of isoflurane (0, 1, and 2.5 MAC) were administered to the left lung in a randomized sequence. The CO was altered by opening and closing surgically produced arteriovenous fistulae, at all isoflurane concentrations, and by hemorrhage at 0 MAC isoflurane. The magnitude of the HPV response was measured by differential CO2 elimination in the absence of isoflurane and by venous admixtures in all phases. During normoxia, the left lung effective flow (QL%) measured from differential CO2 excretion was 39.9 +/- 1.2% of the total blood flow and decreased to 18.8 +/- 2.6% when ventilated with the hypoxic gas mixture. Venous admixture (QVA/QT%) was significantly correlated with QL% during hypoxic ventilation in the absence of isoflurane. QVA/QT% was 22.3 +/- 2.7% during hypoxia with normal CO, and it increased significantly to 27.7 +/- 1.1% when the CO was increased 43%. It was not significantly altered (23.6 +/- 3.6%) when the CO was decreased by 54%. Isoflurane 2.5 MAC significantly increased QVA/QT% during hypoxic ventilation of the left lung to 33.9 +/- 2.6% with low CO and 35.4 +/- 1.7% with normal CO. Isoflurane 1 MAC increased QVA/QT% to 27.2 +/- 2.7% with normal CO and 28.1 +/- 2.6% with high CO.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytoprotective effects of hypoxia against cisplatin-induced tubular cell apoptosis: involvement of mitochondrial inhibition and p53 suppression

Hypoxia that is caused by vascular defects or disruption is commonly associated with renal diseases. During cisplatin nephrotoxicity, hypoxic regions are identified in the outer medulla and the renal cortex. However, the regulation of cisplatin injury by hypoxia is unclear. Previous work has demonstrated the cytoprotective effects of hypoxia against apoptotic injury. This study further examines the cytoprotective mechanisms in models of cisplatin-induced tubular cell apoptosis. In cultured renal tubular cells, 20 microM cisplatin induced approximately 60% apoptosis within 16 h. The rate of apoptosis was suppressed to < 20%, when the incubation was conducted under hypoxia (2% O2). Mitochondrial events of apoptosis, namely Bax accumulation and cytochrome c release, also were ameliorated. During cisplatin treatment, cell ATP was maintained in both normoxic and hypoxic cells. Hypoxic incubation lowered extracellular pH, but prevention of the pH decrease did not restore cisplatin-induced apoptosis. The cytoprotective effects of hypoxia also were independent of hypoxia-inducible factor 1 (HIF-1). Cobalt, as hypoxia, activated HIF-1 yet did not suppress cisplatin-induced apoptosis. Moreover, hypoxia suppressed cisplatin-induced apoptosis in HIF-1-deficient mouse embryonic stem cells and renal proximal tubular cells. Conversely, mitochondrial inhibitors, particularly inhibitors of respiration complex III (antimycin A and myxothiazol), mimicked hypoxia in apoptosis suppression. The effects of hypoxia and mitochondrial inhibitors were not additive. It is interesting that both hypoxia and complex III inhibitors ameliorated cisplatin-induced p53 activation. Therefore, the cytoprotective effects of hypoxia are independent of changes in cell ATP, pH, or HIF but may involve mitochondrial inhibition and the suppression of p53.     

Newly Developed Neutralized pH Icodextrin Dialysis Fluid: Nonclinical Evaluation

A two‐compartment system (NICOPELIQ; NICO, Terumo Co., Tokyo, Japan) has recently been developed to neutralize icodextrin peritoneal dialysis fluid (PDF). In this study, a nonclinical evaluation of NICO was carried out to evaluate biocompatibility as well as water transport ability. Glucose degradation products (GDPs) in the icodextrin PDFs were analyzed via high‐performance liquid chromatography (HPLC). The cell viability of human peritoneal mesothelial cells derived from peritoneal dialysis effluent (PDE‐HPMCs) was evaluated as well as the amount of lactate dehydrogenase (LDH) released after exposure to different PDFs (NICO and EXTRANEAL [EX, Baxter Healthcare Corp., Chicago, IL, USA]) and neutralized pH glucose PDF MIDPELIQ 250 (M250, Terumo). The water transport ability of NICO, EX, and M250 was tested using dialysis tube membranes with various pore sizes: 1, 2, 6–8, and 12–16 kDa. Although cell viability decreased by 30% after 30 min exposure to NICO, it was maintained for 6 h while a significant decrease was observed after 6 h exposure to EX. However, following adjustment of the pH to the same pre‐exposure pH value, there was no significant difference in cell viability within the same pH group despite a doubling of the difference in the total amount of GDPs (44.6 ± 8.6 µM in NICO and 91.9 ± 9.5 µM in EX, respectively). In contrast, a significant decrease in cell viability was observed when the pH decreased to less than pH 6. Levels of released LDH, a cytotoxic marker, were within 5% after a 6‐h exposure of NICO to PDE‐HPMCs. There was no significant difference in water transport ability represented as overall osmotic gradients between NICO and EX. In conclusion, neutralization of icodextrin PDF is beneficial for maintaining cell viability and minimizing LDH release while water transport ability is comparable to the conventional icodextrin PDF.     

Effects of reactive oxygen species from activated leucocytes on human sperm motility, viability and morphology

The accumulated data suggest that inflammation can increase the level of reactive oxygen species (ROS), which contribute to impaired sperm function and male infertility. Therefore, we propose that inflammation-mediated production of ROS in male and female reproductive tracts hinder sperm fertilisation. To test this hypothesis, phorbol myristate acetate (PMA) with polymorphonuclear leucocytes (PMNs) was applied to generate endogenous ROS. We evaluated the time-dependent effects of ROS on human sperm motility, viability and mitochondrial membrane potential (MMP). The results showed that after treatment with PMA and PMNs, the motility of human spermatozoa significantly decreased to 50% on Day 1 and 15% on Day 4 compared with that of the, respectively, negative controls (P = 0.012). The viability of human spermatozoa decreased on Day 4 of PMA + PMNs treatment (P = 0.028). The MMP of human spermatozoa significantly decreased from Day 2 to Day 4 in the PMA + PMN group compared with that of the controls (P = 0.019). Taken together, the 4-day cultivation approach provided an accurate evaluation of sperm quality, especially sperm motility and MMP. Our findings indicated that endogenous inflammation increased ROS levels, which might induce sperm oxidative damage. Additionally, sperm motility might be one of the earliest and most sensitive indicators of this damage.     

Long-term exposure to new peritoneal dialysis solutions: Effects on the peritoneal membrane

BACKGROUND: Chronic exposure to peritoneal dialysis fluid (PDF) affects the peritoneum, but precise causative factors are incompletely understood. We examined the effects of standard and new PDF on peritoneal function and structure. METHODS: Female Wistar rats received twice daily intraperitoneal infusions of a standard lactate-buffered 3.86% glucose PDF at pH 5.5 (Dianeal) (N= 12), a low glucose degradation product (GDP) containing bicarbonate/lactate-buffered 3.86% glucose PDF at pH 7.4 (Physioneal) (N= 12), a lactate-buffered amino acid-based PDF at pH 6.7 (Nutrineal) (N= 12) or Earle's Balanced Salt Solution at pH 7.4 (EBSS) (N= 12) during 12 weeks. RESULTS: Net ultrafiltration was lower after treatment with standard PDF, but not with low-GDP bicarbonate/lactate-buffered and amino acid-based PDF, compared to EBSS. Peritonea exposed to standard PDF were characterized by an increased expression of vascular endothelial growth factor (VEGF), microvascular proliferation as well as submesothelial fibrosis, which were not observed in other groups. Staining for methylglyoxal adducts was prominent in the standard PDF-exposed group, mild in the low GDP bicarbonate/lactate-buffered group and absent in the other groups. Standard PDF induced accumulation of advanced glycation end products (AGEs) and up-regulation of the receptor for AGE (RAGE). AGEs accumulation was absent and RAGE expression was only modestly increased in low-GDP bicarbonate/lactate-buffered and amino acid-based PDF. CONCLUSION: Long-term in vivo exposure to standard PDF adversely affects peritoneal function and structure. A low-GDP bicarbonate/lactate-buffered and amino acid-based PDF better preserved peritoneal integrity and may thus improve the longevity of the peritoneal membrane. GDPs and associated accelerated AGE formation are the main causative factors in PDF-induced peritoneal damage.     

Propofol displays no protective effect against hypoxia/reoxygenation injury in rat liver slices

Using precision-cut liver slices (20-25 mg wet weight) from male Wistar rats, we examined whether clinically relevant propofol concentrations have hepatoprotective or -toxic effects during hypoxia/reoxygenation. Slices were preincubated for 2 h in sealed roller vials (three slices per vial) containing Waymouth's medium (37 degrees C; 95% oxygen/5% CO(2)). Then, propofol or Intralipid was added to create four different groups (control, Intralipid, small-concentration propofol [0.5-1.5 micro g/mL], and large-concentration propofol [2.0-6.0 micro g/mL]). Thereafter, each group was incubated for 4 h under 95% oxygen/5% CO(2) (no hypoxia) or for 2 h under 100% nitrogen plus 2 h under 95% oxygen/5% CO(2) (hypoxia/reoxygenation). Slice viability and hypoxia/reoxygenation injury were assessed at 2, 3, and 4 h after incubation began by using the slice intracellular K(+) concentration, energy status (adenosine triphosphate content, total adenine nucleotides content, and energy charge), and liver enzyme leakage (aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase). Propofol and Intralipid caused a significant delay in energy charge recovery in comparison with the control. There were no significant differences between the propofol groups and the other two groups in intracellular K(+) content or liver enzyme leakage. Propofol had no hepatotoxic effect under no-hypoxia conditions in rat liver slices, nor did it have a protective effect against hypoxia/reoxygenation-induced hepatic injury. IMPLICATIONS: Propofol had no hepatotoxic effect under no-hypoxia conditions in rat liver slices, nor did it have a protective effect against hypoxia/reoxygenation-induced hepatic injury.