Forty-two male Wistar rats (200 to 250 g, 6 to 8 weeks old) were obtained from Tierzucht Schönwalde GmbH, Schönwalde, Germany, housed in chip-bedded cages in an air-conditioned animal quarter, and acclimatized for one week to the institutional animal care unit prior to the experiments. The animals were kept on a 12 hours light/dark cycle with free access to water (drinking bottle) and standard rat chow (Altromin®, Lage, Germany). Eighteen hours prior to each experiment food was withdrawn; water remained accessible. The animal experiments were approved by the Institutional Review Board for the care of animal subjects (protocol G 0133/00) and performed in accordance with German legislation on the protection of animals.
Anesthesia and monitoring
The animals were initially anesthetized with 60 mg/kg pentobarbital (Sigma, Deisenhofen, Germany) intraperitoneally and were supplemented with 20 mg/kg/h pentobarbital intravenously in the course of the experiment. Fixation of the animals was carried out in supine position on a heating pad, keeping a rectal body temperature between 36.5°C (97.7°F) and 37°C (98.6°F). Tracheostomy was performed to maintain airway patency and animals breathed room air spontaneously. The left jugular vein and carotid artery were cannulated with polyethylene catheters (PE50; inner diameter 0.58 mm; outer diameter 0.96 mm; Portex, Hythe, Kent, UK). Arterial pressure and heart rate were recorded continuously (Biomonitor BMT 5231, RFT, Stassfurt, Germany). The animals received 7.5 ml/kg/h crystalloid solution (Thomaejonin®, Thomae, Biberach, Germany).
Experiments started 30 minutes after cannulation (time = 0 h). The rats were divided into 3 groups of 14 animals each. One half of the animals in each group underwent an examination of the submucosa for leukocyte adherence on venular endothelium and functional capillary density (FCD) by intravital fluorescence microscopy (IVM) of the small bowel wall. In the other half of the animals, plasma extravasation in the mesentery was determined by IVM.
The animals of group 1 (control group) did not receive endotoxin. In group 2 (lipopolysaccharide (LPS) group) and group 3 (F XIII group), endotoxemia (endotoxin challenge) was induced by continuous infusion of 2.5 mg/kg/h LPS from Escherichia coli, serotype O55:B5 (Sigma) over 2 hours. The animals of the control group were given an equivalent volume of normal saline (placebo infusion). In the F XIII group we continuously administered 50 U/kg body weight human F XIII (Fibrogammin® HS, Aventis Behring, Marburg, Germany) during the first 30 minutes of endotoxemia.
Blood samples (total volume 0.2 ml) were taken 30 minutes after cannulation (time = 0 h) for white blood cell count determination as well as three hours after the endotoxin challenge (cell counter, Technicon H1, Bayer, Leverkusen, Germany). The F XIII levels were estimated at baseline (0 h) as well as 1.5 hours and 3 hours after the start of the endotoxin challenge.
Laparotomy for IVM was performed before the start of the endotoxin or placebo infusion. The abdomen was opened by a midline incision. A section of the distal small intestine orally from the ileocoecal valve was placed carefully on a specially designed stage attached to the microscope. During the entire in vivo microscopic procedure, the intestine was superfused with thermostat-controlled (37°C/98.6°F) crystalloid solution (Thomaejonin®) to avoid drying and exposure to ambient air . The duration of each experiment, including induction of anesthesia, did not exceed 240 minutes. At the end of the experiments, the animals were euthanized by pentobarbital overdose.
IVM was performed using an epifluorescent microscope (Axiotech Vario, filter block No. 20, Zeiss, Oberkochen, Germany) with a 50-W HBO (Osram, Munich, Germany) short arc mercury lamp and equipped with a 10 × long distance (10/0.5; Fluar, Zeiss) and a 20 × water immersion (20/0.5; Achroplan, Zeiss) objective (mesentery: 40 × water immersion, 40/0,8; Achroplan, Zeiss) and a 10 × eyepiece. The images were transferred to a monitor (LDH 2106/00, Philips Electronics, Eindhoven, The Netherlands) by means of a video camera (FK 6990-IQ, Pieper, Schwerte, Germany) and were recorded on video at the same time using a video cassette recorder (Panasonic AG 6200, Matsushita, Japan) for off-line evaluation.
Functional capillary density
After two hours of endotoxemia, 50 mg/kg bw FITC-labeled BSA (Sigma) was administered intravenously to distinguish plasma from red blood cells (negative contrast). The assessment of FCD in the intestinal mucosa and the circular as well as the longitudinal muscle layer was performed by morphometric determination of the length of red blood cell perfused capillaries per area in accordance with the method of Schmid-Schönbein and colleagues . Five separate fields were examined in each layer.
After two hours of endotoxemia, leukocytes were stained in vivo by intravenous injection of 0.2 ml of 0.017 g % rhodamine 6G (MW 479; Sigma) for contrast enhancement, enabling visualization in the microvasculature. Microvessels in the intestinal submucosal layer were classified by their order of branching according to Gore and Bohlen . Submucosal collecting venules (V 1) as well as postcapillary venules (V 3) were analyzed. The flux of rolling leukocytes was defined as the count of white cells moving at a velocity of less than two-fifths of that of erythrocytes in the centerline of the microvessels  and is quoted as non-adherent leukocytes passing through the observed vessel segment within 30 seconds. Adherent leukocytes (stickers) were defined in each vessel segment as cells that did not move or detach from the endothelial lining within an observation period of 30 seconds, and are quoted as the number of cells per mm2 of endothelial surface, calculated from diameter and length of the vessel segment studied, assuming cylindrical geometry . Seven vessels of each population were evaluated in every animal. The evaluation of leukocyte adherence was performed in a blinded fashion. The values were adjusted to the white blood cell count.
To quantify the plasma extravasation across mesenteric venules, 50 mg/kg bw FITC-BSA (Sigma) was injected 15 minutes before each experiment. The recorded fluorescent images were digitized and the gray levels were measured within five segments of the venule under study (Iv) and in five contiguous areas of the perivenular interstitium (Ip) depending on the fluorescence activity (gray levels range from 0 (black) to 255 (white)). Plasma extravasation (macromolecular leakage) was expressed as the ratio of Ip/Iv after one hour of endotoxemia. Evaluation was performed in a blinded fashion.
Data analysis was performed using a statistical software package (SigmaStat, Jandel Scientific, Erkrath, Germany). All data were expressed as group mean ± standard deviation or standard error of mean and analyzed using a one-way analysis of variance followed by the Bonferroni corrected t test. Plasma extravasation, mean arterial pressure, heart rate and white blood cell count were analyzed by a two-way analysis of variance (repeated measures in the factor of time). This test was followed by the Scheffé test. A p value <0.05 was considered significant.