Ischemia/reperfusion injury in porcine intestine - Viability assessment.

AIM: To investigate viability assessment of segmental small bowel ischemia/reperfusion in a porcine model. METHODS: In 15 pigs, five or six 30-cm segments of jejunum were simultaneously made ischemic by clamping the mesenteric arteries and veins for 1 to 16 h. Reperfusion was initiated after different intervals of ischemia (1-8 h) and subsequently monitored for 5-15 h. The intestinal segments were regularly photographed and assessed visually and by palpation. Intraluminal lactate and glycerol concentrations were measured by microdialysis, and samples were collected for light microscopy and transmission electron microscopy. The histological changes were described and graded. RESULTS: Using light microscopy, the jejunum was considered as viable until 6 h of ischemia, while with transmission electron microscopy the ischemic muscularis propria was considered viable until 5 h of ischemia. However, following ≥ 1 h of reperfusion, only segments that had been ischemic for ≤ 3 h appeared viable, suggesting a possible upper limit for viability in the porcine mesenteric occlusion model. Although intraluminal microdialysis allowed us to closely monitor the onset and duration of ischemia and the onset of reperfusion, we were unable to find sufficient level of association between tissue viability and metabolic markers to conclude that microdialysis is clinically relevant for viability assessment. Evaluation of color and motility appears to be poor indicators of intestinal viability. CONCLUSION: Three hours of total ischemia of the small bowel followed by reperfusion appears to be the upper limit for viability in this porcine mesenteric ischemia model.

Small intestinal ischemia and reperfusion-bioimpedance measurements.

OBJECTIVE: Trans-intestinal bioimpedance measurements have previously been used to investigate changes in electrical parameters during 6 h of ischemia in the small intestine. Knowledge is lacking regarding the time course of trans-intestinal bioimpedance parameters during reperfusion. As reperfusion is an important part in the clinical treatment of intestinal ischemia, we need to know how it affects the bioimpedance measurements. APPROACH: We performed bioimpedance measurements, using a two-electrode setup on selected segments of the jejunum in 15 pigs. A controlled voltage signal was applied while measuring the resulting current. In each pig, five or six 30 cm segments of the jejunum were made ischemic by clamping the mesenteric arteries and veins creating segments with ischemia from 1-16 h duration. Reperfusion was initiated at selected time intervals of ischemia, and measured for 5-15 h afterwards. MAIN RESULTS: The tan δ parameter (loss tangent) was different (p < 0.016) comparing ischemic and control tissue for the duration of the experiment (16 h). Comparing the control tissue 30 cm from the ischemic area with the control tissue 60 cm from the ischemic tissue, we found that the mean tan δ amplitude in the frequency range (3900-6300 Hz) was significantly higher (p < 0.036) in the proximal control after 10 h of experiment duration. After reperfusion, the time development of tanδm (loss tangent maximum over a frequency range) amplitude and frequency overlapped and periodically increased above the tanδm in the ischemic intestine. Dependent on the ischemic duration pre-reperfusion, the initial increase in tan δ stabilizes or increases drastically over time, compared to the tan δ amplitude of the ischemic tissue. SIGNIFICANCE: As during ischemia, the electrical parameters during reperfusion also follow a characteristic time-course, depending on the ischemic exposure before pre-reperfusion. The temporal changes in electrical parameters during small intestinal ischemia followed by reperfusion provides important information for assessment of tissue injury.

Ischemic small intestine-in vivo versus ex vivo bioimpedance measurements.

OBJECTIVE: Bioimpedance has been used to investigate changes in electrical parameters during ischemia in various tissues. The small intestine is a multi-layered structure, with several distinct tissue types, and ischemia related changes occur at different times in the different intestinal layers. When investigating how the electrical properties in the small intestine is affected by ischemia, some researchers have used ex vivo models while others have used in vivo models. In this study, we compare ischemic time development of electrical parameters in ischemic in vivo versus ex vivo small intestine. APPROACH: Measurements were performed using a two-electrode setup, with a Solartron 1260/1294 impedance gain-phase analyser. Electrodes were placed on the surface of ischemic pig jejunum, applying a voltage and measuring the resulting electrical admittance. In each pig, 4 segments of the jejunum were made ischemic by clamping the mesenteric arteries and veins, resulting in a 30 cm central zone of warm ischemia and edema. The in vivo part of the experiment lasted 10 h, after which 3 pieces of perfused small intestine were resected, stored in Ringer-acetat at 38 °C, and measured during a 10 h ex vivo experiment. Main results and significance: We found significant differences (p < 0.0001) between the values of electric parameters when comparing the in vivo and ex vivo measurements as a function of ischemic time development. We also observed some similarities in the trends. In vivo, we measured an overall decrease in impedance during the duration of the experiment, probably as a result from the formation of edema. Ex vivo, the low frequency impedance increased initially for approximately 3 h before starting to decrease.

Systemic administration of enamel matrix derivative to lipopolysaccharide-challenged pigs: effects on the inflammatory response.

BACKGROUND: Periodontitis is the primary clinical indication for enamel matrix derivative (EMD). Recent investigations, showing that EMD inhibits the production of tumor necrosis factor-alpha (TNF-alpha) when added to human whole blood, indicate a novel role for EMD as a modulator of systemic inflammation. In the present study, we investigated the systemic effects of EMD in lipopolysaccharide (LPS)-challenged pigs. METHODS: In a preparatory study, seven pigs received a prophylactic EMD bolus injection (5 mg/kg), followed by a continuous infusion (50 mg/kg/min). Thirty minutes later, a continuous infusion of LPS (1.7 mcg/kg/h) was started. An additional 12 pigs were randomized into two groups. Six of these animals were given the same treatment, except that EMD was administered 30 min after LPS. The remainder served as controls. The groups were compared according to organ injury and function, hemodynamics, and systemic markers of inflammation. RESULTS: Prophylactic administration of EMD triggered transient hemodynamic instability in two of seven pigs. In the randomized pigs, no or only nonspecific changes were observed in biopsies from vital organs, independent of treatment. Enamel matrix derivative did not modify systemic TNF-alpha, interleukin (IL)-1 beta, or IL-6 concentrations. CONCLUSIONS: In the formulation and dosages used, EMD did not modulate the inflammatory response. No true allergic or immunotoxic reactions were seen. To be usable for systemic application, a new formulation should be developed, or the active part of the protein(s) should be identified and produced in a soluble form designed for infusion. The potential of EMD as a systemic immune modulator is still unsettled.

Activation of cytokine synthesis by systemic infusions of lipopolysaccharide and peptidoglycan in a porcine model in vivo and in vitro.

BACKGROUND: The incidence of gram-positive and mixed bacterial infections in surgical patients has increased, and there has been an alarming rise in the number of drug-resistant bacteria. Peptidoglycan (PepG) is a cell wall component of gram-positive bacteria that stimulates inflammatory responses both ex vivo and in vivo. The systemic effects of PepG on inflammation have not been studied in a large animal model. METHODS: Anesthetized pigs were subjected to 8-h continuous intravenous infusions of lipopolysaccharide (LPS) (4 mcg/kg/h), PepG (40 mcg/kg/h), LPS plus PepG, or saline. The concentrations of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, IL-8, and IL-10 were measured in the plasma prior to infusion (time 0) and thereafter every second hour until the end of the experiments. Heparinized whole blood samples drawn at time 0 and after a 6-h infusion of LPS or PepG were incubated ex vivo with PepG (10 mcg/mL), LPS (10 ng/mL), or a combination of PepG and LPS to study the immunologic consequences of systemic inflammation. Concentrations of TNF-alpha, IL-8, and IL-1beta were measured in the supernatant liquids. RESULTS: In vivo, there was transient upregulation of TNF-alpha after infusion of LPS, PepG, or the combination. Interleukin-6 and IL-8 were upregulated by LPS but not by PepG. In vitro studies of whole blood obtained at time 0 revealed a synergistic effect of LPS and PepG on the release of TNF-alpha. Incubation of whole blood obtained after 6 h of infusion of LPS or PepG revealed tolerance and cross-tolerance between the two bacterial components in the induction of TNF-alpha, IL-8, and IL-1beta. CONCLUSIONS: Peptidoglycan is a potent inducer of TNF-alpha in this large animal model. Peptidoglycan and LPS synergized to increase the formation of the proinflammatory cytokine TNF-alpha. The study demonstrates for the first time the development of tolerance and cross-tolerance between LPS and PepG in a large animal model. These phenomena could be of importance for the signs and symptoms of sepsis.