Intestinal mast cells mediate gut injury and systemic inflammation in a rat model of deep hypothermic circulatory arrest.

OBJECTIVE: Cardiac surgery, especially when employing cardiopulmonary bypass and deep hypothermic circulatory arrest, is associated with systemic inflammatory responses that significantly affect morbidity and mortality. Intestinal perfusion abnormalities have been implicated in such responses, but the mechanisms linking local injury and systemic inflammation remain unclear. Intestinal mast cells are specialized immune cells that secrete various preformed effectors in response to cellular stress. We hypothesized that mast cells are activated in a microenvironment shaped by intestinal ischemia/reperfusion, and investigated local and systemic consequences. DESIGN: Rat model of deep hypothermic circulatory arrest. SETTING: University research laboratory. SUBJECTS: Twelve- to 14-week-old male Sprague-Dawley rats. INTERVENTIONS: Rats were anesthetized and cooled to 16°C to 18°C on cardiopulmonary bypass before instituting deep hypothermic circulatory arrest for 45 minutes. Specimens were harvested following rewarming and 2 hours of recovery. MEASUREMENTS AND MAIN RESULTS: Significant intestinal barrier disruption was found, together with macro- and microscopic evidence of ischemia/reperfusion injury in ileum and colon, but not in the lungs or kidneys. Immunofluorescence and toluidine blue staining revealed increased numbers of mast cells and their activation in the gut. In animals pretreated with the mast cell stabilizer, cromolyn sodium, mast cell degranulation was blocked, and intestinal morphology and barrier function were preserved following deep hypothermic circulatory arrest. Furthermore, cromolyn sodium treatment was associated with reduced intestinal neutrophil influx and blunted systemic release of proinflammatory cytokines. CONCLUSION: Our data provide primary evidence that intestinal ischemia/reperfusion is a leading pathophysiologic process in a rat model of deep hypothermic circulatory arrest, and that intestinal injury, and local and systemic inflammatory responses are critically dependent on mast cell activation. This identifies intestinal mast cells as central players in deep hypothermic circulatory arrest-associated responses, and opens novel therapeutic possibilities for patients undergoing this procedure.

[Genotyping and gene nucleotide polymorphism of epidemic strain of Echinococcus granulosus metacestode from humans and sheep in Tianjun region, Qinghai Province].

OBJECTIVE: To understand the genotypes and nucleotide polymorphisms of Echinococcus granulosus metacestode from humans and sheep in Tianjun region, Qinghai Province. METHODS: The specific primers were designed according to the cox1 and nad1 genes of E. granulosus mitochondrial genome sequences accessed by GenBank. The primers were used to detect the cyst samples from 16 sheep and 2 humans infected with E. granulosus in Tianjun region of Qinghai Province by PCR, then the PCR amplification products were sequenced, the genotypes and nucleotide polymorphisms of the cox1 and nad1 genes were analyzed. RESULTS: The 18 isolated samples all belonged to E. granulosus G1 genotype. Among all the isolates, 9 haplotypes existed in the cox1 gene with 16 nucleotide mutation sites, and there were 0 to 5 nucleotide differences with the highest variation rate of 0.31%, whereas 7 haplotypes occurred with 15 nucleotide mutation sites, and there were 1 to 8 nucleotide differences with the highest variation rate of 0.89% for the nad1 gene. CONCLUSIONS: The epidemic genotype of E. granulosus is G1 in humans and sheep in Tianjun region of Qinghai Province, and the nucleotide polymorphisms of the cox1 gene were more abundant than those of the nad1 gene, and the resolution of the nucleotide polymorphisms of cox1 gene is higher than that of the nad1 gene used in E. granulosus isolates.

The use of moderate hypothermia during cardiac surgery is associated with repression of tumour necrosis factor-alpha via inhibition of activating protein-1: an experimental study.

INTRODUCTION: The use of moderate hypothermia during experimental cardiac surgery is associated with decreased expression of tumour necrosis factor (TNF)-alpha in myocardium and with myocardial protection. In order to identify the cellular mechanisms that lead to that repression, we investigated the effect of hypothermia during cardiac surgery on both main signalling pathways involved in systemic inflammation, namely the nuclear factor-kappaB (NF-kappaB) and activating protein-1 pathways. METHOD: Twelve female pigs were randomly subjected to standardized cardiopulmonary bypass with moderate hypothermia or normothermia (temperature 28 degrees C and 37 degrees C, respectively; six pigs in each group). Myocardial probes were sampled from the right ventricle before, during and 6 hours after bypass. We detected mRNA encoding TNF-alpha by competitive RT-PCR and measured protein levels of TNF-alpha, inducible nitric oxide synthase and cyclo-oxygenase-2 by Western blotting. Finally, we assessed the activation of NF-kappaB and activating protein-1, as well as phosphorylation of p38 mitogen-activated protein kinase by electrophoretic mobility shift assay with super shift and/or Western blot. RESULTS: During and after cardiac surgery, animals subjected to hypothermia exhibited lower expression of TNF-alpha and cyclo-oxygenase-2 but not of inducible nitric oxide synthase. This was associated with lower activation of p38 mitogen-activated protein kinase and of its downstream effector activating protein-1 in hypothermic animals. In contrast, NF-kappaB activity was no different between groups. CONCLUSION: These findings indicate that the repression of TNF-alpha associated with moderate hypothermia during cardiac surgery is associated with inhibition of the mitogen-activated protein kinase p38/activating protein-1 pathway and not with inhibition of NF-kappaB. The use of moderate hypothermia during cardiac surgery may mitigate the perioperative systemic inflammatory response and its complications.

Intramyocardial synthesis of pro- and anti-inflammatory cytokines in infants with congenital cardiac defects.

OBJECTIVES: We sought to test the hypothesis that cytokines would be expressed in the myocardium of infants with congenital cardiac defects and to identify the signaling pathways involved. BACKGROUND: Mechanical stress upregulates pro-inflammatory cytokines in the myocardium. METHODS: Fifteen infants with tetralogy of Fallot (TOF) (n = 7) or with ventricular septal defects (VSDs) (n = 8) were investigated. Concentrations of pro- and anti-inflammatory cytokines and of the inducible nitric oxide synthase (iNOS) were measured by enzyme-linked immunosorbent assay and/or Western blotting in the right ventricular myocardium taken during cardiac surgery. Activation of the nuclear factor-kappa-B (NF-kappa-B) and p38 mitogen-activated protein kinase (MAPK) pathways was assessed by electrophoretic mobility shift assay with supershift and/or Western blotting, respectively. RESULTS: The pro-inflammatory cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-1-beta, and IL-6 and the anti-inflammatory cytokine IL-10 were detected in the myocardium of all patients. Concentrations of the pro-inflammatory cytokines and also of phosphorylated p38 MAPK were higher in patients with TOF than in those with VSD and correlated with the degree of pressure overload of the right ventricle. Levels of phosphorylated I-kappa-B-alpha, iNOS, and IL-10 were similar in patients with TOF and in those with VSD. CONCLUSIONS: Our results show intramyocardial synthesis of pro-inflammatory cytokines in infants with congenital cardiac defects. This is associated with activation of both the NF-kappa-B and p38 MAPK pathways. The latter could be particularly important for the transduction of mechanical signals in the infant's myocardium. Synthesis of IL-10 indicates an intramyocardial anti-inflammatory potential in this age group.

Moderate hypothermia during cardiopulmonary bypass increases intramyocardial synthesis of heat shock protein 72.

OBJECTIVES: This study was undertaken to test the hypothesis that the myocardial protective effect of moderate hypothermia during cardiopulmonary bypass involves upward regulation of heat shock protein 72. METHODS: Sixteen young pigs were randomly assigned to a temperature regimen during standardized cardiopulmonary bypass of normothermia or moderate hypothermia (temperatures 37 degrees C and 28 degrees C, respectively, n = 8 per group). Myocardial probes were sequentially sampled from the right ventricle before and during bypass and 6 hours after bypass. Messenger RNA encoding for heat shock protein 72 was assessed by competitive reverse transcriptase-polymerase chain reaction, and heat shock protein 72 synthesis was assessed by Western blot and immunohistochemical methods. Induction of apoptosis was assessed by gene expression of apoptosis-regulating proteins (Bcl-xL, Bak, and Fas) according to competitive reverse transcriptase polymerase chain reaction. Apoptotic cells were identified with an in situ apoptosis-detection kit (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling) in combination with morphologic criteria. Necrotic cells were detected by standard histologic methods. RESULTS: Moderate hypothermia rather than normothermia was associated with earlier and higher gene expression and synthesis of heat shock protein 72 in the myocardium during and after cardiac surgery. In the hypothermia group both heat shock protein 72 and the messenger RNA encoding it were detected as soon as 30 minutes after initiation of bypass and before aortic clamping, whereas in the normothermia group they were not detected before aortic clamping. Immunohistochemical methods showed localization of heat shock protein 72 in the cardiomyocytes, endothelial cells, and macrophages. Although the percentage of necrotic cells in the myocardium was lower in the hypothermic group, the induction of apoptosis regulatory proteins and the percentage of apoptotic cells did not differ between the groups. CONCLUSIONS: These results suggest that the myocardial protective effect of moderate hypothermia during cardiopulmonary bypass involves upward regulation of heat shock protein 72 and inhibition of necrosis but not of apoptosis.

Effect of biologically active coating on biocompatibility of Nitinol devices designed for the closure of intra-atrial communications.

Anti-thrombogenicity and rapid endothelialisation are prerequisites for the use of closure devices of intra-atrial communications in order to reduce the risk of cerebral embolism. The purpose of this study was therefore to assess the effect of bioactive coatings on biocompatibility of Nitinol coils designed for the closure of intra-atrial communications. Nitinol coils (n = 10, each) and flat Nitinol bands (n = 3, each) were treated by basic coating with poly(amino-p-xylylene-co-p-xylylene) and then coated with either heparin, r-hirudin or fibronectin. Anti-thrombogenicity was studied in vitro in a dynamic model with whole blood by partial thromboplastin time (PTT), platelet binding and thrombin generation, respectively, and cytotoxicity by hemolysis. Endothelialisation was studied on Nitinol bands with human umbilical venous endothelial cells (HUVEC) by 3-(4,5-dimethylthiazole-2yl)-2,5-triphenyl tetrazolium (MTT) assay and immnuofluorescence analysis of Ki67, vinculin, fibronectin and von Willebrand Factor. Uncoated or coated devices did not influence hemolysis and PTT. r-Hirudin (but not heparin) and fibronectin coating showed lower platelet binding than uncoated Nitinol (p < 0.005, respectively). Heparin and r-hirudin coating reduced thrombin formation (p < 0.05 versus Nitinol, respectively). HUVEC adhesion, proliferation, and matrix formation decreased in the order: fibronectin coating > uncoated Nitinol > r-hirudin coating > heparin coating > basic coating. MTT assay corroborated these findings. In conclusion, r-hirudin and fibronectin coating, by causing no acute cytotoxicity, decreasing thrombogenicity and increasing endothelialisation improve in vitro biocompatibility of Nitinol devices designed for the closure of intra-atrial communications.

Cytokine balance in infants undergoing cardiac operation.

BACKGROUND: The control of the systemic inflammatory response taking place during cardiac operations depends on adequate antiinflammatory reaction. In this prospective study we tested the hypothesis that cytokine balance during pediatric cardiac surgical procedures would be influenced by the patients' preoperative clinical condition, defined as hypoxemia or heart failure. METHODS: Twenty infants (median age, 8 months) with hypoxemia owing to intracardiac right-to-left shunt (group 1, n = 10) or with heart failure because of intracardiac left-to-right shunt (group 2, n = 10), scheduled for elective primary corrective operation, were enrolled. Plasma levels of the proinflammatory cytokine interleukin (IL) 6, the natural antiinflammatory cytokine IL-10, and the markers of the acute-phase response, C-reactive protein and procalcitonin, were sequentially measured before, during, and after cardiac operation up to the 10th postoperative day. The ratio of IL-10 to IL-6 levels served as a marker for the individual's antiinflammatory cytokine balance. RESULTS: Group 1 showed higher preoperative IL-6 (p < 0.001), lower IL-10 levels (p < 0.02), and lower ratio of IL-10 to IL-6 levels (p < 0.001) than group 2. Preoperative C-reactive protein and procalcitonin were not detectable. In group 1, preoperative IL-6 levels inversely correlated with preoperative oxygen saturation (Spearman correlation coefficient, -0.74, p < 0.02). During cardiopulmonary bypass, IL-6 levels were higher, whereas IL-10 and ratio of IL-10 to IL-6 levels were lower in group 1 than in group 2. In all patients, postoperative IL-6 levels were positively correlated with duration of inotropic support and serum creatinine value and inversely correlated with oxygenation index and diuresis. CONCLUSIONS: Infants with hypoxemia show a preoperative inflammatory state with low antiinflammatory cytokine balance in contrast to those with heart failure. This in turn is associated with lower perioperative antiinflammatory cytokine balance and might contribute to postoperative morbidity.

Tumor necrosis factor-alpha and troponin I release in porcine cardiac lymph and coronary sinus blood before and after cardiopulmonary bypass.

To assess the concentrations of cardiac troponin I (cTnI) and tumor necrosis factor-alpha (TNFalpha) in cardiac lymph compared with coronary sinus (CS) blood and to measure cardiac lymph flow before and after cardiopulmonary bypass (CPB). In 21 pigs, the main cardiac lymph trunk was cannulated before institution of standardized CPB. Lymph flow, cTnI and TNFa in cardiac lymph and CS blood were measured before and after CPB for 6 hours. Before CPB, cTnI concentration was 215 +/- 36 nglml in cardiac lymph and 0.5 +/- 0.1 nglml in CS blood, respectively. After aortic declamping a significant elevation of cTnI values was measured in cardiac lymph and CS blood. cTnl concentration in cardiac lymph and CS blood peaked 6 hrs after CPB (10,556 +/- 4,735 vs. 22.2 +/- 3.7 nglml, p < 0.01). TNFalpha concentration at baseline was 23.2 +/- 5.6 pg/ml in lymph and 18.7 +/- 9.5 pg/ml in CS blood, and there was no significant release of TNFalpha up to the end of the experiment. Baseline cardiac lymph flow was 3.07 +/- 0.35 ml/h and declined after aortic clamping (0.72 +/- 0.16 ml/h; p < 0.01) and peaked one hour after CPB (5.66 +/- 0.97 ml/h; p < 0.01). In conclusion, very high cTnI concentrations in cardiac lymph suggest serious perioperative myocardial damage after CPB with cardioplegia, which is underestimated by cTnI release into the bloodstream. In our study, the myocardium was not a major source of TNFalpha release.

Myocardial cardiotrophin-1 is differentially induced in congenital cardiac defects depending on hypoxemia.

AIM: Cardiotrophin-1 (CT-1) is upregulated by hypoxemia and hemodynamic overload and is characterized by potent hypertrophic and protective properties on cardiac cells. This study aimed to investigate whether CT-1 is differentially induced in the myocardium of infants with congenital cardiac defects depending on hypoxemia. METHODS & RESULTS: Infants with Tetralogy of Fallot (n = 8) or with large nonrestrictive ventricular septal defect (n = 8) undergoing corrective surgery were investigated. Expression of CT-1 was assessed at mRNA and protein levels in the right atrial and ventricular myocardium. The activation of the STAT-3 and VEGF were measured. Degradation of cardiac troponin-I served as a marker of myocardial damage. CT-1 was detected in all patients with levels negatively correlating to the arterial oxygen saturation. Higher CT-1 expression in Tetralogy of Fallot patients was associated with activation of the JAK/STAT pathway and higher cardiac troponin-I degradation. CONCLUSION: CT-1 may mediate myocardial hypertrophy and dysfunction in infants with congenital cardiac defects, particularly in those with hypoxemia.

The effect of blood pressure on cerebral outcome in a rat model of cerebral air embolism during cardiopulmonary bypass.

OBJECTIVE: Higher mean arterial pressure during cardiopulmonary bypass may improve cerebral outcome associated with cerebral air embolism by increasing emboli clearance and collateral flow to salvage the ischemic penumbra. However, this may come at the expense of increased delivery of embolic load. This study was designed to investigate the influence of mean arterial pressures on cerebral functional and histologic outcome after cerebral air embolism during cardiopulmonary bypass in an established rat model. METHODS: Male Sprague-Dawley rats were exposed to 90 minutes of normothermic cardiopulmonary bypass with 10 cerebral air embolisms (0.3 µL/bolus) injected repetitively. Rats were randomized to 3 groups (n = 10, each) that differed in mean arterial pressure management during cardiopulmonary bypass: 50 mm Hg (low mean arterial pressure), 60 to 70 mm Hg (standard mean arterial pressure), and 80 mm Hg (high mean arterial pressure). Neurologic score was assessed on postoperative days 3 and 7 when cerebral infarct volumes were determined. Cognitive function was determined with the Morris water maze test beginning on postoperative day 3 and continuing to postoperative day 7. RESULTS: Neurologic score was better in high and standard mean arterial pressure groups versus low mean arterial pressure groups. High mean arterial pressure resulted in shorter water maze latencies compared with standard and low mean arterial pressure on postoperative days 6 and 7. Total infarct volume and number of infarct areas were not different among groups. CONCLUSIONS: The use of higher mean arterial pressure during cardiopulmonary bypass in a rat model of cerebral air embolism conveyed beneficial effects on functional cerebral outcome with no apparent disadvantage of increased delivery of embolic load. Maintaining higher perfusion pressures in situations of increased cerebral embolic load may be considered as a collateral therapeutic strategy.

Cerebral tumor necrosis factor alpha expression and long-term neurocognitive performance after cardiopulmonary bypass in rats.

OBJECTIVE: Cerebral inflammatory reaction is discussed as a contributor to adverse cerebral outcome after cardiac surgery with cardiopulmonary bypass. This study was designed to determine the effect of cardiopulmonary bypass on both cerebral expression of tumor necrosis factor alpha and neurocognitive outcome in rats. METHODS: With institutional review board approval, 50 rats were randomly assigned to one of 3 groups: rats of the cardiopulmonary bypass group were subjected to 75 minutes of normothermic cardiopulmonary bypass. Sham-operated animals underwent identical preparation but were not connected to cardiopulmonary bypass, whereas rats of the control group were neither anesthetized nor cannulated. Ten rats per group survived 4 hours after cardiopulmonary bypass or the sham operation for immediate postoperative determination of tumor necrosis factor alpha-expressing cells (immunohistochemistry) and cerebral tumor necrosis factor alpha mRNA levels (polymerase chain reaction). The remaining animals survived 10 days for neurocognitive assessment by using the modified hole-board test and for analysis of cerebral tumor necrosis factor alpha activation in the late postoperative period. RESULTS: Expression of tumor necrosis factor alpha mRNA was increased 4 hours after cardiopulmonary bypass and the sham operation, with higher expression in the cardiopulmonary bypass group (chi(2) [2] = 25.08, P < .001). Both experimental groups demonstrated larger numbers of tumor necrosis factor alpha-positive cells in the early and late postoperative periods (F [1] = 13.08, P < or = .001) and an impaired neurocognitive performance on the first postoperative days compared with that seen in the control group (F [2, 24] = 4.26, P = .02). CONCLUSIONS: Cerebral tumor necrosis factor alpha activation in both experimental groups during the early postoperative period was accompanied by transient neurocognitive impairment. Therefore cardiopulmonary bypass alone demonstrated no effect on cerebral inflammation and neurocognitive outcome.

The hypoxia-inducible factor HIF-1 promotes intramyocardial expression of VEGF in infants with congenital cardiac defects.

OBJECTIVES: The response to hypoxia is primarily mediated by the transcription factor hypoxia-inducible factor-1 (HIF-1) which leads to the induction of a variety of adaptive gene products including vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS). This study was designed to test the hypothesis that HIF-1 and its target genes would be upregulated in the ventricular myocardium of infants with cyanotic congenital cardiac defects. METHODS: 14 infants with cyanotic (n = 7) or acyanotic cardiac defects (n = 7) were investigated. Samples from the right ventricular myocardium taken immediately after aortic clamping were studied for protein expression and DNA-binding activity. RESULTS: Protein levels of HIF-1alpha were significantly elevated in patients with cyanotic compared to acyanotic congenital heart disease and inversely correlated with the degree of hypoxemia. This response was accompanied by significantly enhanced HIF-1 DNA binding activity. Furthermore, protein levels of VEGF and eNOS were significantly higher in the myocardium of cyanotic than of acyanotic infants. To test the potential involvement of upstream regulatory pathways, activation of MAP kinases was determined. Intramyocardial levels of phosphorylated p38 MAP kinase, but not of ERK1/2 were significantly higher in infants with cyanotic compared to those with acyanotic congenital heart disease and inversely correlated to hypoxemia. CONCLUSIONS: These findings show that chronic hypoxemia is associated with the induction and stabilization of the transcription factor HIF-1 as well as its target genes VEGF and eNOS in the myocardium of infants with cyanotic cardiac defects. Thus, stabilization of HIF-1 and induction of the adaptive hypoxia response could particularly participate in myocardial remodeling in children with congenital cardiac defects and chronic hypoxemia.

Preliminary application of neural network in differentiating benign from malignant solitary pulmonary nodule on HRCT.

In this study, the performance of artificial neural network (NN) in diagnosis of solitary pulmonary nodule (SPN) on HRCT images was evaluated. One hundred and forty-five cases of SPN, including 86 cases of pulmonary carcinoma, 18 cases of tuberculoma, 29 cases of inflammatory nodule and 12 cases of benign tumor, were collected, which were all confirmed by pathology or biopsy and over-two-year clinical treatment. Five clinical parameters and 10 radiological characteristics were observed and quantified for qualitative characteristics. About 70 percent of all cases (up to 103 cases) were selected randomly to form training samples set, on which BP neural network and Logistic regression model were built. The total consistent rate 98.6% of BP NN was greater than that of Logistic model, which is 88.3% (P=0.0007). Areas under ROC curve were 0.997+/- 0.004 and 0.959+/-0.016 respectively, and the difference between the two was significant statistically (P=0.009). NN showed high performance in diagnosis of SPN on HRCT images. It was worthy of further study.

Intrahepatic synthesis of tumor necrosis factor-alpha related to cardiac surgery is inhibited by interleukin-10 via the Janus kinase (Jak)/signal transducers and activator of transcription (STAT) pathway.

SUMMARY: OBJECTIVES To identify the signaling pathways involved in the anti-inflammatory shift of the cytokine balance due to hypothermia during cardiopulmonary bypass. DESIGN Experimental animal study. SETTING Department of experimental surgery of a university hospital. SUBJECTS Young pigs. INTERVENTIONS Animals underwent normothermic (37 degrees C) or hypothermic (28 degrees C) cardiopulmonary bypass (n = 6 each). Samples of liver tissue were taken before and 6 hrs after cardiopulmonary bypass. MEASUREMENTS AND MAIN RESULTS Intrahepatic expression of tumor necrosis factor-alpha, interleukin-10, inducible nitric oxide synthase, and suppressor of cytokine signaling-3 was detected by reverse transcriptase polymerase chain reaction and/or Western blotting. Concentrations of the inhibitory protein of nuclear factor-kappaB, IkappaB, and of the signal transducer and activator of transcription (STAT)-3 were measured by Western blotting. The DNA-binding activity of nuclear factor-kappaB and STAT-3 was assessed by electrophoretic mobility shift and supershift assays. Liver cell necrosis and apoptosis were assessed by histology and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay, respectively. Pigs operated on in hypothermia showed significantly higher intrahepatic concentrations of interleukin-10 and lower concentrations of tumor necrosis factor-alpha than the others. They also showed a lower percentage of hepatic cell necrosis but not of apoptosis. This anti-inflammatory reaction observed in the hypothermic group was associated with a higher expression of suppressor of cytokine signaling-3 and with increased activation of STAT-3. Activation of nuclear factor-kappaB and expression of inducible nitric oxide synthase, however, were not significantly different between both groups. CONCLUSION Our results show that hypothermia during cardiopulmonary bypass up-regulates interleukin-10 via STAT-3 activation, which in turn leads to the attenuation of tumor necrosis factor-alpha expression and to hepatic protection.