Microscopy of bacterial translocation during small bowel obstruction and ischemia in vivo – a new animal model

BACKGROUND: Existing animal models provide only indirect information about the pathogenesis of infections caused by indigenous gastrointestinal microflora and the kinetics of bacterial translocation. The aim of this study was to develop a novel animal model to assess bacterial translocation and intestinal barrier function in vivo. METHODS: In anaesthetized male Wistar rats, 0.5 ml of a suspension of green fluorescent protein-transfected E. coli was administered by intraluminal injection in a model of small bowel obstruction. Animals were randomly subjected to non-ischemic or ischemic bowel obstruction. Ischemia was induced by selective clamping of the terminal mesenteric vessels feeding the obstructed bowel loop. Time intervals necessary for translocation of E. coli into the submucosal stroma and the muscularis propria was assessed using intravital microscopy. RESULTS: Bacterial translocation into the submucosa and muscularis propria took a mean of 36 ± 8 min and 80 ± 10 min, respectively, in small bowel obstruction. Intestinal ischemia significantly accelerated bacterial translocation into the submucosa (11 ± 5 min, p < 0.0001) and muscularis (66 ± 7 min; p = 0.004). Green fluorescent protein-transfected E. coli were visible in frozen sections of small bowel, mesentery, liver and spleen taken two hours after E. coli administration. CONCLUSIONS: Intravital microscopy of fluorescent bacteria is a novel approach to study bacterial translocation in vivo. We have applied this technique to define minimal bacterial transit time as a functional parameter of intestinal barrier function

Network Physiology reveals relations between network topology and physiological function

The human organism is an integrated network where complex physiologic systems, each with its own regulatory mechanisms, continuously interact, and where failure of one system can trigger a breakdown of the entire network. Identifying and quantifying dynamical networks of diverse systems with different types of interactions is a challenge. Here, we develop a framework to probe interactions among diverse systems, and we identify a physiologic network. We find that each physiologic state is characterized by a specific network structure, demonstrating a robust interplay between network topology and function. Across physiologic states the network undergoes topological transitions associated with fast reorganization of physiologic interactions on time scales of a few minutes, indicating high network flexibility in response to perturbations. The proposed system-wide integrative approach may facilitate the development of a new field, Network Physiology.Comment: 12 pages, 9 figure

Comparison of ultrasonography with computed tomography in the diagnosis of incisional hernias

Background: The objective of this study is to determine the reliability and validity of ultrasonography (US) in diagnosing incisional hernias in comparison with computed tomography (CT). The CT scans were assessed by two radiologists in order to estimate the inter-observer variation and twice by one radiologist to estimate the intra-observer variation. Patients were evaluated after reconstruction for an abdominal aortic aneurysm or an aortoiliac occlusion. Methods: Patients with a midline incision after undergoing reconstruction of an abdominal aortic aneurysm or aortoiliac occlusion were examined by CT scanning and US. Two radiologists evaluated the CT scans independently. One radiologist examined the CT scans twice. Discrepancies between the CT observations were resolved in a common evaluation session between the two radiologists. Results: After a mean follow-up of 3.4 years, 40 patients were imaged after a reconstructed abdominal aortic aneurysm (80% of the patients) or aortoiliac occlusion. The prevalence of incisional hernias was 24/ 40 = 60.0% with CT scanning as the diagnostic modality and 17/40 = 42.5% with US. The measure of agreement between CT scanning and US expressed as a Kappa statistic was 0.66 (95% confidence interval [CI] 0.45-0.88). The sensitivity of US examination when using CT as a comparison was 70.8%, the specificity was 100%, the predictive value of a positive US was 100%, and the predictive value of a negative US was 69.6%. The likelihood ratio of a positive US was infinite and that of a negative US was 0.29. The inter- and intra-observer Kappa statistics were 0.74 (CI 0.54-0.95) and 0.80 (CI 0.62-0.99), respectively. Conclusions: US imaging has a moderate sensitivity and negative predictive value, and a very good specificity and positive predictive value. Consistency of diagnosis, as determined by calculating the inter- and intra-observer Kappa statistics, was good. The incidence of incisional hernias is high after aortic reconstructions

Trauma Hemorrhagic Shock-Induced Lung Injury Involves a Gut-Lymph-Induced TLR4 Pathway in Mice

Injurious non-microbial factors released from the stressed gut during shocked states contribute to the development of acute lung injury (ALI) and multiple organ dysfunction syndrome (MODS). Since Toll-like receptors (TLR) act as sensors of tissue injury as well as microbial invasion and TLR4 signaling occurs in both sepsis and noninfectious models of ischemia/reperfusion (I/R) injury, we hypothesized that factors in the intestinal mesenteric lymph after trauma hemorrhagic shock (T/HS) mediate gut-induced lung injury via TLR4 activation.The concept that factors in T/HS lymph exiting the gut recreates ALI is evidenced by our findings that the infusion of porcine lymph, collected from animals subjected to global T/HS injury, into naïve wildtype (WT) mice induced lung injury. Using C3H/HeJ mice that harbor a TLR4 mutation, we found that TLR4 activation was necessary for the development of T/HS porcine lymph-induced lung injury as determined by Evan's blue dye (EBD) lung permeability and myeloperoxidase (MPO) levels as well as the induction of the injurious pulmonary iNOS response. TRIF and Myd88 deficiency fully and partially attenuated T/HS lymph-induced increases in lung permeability respectively. Additional studies in TLR2 deficient mice showed that TLR2 activation was not involved in the pathology of T/HS lymph-induced lung injury. Lastly, the lymph samples were devoid of bacteria, endotoxin and bacterial DNA and passage of lymph through an endotoxin removal column did not abrogate the ability of T/HS lymph to cause lung injury in naïve mice.Our findings suggest that non-microbial factors in the intestinal mesenteric lymph after T/HS are capable of recreating T/HS-induced lung injury via TLR4 activation

Protease Activity Increases in Plasma, Peritoneal Fluid, and Vital Organs after Hemorrhagic Shock in Rats

Hemorrhagic shock (HS) is associated with high mortality. A severe decrease in blood pressure causes the intestine, a major site of digestive enzymes, to become permeable – possibly releasing those enzymes into the circulation and peritoneal space, where they may in turn activate other enzymes, e.g. matrix metalloproteinases (MMPs). If uncontrolled, these enzymes may result in pathophysiologic cleavage of receptors or plasma proteins. Our first objective was to determine, in compartments outside of the intestine (plasma, peritoneal fluid, brain, heart, liver, and lung) protease activities and select protease concentrations after hemorrhagic shock (2 hours ischemia, 2 hours reperfusion). Our second objective was to determine whether inhibition of proteases in the intestinal lumen with a serine protease inhibitor (ANGD), a process that improves survival after shock in rats, reduces the protease activities distant from the intestine. To determine the protease activity, plasma and peritoneal fluid were incubated with small peptide substrates for trypsin-, chymotrypsin-, and elastase-like activities or with casein, a substrate cleaved by multiple proteases. Gelatinase activities were determined by gelatin gel zymography and a specific MMP-9 substrate. Immunoblotting was used to confirm elevated pancreatic trypsin in plasma, peritoneal fluid, and lung and MMP-9 concentrations in all samples after hemorrhagic shock. Caseinolytic, trypsin-, chymotrypsin-, elastase-like, and MMP-9 activities were all significantly (p<0.05) upregulated after hemorrhagic shock regardless of enteral pretreatment with ANGD. Pancreatic trypsin was detected by immunoblot in the plasma, peritoneal space, and lungs after hemorrhagic shock. MMP-9 concentrations and activities were significantly upregulated after hemorrhagic shock in plasma, peritoneal fluid, heart, liver, and lung. These results indicate that protease activities, including that of trypsin, increase in sites distant from the intestine after hemorrhagic shock. Proteases, including pancreatic proteases, may be shock mediators and potential targets for therapy in shock