Liver as a focus of impaired oxygenation and cytokine production in a porcine model of endotoxicosis

OBJECTIVES: To determine whether the liver is a focus of insufficient oxygenation and whether liver is a source of tumor necrosis factor (TNF) and interleukin-6 (IL-6) in a porcine model of endotoxicosis. DESIGN: In vivo, prospective, controlled, repeated-measures, experimental study. SETTING: Experimental physiology laboratory in a university. SUBJECTS: Juvenile pigs, weighing 22 to 35 kg. INTERVENTIONS: Catheters for blood sampling were inserted into the carotid artery, portal vein, hepatic vein, and pulmonary artery of anesthetized animals. Ultrasonic flow probes were placed on the portal vein and the hepatic artery. During surgery, normal saline was infused intravenously at 25 mL/kg/hr. Following stabilization, animals were allocated randomly to one of two groups. The endotoxemic group (n = 6) received 50 mg/kg of purified Escherichia coli lipopolysaccharide infused into the external jugular vein over 1 hr. The control group (n = 6) received a sham saline infusion infused over 1 hr. Once the endotoxin or sham infusion was initiated, the rate of the intravenous saline infusion was increased to 48 mL/kg/hr for the remainder of the experiment. Measurements were obtained before the endotoxin or sham infusion, immediately after the infusion, and every 30 mins thereafter for 4 hrs. MEASUREMENTS AND MAIN RESULTS: Blood gases, lactate, and bioactive TNF and IL-6 concentrations were measured from the carotid artery, portal vein, hepatic vein, and pulmonary artery. The porcine model is characterized by systemic hypotension, pulmonary hypertension, and maintenance of cardiac output. Despite decreased hepatic oxygen delivery in endotoxemic animals (p < .02), there was no change in hepatic oxygen consumption compared with controls. Throughout the experiment, there was net hepatic consumption of lactate in both groups. There was no significant hepatic production (or consumption) of TNF or IL-6 in either group. CONCLUSIONS: In this porcine model of endotoxicosis, there is a reduction of hepatic oxygen delivery but dysoxia is not present. The liver is not a source of TNF or IL-6 in this model of endotoxicosis.     

Is reperfusion injury an important cause of mucosal damage after porcine intestinal ischemia?

BACKGROUND: Intestinal ischemic injury is exacerbated by reperfusion in rodent and feline models because of xanthine oxidase-initiated reactive oxygen metabolite formation and neutrophil infiltration. Studies were conducted to determine the relevance of reperfusion injury in the juvenile pig, whose low levels of xanthine oxidase are similar to those of the human being. METHODS: Ischemia was induced by means of complete mesenteric arterial occlusion, volvulus, or hemorrhagic shock. Injury was assessed by means of histologic examination and measurement of lipid peroxidation. In addition, myeloperoxidase, as a marker of neutrophil infiltration, and xanthine oxidase-xanthine dehydrogenase were measured. RESULTS: Significant ischemic injury was evident after 0.5 to 3 hours of complete mesenteric occlusion or 2 hours of shock or volvulus. In none of these models was the ischemic injury worsened by reperfusion. To maximize superoxide production, pigs were ventilated on 100% O2, but only limited reperfusion injury (1.2-fold increase in histologic grade) was noted. Xanthine oxidase-xanthine dehydrogenase levels were negligible (0.4 +/- 0.4 mU/gm). CONCLUSIONS: Reperfusion injury may not play an important role in intestinal injury under conditions of complete mesenteric ischemia and low-flow states in the pig. This may result from low xanthine oxidase-xanthine dehydrogenase levels, which are similar to those found in the human being.     

The endothelin receptor antagonist bosentan restores gut oxygen delivery and reverses intestinal mucosal acidosis in porcine endotoxin shock

BACKGROUND: Endothelin-1, the most potent vasoconstrictor known, is produced in septic states and may be involved in the pathophysiology of the deteriorated splanchnic circulation seen in septic shock. AIMS: To elucidate the capability of bosentan, a non-peptide mixed endothelin receptor antagonist, to attenuate splanchnic blood flow disturbances and counteract intestinal mucosal acidosis in endotoxic shock. METHODS: In 16 anaesthetised pigs, central and regional haemodynamics were monitored by thermodilution and ultrasonic flow probes, respectively. A tonometer in the ileum was used for measurement of mucosal pH. Onset of endotoxin challenge was followed by bosentan administration (to eight pigs) two hours later. RESULTS: Endotoxin infusion reduced cardiac index and systemic oxygen delivery; bosentan restored these parameters. The reduced mean arterial blood pressure and renal blood flow remained unaffected by bosentan. The profound reduction in gut oxygen delivery in response to endotoxin was completely abolished by bosentan. Bosentan significantly improved the notably deteriorated intestinal mucosal pH and mucosal-arterial PCO2 gap. The mucosal-portal vein PCO2 gap, used to monitor the mucosa in relation to the gut as a whole (including the spleen and pancreas), was also greatly increased by endotoxaemia and significantly reversed by bosentan. CONCLUSION: Bosentan completely restored the profound endotoxin induced reductions in systemic and gut oxygen delivery with a concomitant reversal of intestinal mucosal acidosis. Results suggest that endothelin is involved in the pronounced perfusion disturbances seen in the gut in endotoxic shock. Bosentan may prove useful in reducing gut ischaemia in septic shock.     

Near-infrared spectroscopy for monitoring of tissue oxygenation of exercising skeletal muscle in a chronic compartment syndrome model

Variations in the levels of muscle hemoglobin and of myoglobin oxygen saturation can be detected non-invasively with near-infrared spectroscopy. This technique could be applied to the diagnosis of chronic compartment syndrome, in which invasive testing has shown increased intramuscular pressure associated with ischemia and pain during exercise. We simulated chronic compartment syndrome in ten healthy subjects (seven men and three women) by applying external compression, through a wide inflatable cuff, to increase the intramuscular pressure in the anterior compartment of the leg. The tissue oxygenation of the tibialis anterior muscle was measured with near-infrared spectroscopy during gradual inflation of the cuff to a pressure of forty millimeters of mercury (5.33 kilopascals) during fourteen minutes of cyclic isokinetic dorsiflexion and plantar flexion of the ankle. The subjects exercised with and without external compression. The data on tissue oxygenation for each subject then were normalized to a scale of 100 per cent (the baseline value, or the value at rest) to 0 per cent (the physiological minimum, or the level of oxygenation achieved by exercise to exhaustion during arterial occlusion of the lower extremity). With external compression, tissue oxygenation declined at a rate of 1.4 +/- 0.3 per cent per minute (mean and standard error) during exercise. After an initial decrease at the onset, tissue oxygenation did not decline during exercise without compression. The recovery of tissue oxygenation after exercise was twice as slow with compression (2.5 +/- 0.6 minutes) than it was without the use of compression (1.3 +/- 0.2 minutes).     

Long-term composite tissue allograft survival in a porcine model with cyclosporine/mycophenolate mofetil therapy

BACKGROUND: Low-dose cyclosporine (CsA)/mycophenolate mofetil (MMF) therapy has significantly reduced the frequency of rejection and drug-induced side effects in rat hindlimb allograft recipients. With an eye toward direct clinical application, we developed a large-animal extremity composite tissue allograft model to assess the antirejection efficacy and systemic toxicity of combination CsA/MMF treatment. METHODS: Radial forelimb osteomyocutaneous flap transplants were performed between size-matched, outbred pigs assigned to one of two groups: 5 control pigs received no immunosuppression, and 10 pigs received a once-daily oral CsA/MMF/prednisone regimen. Rejection was assessed by visual inspection of flap skin and correlated with serial histopathologic examination of skin biopsies. RESULTS: In all control pigs, the flap was completely rejected on day 7. Of the 10 pigs receiving treatment, one died from pneumonia and an another from an anesthetic complication on days 19 and 30, respectively, without signs of rejection. Two flaps were lost on days 25 and 29 from severe rejection. Three pigs were free of rejection at the end of the 90-day follow-up period, and three had stable mild-to-moderate rejection at 90 days (P= 0.0007 vs. controls). White blood cell and platelet counts, serum creatinine values, and liver function tests remained normal in all animals receiving immunosuppressive therapy. CONCLUSIONS: Our results, to our knowledge, demonstrate for the first time that rejection can be significantly delayed in a large-animal composite tissue allograft model including skin using only orally administered agents dosed according to clinically relevant strategies without significant drug-specific systemic side effects.     

Lipoprotein and hormone-sensitive lipases in porcine adipose tissue

Lipoprotein lipase (LPL) is an adipocyte enzyme that cleaves fatty acids from circulating lipoproteins. Fatty acids enter the cell to be oxidized or esterified. Hormone-sensitive lipase (HSL) is an adipocyte enzyme that cleaves fatty acids from intracellular triacylglycerol. The HSL is activated by phosphorylation. Assays for the two lipases are complex because the hydrophobic substrate, triacylglycerol, must be presented as a gum-based suspension or as a detergent-based emulsion to a relatively hydrophilic enzyme. A convenient, stable glycerol/phospholipid suspension of the substrate was used for measurement of porcine adipose tissue LPL and HSL in vitro. This substrate was excellent for LPL. It produced rates five times those using a more complex and less convenient gum-based substrate suspension. The LPL activity was released by heparin, had a pH optimum of approximately 8.5, was activated by serum, and was inhibited by NaCl and protamine. This LPL assay measures enzyme capacity. The same substrate was used to measure an adipose tissue lipase activity that had a pH optimum below 7, was not activated by serum, and was not inhibited by NaCl or protamine. These are all characteristics of HSL. Despite the convenience, this substrate was not appropriate for porcine adipose tissue HSL because the rates were only 30 to 50% of those produced with a more complex, less convenient gum-based substrate suspension. Furthermore, incubation of enzyme or tissue slices with insulin, or agents that elevate cAMP concentration, did not modulate this lipase activity, as expected. These incubations poorly modulated LPL activity.     

Use of jugular venous blood, compared with mixed venous blood, for measurement of venous oxygenation indices in a porcine model of endotoxic shock

OBJECTIVE: To evaluate the reliability of oxygen saturation and oxygen content values measured from jugular venous blood in estimating values measured from mixed venous blood during endotoxic shock. ANIMALS: 14 random-bred 10- to 15-kg Yorkshire pigs. PROCEDURE: 60 pairs of heparinized blood samples were simultaneously collected from the pulmonary artery and right jugular vein during an independent study, using a porcine model of endotoxic shock. Endotoxic shock was induced by infusion of Escherichia coli endotoxin. Eighteen of the sample pairs were obtained from pigs prior to infusion of endotoxin or from control pigs. Oxygen saturation and venous oxygen content were measured by direct oximetry. Analysis of bias and precision was used to compare jugular venous blood values with values obtained from mixed venous blood. Samples from endotoxemic pigs were subclassified on the basis of abnormal states of global oxygen imbalance associated with septic shock. RESULTS: Indices of venous oxygenation measured from jugular venous blood were an imprecise method of estimating values measured from mixed venous blood. There was no significant difference in bias between nonendotoxemic and endotoxemic pigs, regardless of abnormal hemodynamic states. CONCLUSION: Jugular venous blood oxygen saturation and oxygen content values should not be used to assess global oxygen transport during endotoxic shock.     

Varioliform mucosal polypoid lesions in intestinal tract in a patient with adult T-cell leukemia

We have investigated the significance of local recurrence on survival in 173 patients with localised soft-tissue sarcomas of the limbs and of the trunk. The overall survival rates at five and ten years were 75.2% and 68.0%, respectively. After definitive surgery at our hospitals, there was local recurrence in 25 patients (14.5%). After inadequate operations elsewhere, there was a higher incidence of late local recurrence (28.3%), in comparison with those with primary tumours treated by us (9.0%), or patients referred to us immediately after inadequate surgery elsewhere (10.2%). Because of small numbers these differences in the survival rates were not statistically significantly different. Univariate survival analysis showed that local recurrence after definitive surgery (p = 0.006) together with the histological grade (p = 0.0002), the size of the tumour (p = 0.002), its depth in relation to deep fascia (p = 0.003), and the surgical margin (p = 0.0001) were the significant prognostic factors. Local recurrence at the initial presentation did not affect survival. Multivariate analysis showed that local recurrence after definitive surgery also lost its apparent prognostic significance.     

Protein turnover in intestinal mucosal villus and crypt brush border membranes

Following a concomitant oral dose of salicylamide and acetaminophen (5 mg/kg of each) the urinary excretion of glucuronide and sulfate conjugates of the drugs were followed in children (ages seven to ten years) and adults. No significant difference were observed between the two age groups in the half-lives for appearance of salicylamide conjugates in urine. Age-related changes in the metabolic pathways, however, were observed. The mean percentage of dose excreted as salicylamide sulfate was significantly higher in children (78%) than in adults (36%). In contrast, salicylamide glucuronide was the major excretory product in adults. Similar age-related differences were observed for acetaminophen conjugation. Pharmacokinetic analysis indicated that the deficiency in glucuronide conjugation of these drugs in children is accompanied by a higher rate of sulfate formation.     

CT and MRI of experimentally induced mesenteric ischemia in a porcine model

OBJECTIVE: Our goal was to assess the value of CT and MRI for the detection of bowel wall changes in experimentally induced mesenteric ischemia. METHODS: in 18 female pigs, a percutaneous embolization of the superior mesenteric artery was performed with buthyl-2-cyanoacrylate and Lipiodol (1:1) (experimental group). In six animals, only diagnostic imaging and histologic evaluation were performed (control group). CT was carried out 3, 6, and 12 h after occlusion. Incremental CT (1 s scan time, 5 mm slice thickness, 7 mm increment, 120 kV/290 mAs) and spiral CT (slice thickness 5 mm, pitch 1.5, 120 kV/165 mA) were performed pre and post contrast injection (Somatom Plus/Siemens). Serial CT was carried out after intravenous contrast injection (1 ml/kg, 2 ml/s). MRI (Magnetom 1.5 T; Siemens) was performed with T1 (pre and post 0.01 mmol/kg Gd-DTPA; Magnevist; Schering, Germany), T2, and proton density images in axial orientation. Slice thickness was 3 mm and slice gap 1 mm. Additionally, a T1-weighted GE sequence (multislice FLASH 2D) was obtained in dynamic technique (before and 30, 60, and 90 s after contrast agent injection) with a slice thickness of 5 mm. Biometrical monitoring included blood pressure, heart frequency, blood cell count, electrolyte status, blood gas analysis, and determination of serum lactate. Image evaluation included morphological analysis and determination of the enhancement pattern. Histological specimens were obtained and analyzed according to the Chiu classification. RESULTS: The histologic workup of the specimen 3, 6, and 12 h after vascular occlusion revealed an average Chiu state 3, 4, and 5. On CT, the bowel wall had a thickness of 4.7 mm on average in the ischemic segments. There was a significant difference from the control group (average 3 mm). Free intraperitoneal fluid and intramural gas were seen after 12 h of ischemia in 80%. In ischemic bowel segments, no mural enhancement was seen. Normal segments and the bowel of the control animals showed an enhancement of 34 HU on average (SD = 3.1 HU; p.<0.01). In MRI, S/N and C/N differed significantly between experimental and control groups in T1 and proton density images. In ischemic segments of all phases, the bowel wall did not show contrast enhancement. Healthy segments and bowel of control animals showed a significant enhancement (p<0.01). CONCLUSION: Cross-sectional imaging has a high sensitivity for delineation of ischemic bowel wall segments. The enhancement pattern of the bowel wall enables detection of location, extent, and cause of a acute arterial mesenteric ischemia with high accuracy in an early phase.     

Characterization of a polarized porcine uterine epithelial model system

The aim of this study was to elucidate the possible causes of elevated low-density lipoprotein (LDL)-cholesterol levels in transplanted patients treated with the immunosuppressant drug, cyclosporine. HepG2 cells, from a well-differentiated cell-line of hepatoma cells, were cultured and used as a model for in vitro hepatocytic LDL uptake. Different concentrations of cyclosporine, which were within the range of concentrations found in humans treated with cyclosporine, were added to tissue culture medium together with 125I-LDL. The results showed that cyclosporine reduced LDL uptake and degradation in HepG2 cells by about 25%. The cells were also pretreated with cyclosporine for 1 to 24 hours and then incubated with new medium containing labeled LDL for 2 hours at 4 degrees C in an LDL-binding assay. The data showed that cyclosporine reduced the subsequent LDL binding. Cyclosporine has no toxic effects on HepG2 cells, as shown by unchanged growth capacity of the cells. By means of a 50-fold excess of unlabeled LDL, a monoclonal anti-LDL receptor antibody, and dextran sulfate, we also evaluated if this inhibition of LDL binding occurred through the LDL receptor-mediated pathway, through non-LDL receptor-mediated pathways, or through both. The results show that cyclosporine reduces LDL binding and uptake by mainly inhibiting the LDL receptor-mediated pathway. We also studied the effect of the LDL-cyclosporine complex on the binding of labelled LDL. The presence of cyclosporine in the LDL particle does not influence the binding behaviour of LDL to its receptor. We also found that cyclosporine reduces the expression of the LDL receptor messenger RNA (mRNA) by about 40%. Thus, the interpretation of this study is that cyclosporine can cause an increase in LDL-cholesterol in the plasma of transplantation patients by reducing the catabolism of LDL in the liver by inhibiting mainly the LDL receptor-mediated catabolism through an effect on LDL receptor synthesis.     

Cholesterol biosynthesis in stomach and intestinal tissue of rat

Biosynthesis of cholesterol and its precursors (metastenol, latosterol and 7-dehydrocholesterol) was studied in the mucous and serous membranes of small intestine, secretory and esophageal regions of the normal rat stomach. The content of these sterols was also determined. The intensity of sodium 2-14C-acetate incorporation into cholesterol and its precursors in the mucous membrane of the small intestine and stomach secretory region is considerably higher than into the same sterols of the serous membrane and esophageal region. Cholesterol synthesis is most intensive in the small intestine mucous membrane and stomach secretory region.     

Gut is not a source of cytokines in a porcine model of endotoxicosis

BACKGROUND: We sought to determine whether ischemic gut is a source of endotoxin, tumor necrosis factor (TNF), and interleukin-6 (IL-6) in a porcine model of endotoxicosis. METHODS: Under general anesthesia pigs underwent neck dissection and laparotomy for placement of catheters in the carotid artery and portal vein and application of an ultrasonic flow probe around the portal vein. Endotoxin, TNF, and IL-6 levels were measured from the carotid artery and the portal vein during a 4 hour period in animals given endotoxin (50 mg/kg; n = 6) and in animals in the control group (n = 6). Gut fluxes of the substances of interest were calculated as the product of concentration and portal venous flow. A tonometer placed in the terminal ileum was used to monitor mucosal pH. RESULTS: Small bowel mucosal pH was significantly depressed in endotoxemic animals (6.8 +/- 0.1) when compared with baseline (7.1 +/- 0.1; p < 0.05) and control levels. In the control group portal venous levels of endotoxin, TNF, and IL-6 did not change significantly from baseline levels (1.5 +/- 0.4 endotoxin units (EU)/ml, 24 +/- 3 pU/ml, and 1.3 +/- 0.4 nU/ml, respectively). In the endotoxemic animals portal venous endotoxin and TNF levels peaked immediately after the endotoxin infusion (2186 +/- 437 EU/ml, and 293 +/- 125 pU/ml, respectively), and portal venous IL-6 levels peaked at 180 minutes (168 +/- 21 nU/ml). At no time did endotoxin, TNF, or IL-6 levels differ between arterial and portal venous blood, and at no time did efflux from the gut significantly exceed gut influx in either the control or endotoxemic animals. CONCLUSIONS: Ischemic gut as indicated by decreased mucosal pH is not associated with gut release of endotoxin, IL-6, or TNF in this porcine model of endotoxicosis.     

The metabolism of prostaglandins F2alpha and E2 by non-pregnant porcine endometrial and luteal tissue and early pregnant porcine endometrial tissue, luteal tissue and conceptuses in vitro

Slices of porcine endometrium and corpus luteum from both early pregnant and non-pregnant sows and conceptuses from early pregnant sows were incubated with radioactive PGF2alpha and PGE2 and the degree of metabolism of the prostaglandins measured. Prostaglandins and a metabolites were separated by TLC, radioactive bands located with a Panax scanner and the radioactivity measured in a scintillation counter. Endometrial and luteal tissue from non-pregnant sows gave no significant metabolism at any stage of the oestrous cycle, and while similar tissue from pregnant sows metabolised both prostaglandins slightly, only the conceptuses gave any significant metabolism. The possible physiological significance of these results is discussed.     

Nutrition, tissue oxygenation, and healing of venous leg ulcers

Little research has examined the healing and pain associated with saphenous vein (SV) harvest incisions, and no literature has addressed the associated distress and cosmetic result. The purpose of this study was to determine whether dry sterile gauze dressings, transparent film dressings or no dressings were more effective in hospitalized patients undergoing coronary artery bypass graft (CABG), in terms of minimizing leg incisional pain, minimizing the distress of a leg scar and improving the cosmetic appearance of the leg incision scar. Patients were randomized to dressing type on postoperative day (POD) 1, completed a pain and distress visual analogue scale (VAS) on PODs 1, 3 and 5, and a cosmetic result VAS upon discharge. Overall, leg incisional pain decreased over time (p < 0.05). Females reported decreasing pain between PODs 1 and 3, while males reported increasing pain between PODs 1 and 3 (p < 0.05). The film-dressing group reported decreasing pain from PODs 1 to 3, while the no-dressing and gauze-dressing groups reported increasing pain from PODs 1 to 3 (p < 0.05). Pain on POD 5 was positively correlated with an unfavorable cosmetic result (r = 0.42, p < 0.05), and distress on POD 5 was positively correlated with an unfavorable cosmetic result (r = 0.44, p < 0.05). The results of this study are encouraging and support the continued testing of dressings to minimize pain and distress, as well as enhancing cosmetic result.