Stephan T. Samel

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

BackgroundExisting 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.MethodsIn 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.ResultsBacterial 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.ConclusionsIntravital 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.

A new abdominal cavity chamber to study the impact of increased intra-abdominal pressure on microcirculation of gut mucosa by using video microscopy in rats

ObjectiveIn experimental studies of capillary blood flow that use intravital video microscopy, organs are exposed in observation chambers implanted into the animal. In this article we describe an abdominal cavity chamber for intravital video microscopy of gut mucosa microcirculation during increased intra-abdominal pressure. DesignProspective, experimental animal study. SettingResearch laboratory at a university hospital. SubjectsMale Wistar rats. InterventionsThe abdominal cavity chamber was designed for implantation into the abdominal wall of rats after laparotomy, thus creating an expanded hermetic, abdominal cavity volume. Animals were assigned to three levels of intra-abdominal pressure: controls (group 1), 10 mm Hg (group 2), and 15 mm Hg (group 3). Intra-abdominal pressure was increased by intra-abdominal insufflation of gas. By using a fluorescent marker, we quantitatively assessed mucosa perfusion index, functional capillary density, red blood cell velocity, capillary diameters, and flow motion during increased intra-abdominal pressure by intravital video microscopy. Results were expressed as mean ± sem. Significance of differences was determined by analysis of variance and multiple comparison of means with post hoc test (*p < .05 groups vs. control; †p < .05 group 3 vs. group 2). Measurements and Main ResultsWhen compared with controls, animals subjected to an intra-abdominal pressure of 10 and 15 mm Hg showed a significant stepwise decrease in mucosa perfusion index (88%, 71%*, 22%*†), functional capillary density (665.4 ± 71.7, 461.6 ± 71.9*, 375.1 ± 2.0*† cm−1), and red blood cell velocity (0.50 ± 0.04, 0.33 ± 0.03*, 0.04 ± 0.06*† mm/sec), indicating a stepwise impairment of mucosal microcirculation. Capillary diameters and flow motion did not change with respect to intra-abdominal pressure. ConclusionsThis novel animal model of intravital intestinal video microscopy that uses an abdominal cavity chamber is a feasible and sensitive experimental tool to study intestinal microcirculation during increased intra-abdominal pressure. Intra-abdominal pressure likely results in a severe impairment of mucosal microcirculation.

The Gut Origin of Bacterial Pancreatic Infection during Acute Experimental Pancreatitis in Rats

Background: Infections are frequent complications and determine clinical course and outcome in severe pancreatitis. A novel animal model was used to assess minimal transit time of bacterial translocation (BT) across the gut mucosa in vivo using green fluorescent protein-transfected Escherichia coli and intravital video microscopy. Methods: Three hours after induction of acute pancreatitis by i.p. injection of 40 µg/kg cerulein, 0.5 ml of a suspension of green fluorescent protein-transfected E. coli were injected into the lumen of a small bowel reservoir formed by ligature in anesthetized Wistar rats. Translocation of E. coli was assessed by intravital microscopy. Animals were sacrificed 5 h after induction of pancreatitis. Results: BT across the mucosa and into the muscularis propria took a mean ± SD of 36.4 ± 8 min and 80.9 ± 9.5 min, respectively, in sham animals. Pancreatitis resulted in a significantly shorter minimal transit time across the mucosa (16.4 ± 4.9 min, p = 0.007) and into the muscularis propria (47.7 ± 2.5 min, p = 0.001). E. coli were detected on frozen cross-sections and on bacteriological examination of pancreatic tissue in animals with acute pancreatitis but not in controls. Discussion: Intravital microscopy of fluorescent bacteria is a new approach towards studying BT in vivo. Minimal transit time of BT serves as a novel functional aspect of mucosal barrier function during acute pancreatitis. The observation of fluorescent bacteria translocating from the small bowel lumen into the pancreas provides substantial experimental proof for the gut-origin-hypothesis of infectious complications in pancreatitis.

Peritoneal cancer treatment with CYP2B1 transfected, microencapsulated cells and ifosfamide

The prognosis of peritoneal spread from gastrointestinal cancer and subsequent malignant ascites is poor, and current medical treatments available are mostly ineffective. Targeted chemotherapy with intraperitoneal prodrug activation may be a beneficial new approach. L293 cells were genetically modified to express the cytochrome P450 enzyme 2B1 under the control of a cytomegalovirus immediate early promoter. This CYP2B1 enzyme converts ifosfamide to its active cytotoxic compounds. The cells are encapsulated in a cellulose sulfate formulation (Capcell™). Adult Balb/c mice were inoculated intraperitoneally with 1 × 106 colon 26 cancer cells, previously transfected with GFP to emit a stable green fluorescence, by injection into the left lower abdominal quadrant. Two or five days later animals were randomly subjected to either i.p. treatment with ifosfamide alone or ifosfamide combined with microencapsulated CYP2B1-expressing cells. Peritoneal tumor volume and tumor viability were assessed 10 days after tumor inoculation by means of fluorescence microscopy, spectroscopy and histology. Early i.p. treatment with ifosfamide and CYP2B1 cells resulted in a complete response. Treatment starting on day 5 and single-drug treatment with ifosfamide resulted in a partial response. These results suggest that targeted i.p. chemotherapy using a combination of a prodrug and its converting enzyme may be a successful treatment strategy for peritoneal spread from colorectal cancer.

Supplementation and inhibition of nitric oxide synthesis influences bacterial transit time during bacterial translocation in rats

In the obstructed gut, nitric oxide (NO) may influence intestinal barrier function and translocation of bacteria. By using a novel experimental approach, we investigated the effect of supplementation and inhibition of NO synthesis on the time interval necessary for translocation of green fluorescent protein-transfected Escherichia coli (GFP-uv E. coli) in a rat model of small bowel obstruction. In anesthetized Wistar rats, 4 x 108 GFP-uv E. coli were administered into a reservoir of terminal ileum formed by ligature. Animals were randomized to receive either i.v. arginine (10 mg/kg), aminoguanidine (300 mg/kg), l-NAME (25 mg/kg), or saline (control). Translocation of GFP-uv E. coli was assessed using intravital video microscopy. Minimal transit time of translocation was measured as time from injection of GFP-uv E. coli into the gut lumen until bacteria were observed in the lamina submucosa and as time from injection of bacteria into the gut lumen until bacteria were observed in the lamina muscularis propria. Minimal transit times were expressed as mean ± SD. Bacterial translocation into the submucosa and muscularis propria took 36 ± 7 min and 81 ± 9 min, respectively in control animals receiving saline. Aminoguanidine and l-NAME caused a marked delay of minimal transit time into the submucosa (63 ± 5 min and 61 ± 7 min, respectively;P < 0.05). Arginine significantly accelerated bacterial translocation into the muscularis propria (61 ± 9 min, P < 0.05). GFP-uv E. coli were detected on frozen sections of small bowel, mesentery, liver, and spleen 2 h after GFP-uv E. coli administration in all animals. A marked upregulation of inducible NO synthase (NOS) in the obstructed bowel segment was demonstrated on immunohistochemistry. The assessment of a newly defined parameter, minimal bacterial transit time, may serve as an additional functional aspect of intestinal barrier function for pathophysiological and pharmacological studies. Aminoguanidine, l-NAME, and arginine were effective in influencing minimal transit time of E. coli during small bowel obstruction.

Malignant intestinal non-Hodgkin's lymphoma from the surgical point of view.

Background: Primary intestinal non-Hodgkin’s lymphoma (I-NHL) is much less frequent than gastric lymphoma and has hardly been studied in prospective trails. To the surgeon, patients frequently present with abdominal emergencies. Patients and Methods: A consecutive series of patients subjected to surgery because of I-NHL between 1998 and 1999 was evaluated retrospectively for characteristic clinical, radiographic and intraoperative findings. Patients with gastric lymphoma were not considered. Results: 10 patients, 8 males and 2 females, with I-NHL were subjected to first-line surgery because of painful abdominal tumor, intestinal hemorrhage, obstruction or perforation. I-NHL was located most often in the small bowel (n = 7). It was rare in the colon (n = 2) and the duodenum (n = 1). Median postoperative follow-up was 28 months. Perioperative mortality was 10% (n = 1). Probability of survival 3 years after surgery was 60%. Conclusions: Patients with I-NHL frequently present with complications of tumor growth, requiring urgent surgical treatment. Irrespective of surgical complications we advocate surgery in cases of resectable disease as first-line treatment. Adjuvant treatment is indicated with respect to resection status and histopathological staging.

Alterations of Systemic Endotoxemia over the Course of Acute Edematous Pancreatitis

Background/Aims: To define whether bacterial translocation occurs over the course of acute edematous pancreatitis and to correlate its presence to the advent of an infection since data in humans are lacking. Methods: Thirty-three patients hospitalized over the period January 2000–January 2001 were subjected to venipuncture at regular time intervals for the collection of blood samples for blood culture and for determination of endotoxins and of C-reactive protein. Endotoxins were measured by the Limulus assay and C-reactive protein by nephelometry. Results: A wide range of concentrations of endotoxins was observed over the first 3 days of the disease. Mean (±SE) of endotoxins was 4.01 ± 1.36 and 2.42 ± 0.95 EU/ml 3 and 6 h, respectively, after admission of afebrile patients. Respective values 3 and 6 h after admission of febrile patients were 3.03 ± 1.14 and 5.84 ± 2.28 EU/ml (normal <0.1 EU/ml); these values gradually decreased after the second day. No correlation was found between endotoxins and C-reactive protein. Endotoxins were increased as a result of the occurrence of an infection on the third day. Conclusions: A significant level of endotoxemia is observed over the course of acute edematous pancreatitis, which might be correlated to the advent of the systemic inflammatory response.

CT or MRI for predicting vascular involvement in pancreatic cancer

A 75-year-old male patient with cancer of the pancreatic head was evaluated for resectability by UMRI, including echoplanar sequences, MR cholangiopancreaticography and gadolinium-enhanced dual-phase three-dimensional MR angiography (MRA), as well as dual-phase helical computed tomography. CT demonstrated semicircular encasement and an irregular shape of the portal vein distal to the venous confluence (panel A) suggesting infiltration by cancer. On MRA, however, the appearance of the portal vein was smooth (panel B), denying involvement of the vessel. On laparotomy, locally advanced can-

Alternative Approaches to the Treatment of Fecal Incontinence

Biofeedback and anal electrostimulation are considered to be established techniques for the treatment of chronic fecal incontinence when the involuntary loss of stool cannot be stopped by surgery, by general measures such as optimizing stool consistency, or by the treatment of an underlying proctological condition. In fact the terms “biofeedback” and “electrical stimulation” cover a wide range of therapies, which have been subjected to varying methods of evaluation. The available literature on the subject does not allow a systematic comparison of different forms of treatment (1). No general recommendation to treat fecal incontinence in any particular way can be derived from the individual studies published to date (2). Despite this, certain types of treatment without any clear evidence of efficacy are now reimbursable by the statutory health insurance carriers in Germany, while others cannot be ordered. The frequent result is a fruitless struggle with the Medical Service of Health Insurance Companies in Germany (Medizinischer Dienst der Krankenversicherung, MDK) and with the statutory health insurance carriers (Gesetzliche Krankenversicherungen, GKV), ending in the refusal of payment for indicated treatment. Such conflicts are annoying, and the denial of treatment for our patients is regrettable. Yet there is a real lack of evidence-based treatment due to the inadequacy of scientific study in this area.

Leberregeneration in der FGF-2 defizienten Maus: funktionelle und morphologische Aspekte

Aims We have previously shown that liver regeneration is not impaired in FGF-2 mice. Here we investigate a potential functional substitution of FGF-2 by other growth factors and characterize apoptosis and morphological changes in the liver of FGF-2 mice.