Chronic alcohol intake increases the severity of pancreatitis induced by acute alcohol administration, hyperlipidemia and pancreatic duct obstruction in rats.

BACKGROUND: The mechanism of alcoholic pancreatitis is still unknown. It is of special interest why only about 5% of all alcoholics develop an episode of pancreatitis. We evaluated the role of long-term alcohol intake in the pathogenesis of alcoholic pancreatitis in rats. METHODS: To evaluate the effect of long-term alcohol intake, rats were fed either a Lieber-DeCarli control diet (CD) or a Lieber-DeCarli alcohol diet (AD) for 6 weeks. Then, rats were infused over 2 h with either Ringer's solution (CO) or ethanol (E). In additional animals, alcoholic pancreatitis was induced by ethanol combined with hyperlipidemia and temporary pancreatic duct obstruction (EFO). Controls received Ringer's solution combined with hyperlipidemia and temporary pancreatic duct obstruction (RFO). Intravital microscopy (pancreatic perfusion and leukocyte adhesion), alcohol concentrations, amylase, lipase, cholesterine and triglyceride levels in plasma, myeloperoxidase activity and histology were evaluated at different time intervals. RESULTS: In those animals which received the Lieber-DeCarli control diet, capillary perfusion was reduced in the E group and further reduced in the EFO group as compared to the controls (CO, RFO; p < 0.01). Leukocyte adhesion was significantly increased in rats receiving E (p < 0.01), and was further increased in the combination group EFO (p < 0.01). EFO induced histologically evident acute pancreatitis. The additional administration of a long-term alcohol diet further increased microcirculatory disturbances and pancreatic injury significantly (EFO-AD > EFO-CD). CONCLUSIONS: This study shows that alcoholic pancreatitis is induced by the combination of ethanol and individual cofactors. Chronic alcohol abuse intensifies these changes. Therefore, long-term alcohol intake seems to be a major factor in the pathogenesis of alcoholic pancreatitis.

Bradykinin in ischemia-reperfusion injury of the rat lung.

Recent studies described possibilities to reduce lung damage after intestinal ischemia by application of a selective bradykinin-2 receptor antagonist (HOE 140). In contrast, it has been shown that the preischemic application of bradykinin (BK) reduced ischemic damage of the myocardium. In the present study, to evaluate the effects of BK and HOE 140 in lung ischemia-reperfusion injury we used a standardized in vivo ischemia-reperfusion model of the right rat lung. Ischemia of 60 min was induced by cross-clamping of the right hilus followed by 120 min of reperfusion. During reperfusion, the left hilus was ligated. In Group 1 (n=5), the animals were sham operated without induction of ischemia under ligation of the left lung hilus. Group 2 (n=5) was operated as described, Group 3 (n=5) received 100 microg bradykinin (BK) before reperfusion, Group 4 (n=5) was given a B2-agonist before reperfusion, and Group 5 (n=5) was given 100 microg HOE140/kg body weight before reperfusion. Blood pressure and arterial oxygenation were monitored. As a marker of endothelial damage, angiotensin-converting-enzyme activity (ACE) in serum and RT-PCR of ACE and angiotensin-2 in lung tissue were determined in all groups. Two of the HOE140-treated animals died within 30 min of reperfusion. During reperfusion, significantly higher PaO2 values (P<0.01) have been observed in BK treated animals of Group 3 (214+/-22 mmHg) and sham operated controls of Group 1 (233+/-26 mmHg) compared with Groups 2 (132+/-13 mmHg) and Group 5 (125+/-50 mmHg; P<0.01). Serum ACE activity after reperfusion was significantly lower in Group 1 (3.5+/-0.5 IU/l), Group 3 (3.8+/-1.1 IU/l), and Group 4 (2.2+/-0.5 IU/l; P<0.05) vs. Group 2 (4.8+/-0.9 IU/l), whereas Group 5 (6.2+/-5.4 IU/l) did not differ from Group 2. mRNA expressions of ACE was lower in Group 1 and Group 3 compared with Group 2 (P<0.01). AT-2 mRNA expression did not show any differences between the investigated groups. A significantly lower ACE activity and expression and a significantly higher oxygenation after BK application in Group 3 strongly suggest a positive influence of bradykinin on ischemic preconditioning of the pulmonary endothelium. Positive effects of application of bradykinin-receptor antagonists could not be proved in this study.

Alterations of tumor and normal tissue of human lung cancer resection specimens after isolation perfusion.

The isolation perfusion model, including transbronchial ventilation of human lung, offers the possibility to study pharmacological interactions under physiological conditions. In view of the increasing importance of targeted therapy of lung diseases, this model of perfusion might attract major interest, particularly, in lung cancer. Our study investigated physiological, histological, and immunohistochemical alterations of lung and tumor tissue during isolated perfusion of lung lobectomy specimens to explore potential limitations of this model. Right after resection, 16 human lung resection specimens for primary lung cancer were isolated, ventilated, and perfused under physiological conditions with a modified Krebs-Henseleit solution over a period of 10, 60, 90, 120, and 240 min. Perfusion pressure, pH, lung weight gain, and histological edema formation were measured continuously before and during perfusion. After perfusion, lung and tumor tissue was investigated by hematoxylin-and-eosin stained sections. Immunohistochemistry of NADH, PECAM-1, angiotensin-converting-enzyme and NF-kappabeta were performed to determine lung tissue viability and changes at the endothelial layer. We found that perfusion up to 120 min could be performed with completely stable physiological conditions. Beyond that time span, edema formation and weight gain of the resection specimen started and were followed by an increase in inspiratory pressure and pulmonary artery pressure. Perfusion of more than 4 h led to a significant edema formation in lung tissue accompanied by loss of viability and significant histological alterations. We conclude that isolated ventilation and perfusion of human lung resections within the setup chosen is reliable for pharmacological studies up to a period of 120 min. Thereafter, edema formation and endothelial damage develop and limit the interpretation and reliability of drug delivery studies.

Taurine protects from liver injury after warm ischemia in rats: the role of kupffer cells.

BACKGROUND/AIMS: Warm ischemia to liver with subsequent Kupffer cell-dependent pathology is associated with many clinical conditions. Taurine prevents Kupffer cell activation and improves graft survival after experimental cold ischemia and liver transplantation. Thus this study was designed to assess its effects after warm hepatic ischemia. METHODS: The left liver lobe of female Sprague-Dawley rats (170-210 g) underwent 60 min of warm ischemia. Animals were given either intravenous taurine or Ringer's solution 10 min prior to warm ischemia. Transaminases, histology, in vivo microscopy, intercellular adhesion molecules-1 (ICAM-1) expression, TNF-alpha and tissue hydroperoxide were compared between groups using analysis of variance (ANOVA) or ANOVA on ranks as appropriate. RESULTS: Taurine significantly decreased transaminases and improved histologic outcome. Phagocytosis of latex beads, serum TNF-alpha levels and tissue hydroperoxide concentrations were also significantly reduced. Stickers in sinusoids and post-sinusoidal venules significantly decreased. In parallel, both leukocyte infiltration and ICAM-1 expression decreased (p < 0.05), while flow velocity of red blood cells as well as sinusoidal perfusion rate were improved (p < 0.05). CONCLUSION: This study demonstrates that taurine blunts Kupffer cell-dependent hepatic pathology after warm ischemia in vivo via mechanisms including leukocyte-endothelial interaction, microcirculation disturbances and protection against lipid peroxidation.

Development and evaluation of a training module for the clinical introduction of the da Vinci robotic system in visceral and vascular surgery.

BACKGROUND: With the increasing use of the surgical robotic system in the clinical arena, appropriate training programs and assessment systems need to be established for mastery of this new technology. The authors aimed to design and evaluate a clinic-like training program for the clinical introduction of the da Vinci robotic system in visceral and vascular surgery. METHODS: Four trainees with different surgical levels of experience participated in this study using the da Vinci telemanipulator. Each participant started with an initial evaluation stage composed of standardized visceral and vascular operations (cholecystectomy, gastrotomy, anastomosis of the small intestine, and anastomosis of the aorta) in a porcine model. Then the participants went on to the training stage with the rat model, performing standardized visceral and vascular operations (gastrotomy, anastomosis of the large and small intestines, and anastomosis of the aorta) four times in four rats. The final evaluation stage was again identical to the initial stage. The operative times, the number of complications, and the performance quality of the participants were compared between the two evaluation stages to assess the impact of the training stage on the results. RESULTS: The operative times in the final evaluation stage were considerably shorter than in the initial evaluation stage and, except for cholecystectomies, all the differences reached statistical significance. Also, significantly fewer complications and improved quality for each operation in the final evaluation stage were documented, as compared with their counterparts in the initial evaluation stage. These improvements were recorded at each level of experience. CONCLUSIONS: The presented experimental small and large animal model is a standardized and reproducible training method for robotic surgery that allows evaluation of the surgical performance while shortening and optimizing the learning-curve.

Intravital microscopy of pulmonary microcirculation after single lung transplantation in pigs.

BACKGROUND: Pulmonary reperfusion injury is a significant risk factor following lung transplantation (LTx). Unfortunately, in vivo observations and quantitative analyses of the pulmonary microcirculation following LTx are technically demanding. METHODS: Pigs, weighing 18 to 22 kg, served as the laboratory animals. The left lung was harvested and preserved using donor aortic vessel segments, the pulmonary artery, and the cuff of the lung veins were extended. After 4 hours of ischemia, the lungs were transplanted by direct connection of the conduits to the left atrial appendage and the left pulmonary artery of the recipient. The lungs were placed extrathoracically and ventilated. The recipient left lung was excluded. With this procedure, mechanical trauma to the lung and moving artefacts were avoided. Intravital microscopic observation became feasible. RESULTS: Following reperfusion, oxygenation of pulmonary venous blood was excellent. However, blood flow distribution was significantly reduced to the transplanted lung compared with the native right recipient lung. Pulmonary vascular resistance was significantly increased, dropping from 3500 to 1000 dynes x s x cm(-5) during reperfusion compared to a value of 500 for the native right lung. The pulmonary microcirculation showed a significant number of no-reflow areas with extremely reduced red blood cell velocities. Greater than 90% of microvessels (<30 microm) showed velocities below 0.1 mm/sec. In conclusion, microvascular injury seems to be a major pathogenic factor for the development reperfusion failure. Quantification of alterations within the microvasculature may shed light on various treatment modalities that reduce perfusion failure.

Evaluation of microperfusion disturbances in the transplanted liver after Kupffer cell destruction using GdCl3: an experimental porcine study.

BACKGROUND: Organ function after liver transplantation is determined by ischemia-reperfusion injury. Destruction of Kupffer cells with gadolinium chloride (GdCl3) has been shown to have a possible preventive effect on the extent of this injury, which can be extrapolated by analyzing the distribution of hepatic microperfusion. The aim of this study was to evaluate the protective effect of GdCl3 on disturbances of microperfusion in the transplanted liver. METHODS: Landrace pigs were randomly divided into three groups. In the control group (CG; n=6) a mapping of the native liver was conducted. For mapping, the four hepatic liver lobes were named from right to left with A to D and every lobe was divided into three vertical segments (cranial, medial, and caudal). In each of these 12 areas, microperfusion was quantified using a thermodiffusion probe (TD [mL/100 g/min]). The other two groups were considered as transplanted treated group (TTG; n=10) and transplanted nontreated group (TnTG; n=10). The TTG received an infusion of 20 mg/kg GdCl3 intravenously 24 hours before organ harvesting. Then standardized orthotopic liver transplantation was performed. In TnTG, standardized orthotopic liver transplantation was carried out without prior GdCl3 injection. In the recipients, the microperfusion of transplanted livers were mapped in both TnTG and TTG, in two different time points (1 hour [n=5] and 24 hours (n=5]) after reperfusion. RESULTS: A significant reduction of macrophages in the TTG livers in comparison to the CG and TnTG livers was observed (P<.05). However, the number of macrophages in CG and TnTG livers showed no significant difference (P>.05). Regarding liver microperfusion, in TnTG, a marked heterogeneity was detected in the livers after reperfusion. Significant differences between liver lobes (horizontal planes; P=.032) and vertical layers of intralobar liver parenchyma (P=.029) were observed. The same pattern was seen in TTG livers after reperfusion and a significant difference between horizontal (P=.024) and vertical layers (P=.018) of liver tissue were observed. Comparing intralobar regional flow data between vertical planes 24 hours after reperfusion still showed a prominent variation of hepatic tissue perfusion in TnTG livers (P=.028). Within the same horizontal layers, no significant differences between lobes were measured anymore (P=.16). Contrary to TnTG, in TTG, a homogenous pattern of regional liver tissue perfusion was recorded 24 hours after reperfusion. Comparison of TD data on the liver regions showed no significant microperfusion differences in either horizontal (P=.888) or vertical (P=.841) layers. CONCLUSIONS: Application of GdCl3 resulted in a significant reduction of Kupffer cells. Twenty four hours after transplantation microperfusion showed a homogeneous pattern, which constituted an earlier and better recovery of the transplanted liver. Therefore, destruction of Kupffer cells reduced ischemia-reperfusion injury and seemed to be responsible for the early recovery of microperfusion disturbances and thus for an improvement of graft function.

Surface contact measurement of electrical cell uncoupling in the mouse heart during ischemia.

Electrical cell uncoupling via gap junction closure is assumed to cause characteristic changes of the passive dielectric spectrum of ischemic heart tissue. In order to find an independent evidence for this assumption, we analysed heart tissue during ischemia, measured the open state of gap junctions by means of dye transfer and correlated this parameter with the time course of the dielectric permittivity. The hearts were pre-ischemically arrested by perfusion with Ringer solution containing 20 mmol/L of potassium (group KCL, n=10). This solution was also used with the addition of two gap junction blockers, either 3 mmol/L heptanol (group HEP, n=4) or 20 micromol/L palmitoleic acid (group PA, n=7). During subsequent ischemia at 21.0+/-0.5 degrees C, we monitored the passive dielectric permittivity spectrum and the spread of dye. After a sigmoidal increase the dielectric permittivity reached an upper plateau at 61+/-22 min of ischemia in KCL, at 45+/-7 min in PA, and already during perfusion at 2+/-1 min in group HEP. At the beginning of ischemia, dye migrated to neighbouring cells in groups KCL and PA but not in HEP. In KCL and PA, the intercellular diffusion of dye stopped after 64+/-26 and 40+/-11 min of ischemia, respectively. Our results suggest that the sigmoidal increase in dielectric permittivity and the reduction of dye diffusion depend on a common mechanism, namely gap junction closure.

A review of various techniques of orthotopic liver transplantation in the rat.

Orthotopic liver transplantation (OLT) in rat is a demanding procedure, which has become a popular model to investigate various problems. Our aim was to review and analyze the various techniques of experimental OLT in the rat. A review of the literature revealed 30 techniques or technical modifications. Each modification represented a change or a simplification of the reconstruction method of five anatomical structures, which are cornerstones of a successful OLT: the suprahepatic inferior vena cava (SHVC), portal vein (PV), infrahepatic inferior vena cava (IHVC), hepatic artery (HA), and bile duct (BD). SHVC is anastomosed via microsuture or cuff. The PV anastomosis is performed by microsuture, cuff, or a microsuture-temporary splint technique. IHVC is reconstructed by a microsuture, cuff, or microsuture-temporary splint technique. Arterialization has been accomplished via microsuture (aortic segment, celiac segment, or aortic patch), cuff, splint, sleeve, or telescopic method. Nonarterialization of the graft has also been described. Methods for BD reconstruction include pull-through, telescopic, splint, and T-tube. Although a high level of microsurgical skill is the basic requirement in the microsuture technique which provides the most physiological situation and concomitantly reduces thrombosis, it increases anhepatic time compared to the cuff procedure. The learning curve of microsuture techniques is flat; beginners need much practice to become expert. The most physiologic techniques for anastomoses are preferred for long-term survival studies, while the faster techniques are options for short-term survival studies. Each research group must choose techniques according to study defined aims.

Laparoscopic organ retrieval for living donor liver transplantation does not prevent graft injury.

The cause of transplant failure may be difficult to define. However, organ retrieval before preservation and transplantation is an important factor. Organ manipulation during harvesting, which is inevitable with most techniques, leads to injury upon reperfusion including microcirculatory disturbances. Recently, laparoscopic organ retrieval has been successfully performed for human living donor liver transplantation (LDLT). Pneumoperitoneum for laparoscopy changes the pattern of hepatic blood flow. To study the effects of pneumoperitoneum on the graft prior to cold storage, livers from Sprague-Dawley rats underwent pneumoperitoneum with an intra-abdominal pressure of 8 mm Hg for 90 minutes. Subsequently, intravital microscopy was performed to assess intrahepatic microcirculation and transaminases were measured. Serum transaminases increased 1.5-fold compared with sham controls (P < .05). Intrahepatic microcirculation was significantly disturbed immediately after pneumoperitoneum. If this is confirmed in humans, laparoscopic organ retrieval for LDLT would be expected to decrease graft quality and not be beneficial in liver transplantation.

A novel model for the investigation of orthotopically growing primary and secondary bone tumours using intravital microscopy.

Here is reported the development of an experimental model using intravital microscopy as a tool to orthotopically investigate malignant bone tumours. Although up to 85% of the most frequently occurring malignant solid tumours, such as lung and prostate carcinomas, metastasize into the bone, and despite the knowledge that a tumour's course may be altered by its surrounding tissue, there is no adequate experimental model available enabling the investigation of orthotopically grown bone tumours in vivo. Intravital microscopy is an internationally accepted experimental method, used in various acute and chronic animal models, that enables qualitative and quantitative analysis of the angiogenesis, microcirculation, growth behaviour, etc. of various benign and malignant tissues. Non-invasive investigations of up to several weeks are possible. Additionally, tissue samples can be taken after termination of the in vivo experiments for further ex vivo investigation (histology, immunohistochemistry, molecular biology, etc.), elucidating the mechanisms that underlie the in vivo observations. Severe combined immunodeficient mice were fitted with a cranial window preparation where the calvaria served as the site for orthotopic implantation of the solid human tumours Saos-2 osteosarcoma (primary) and A 549 lung carcinoma and PC-3 prostate carcinoma (secondary). In all preparations, the take rate was 100%. Histological assessment confirmed the data obtained in vivo, showing typical tumour growth with infiltration of the surrounding osseous and soft tissues. This novel model serves as a valuable tool in understanding the biology of primary and secondary bone tumours in physiological and pathophysiological situations, with implications for the most areas of tumour therapy such as chemotherapy, radiation and antiangiogenesis.

Desmopressin impairs microcirculation in donor pancreas and early graft function after experimental pancreas transplantation.

BACKGROUND: Protective effects of desmopressin in brain dead organ donors oppose reports on a hypercoagulatory potential and an increased leukocyte-endothelial interaction (LEI) after application of the drug. The aim was to evaluate the effect of desmopressin on organ donor's pancreas and early graft function. METHODS: Donor microcirculation was evaluated via intra-vital microscopy (IVM) in 24 BR (di/di) rats with central diabetes insipidus, randomly assigned to groups I (control without desmopressin application), II (single i.v. application, no pretreatment) or group III (single i.v. desmopressin application, s.c. pretreatment for 3 days). Microcirculation in recipients was evaluated 1 hr and 6 hr after syngenic pancreas transplantation. Groups III and I served as organ donors. After IVM specimens were taken for histology and immunohistochemistry. RESULTS: Desmopressin in II vs. I led to temporarily (30') increased LEI (Sticker 274.3+/-87.7 vs. 76.5+/-31.1/mm2 endothelial surface; P<0.01) and impaired microcirculation (MCEV 0.43+/-0.07 vs. 0.99+/-0.06 mm/s; P<0.01). Repeated application reduced MCEV and increased LEI for up to 12 hr. Histology in I vs. III showed increased inflammation (n.s.), necrosis (P<0.05) and vacuolization (P<0.01). Immunohistochemistry revealed increased endothelial P-selectin 20' after application. 6 hr after reperfusion organs from III showed reduced MCEV and increased LEI (P<0.01). CONCLUSION: Repeated application of desmopressin impairs graft microcirculation. Perfusion of the pancreas is significantly reduced at the beginning of organ tissue conservation as well as after reperfusion. These disturbances might partly be due to observed endothelial P-selectin expression. Application of desmopressin up to 12 hr prior to organ explantation may impact graft quality.

Impact of N-acetylcysteine on the hepatic microcirculation after orthotopic liver transplantation.

Recent observations showed an improvement of hepatic macro- and microhemodynamics as well as survival rates after warm ischemia of the liver following treatment with N-acetylcysteine (NAC). In this study we assessed the influence of NAC on the hepatic microcirculation after orthotopic liver transplantation (OLT) using intravital fluorescence microscopy. OLT with simultaneous arterialization was performed in 16 male Lewis rats following cold storage in University of Wisconsin solution for 24 hr. Within the experimental group (n = 8) donors received NAC (400 mg/kg) 25 min before hepatectomy. In addition, high-dose treatment of recipients with NAC (400 mg/kg) was started with reperfusion. Control animals (n = 8) received an equivalent amount of Ringer's solution. Intravital fluorescence microscopy was performed 30-90 min after reperfusion assessing acinar and sinusoidal perfusion, leukocyte-endothelium interaction, and phagocytic activity. Treatment with NAC reduced the number of nonperfused sinusoid from 52.4 +/- 0.8% to 15.7 +/- 0.5% (p = 0.0001) (mean +/- SEM). Furthermore, we achieved a significant reduction of leukocytes adhering to sinusoidal endothelium (per mm2 liver surface) from 351.9 +/- 13.0 in controls to 83.6 +/- 4.2 in the experimental group (P = 0.0001). In postsinusoidal venules, treatment with NAC decreased the number of sticking leukocytes (per mm2 endothelium) from 1098.5 +/- 59.6 to 425.9 +/- 37.7 (P = 0.0001). Moreover, bile flow was significantly increased after therapy with NAC (4.3 +/- 1.2 vs. 2.2 +/- 0.7 ml/90 min x 100g liver) (P < 0.05). Phagocytic activity was not influenced by application of NAC. We conclude that high-dose therapy with NAC in OLT attenuates manifestations of microvascular perfusion failure early after reperfusion and should be considered as a means to reduce reperfusion injury.

Complement inhibition by soluble complement receptor type 1 improves microcirculation after rat liver transplantation.

BACKGROUND: Recent observations provide evidence that complement is involved in the pathophysiology of ischemia/reperfusion injury. In this study, we assessed the impact of complement inhibition on hepatic microcirculation and graft function using a rat model of liver transplantation. METHODS: Arterialized orthotopic liver transplantation was performed in Lewis rats after cold preservation (University of Wisconsin solution, 4 degrees C, 24 h). Eight animals received the physiological complement regulator soluble complement receptor type 1 (sCR1) intravenously 1 min before reperfusion. Controls received Ringer's solution (n=8). Microvascular perfusion, leukocyte adhesion, and Kupffer cell phagocytic activity were studied 30-100 min after reperfusion by in vivo microscopy. RESULTS: Microvascular perfusion in hepatic sinusoids was improved in the sCR1 group (87+/-0.7% vs. 50+/-1%; P < 0.001). The number of adherent leukocytes was reduced in sinusoids (68.3+/-4.7 vs. 334.1+/-15.8 [adherent leukocytes per mm < or = liver surface]; P < 0.001) and in postsinusoidal venules after sCR1 treatment (306.6+/-21.8 vs. 931.6+/-55.9 [adherent leukocytes per mm < or = endothelial surface]; P < 0.001). Kupffer cell phagocytic activity was decreased in the sCR1 group compared to controls. Postischemic bile production reflecting hepatocellular function was increased by almost 200% (P = 0.004) after complement inhibition. Plasmatic liver enzyme activity was decreased significantly upon sCR1 treatment, indicating reduced parenchymal cell injury. CONCLUSIONS: Our results provide further evidence that the complement system plays a decisive role in hepatic ischemia/reperfusion injury. We conclude that complement inhibition by sCR1 represents an effective treatment to prevent reperfusion injury in liver transplantation.

Dielectric properties of skeletal muscle during ischemia in the frequency range from 50 Hz to 200 MHz.

The complex dielectric properties of canine skeletal muscles were measured at 25 degrees C during ischemia in the frequency range from 50 Hz to 200 MHz. The dielectric spectrum of skeletal muscle shows an alpha-dispersion below 1 kHz and a beta-dispersion with a relaxation frequency of about 100 kHz. The alpha-dispersion disappears between 450 and 500 min of ischemia time, the same time during which mechanical contraction was observed, and was restored later. During ischemia, the beta-dispersion is shifted continuously to higher frequencies; and at frequencies above 50 MHz, a decrease of the real part of the dielectric permittivity was measured. The dielectric loss factor decreases during ischemia at frequencies below 500 kHz, only interrupted by a short increase, coinciding with the disappearance of the alpha-dispersion. The principal processes that happen during ischemia inside the skeletal muscle tissues were studied with the help of a model especially designed to simulate membrane effects on the dielectric spectrum. The disappearance of the alpha-dispersion is explained by an increase of conductivity in the membrane of the sarcoplasmic reticulum. Shifting beta-dispersion to higher frequencies is a result of metabolically produced ions and therefore increasing conductivity of the intracellular medium. Decreasing dielectric permittivity at frequencies above 50 MHz and decreasing dielectric loss factor at low frequencies are caused by the cell edema.

C1-esterase inhibitor and its effects on endotoxin-induced leukocyte adherence and plasma extravasation in postcapillary venules.

BACKGROUND: C1-esterase inhibitor (C1-INH) has been shown to have beneficial effects in patients with sepsis. However, the microcirculatory effects of C1-INH during sepsis are unknown. This study investigated the influence of C1-INH on leukocyte-endothelial cell adhesion, vascular leakage, and venular microhemodynamics in postcapillary venules of rat mesentery during endotoxemia. METHODS: Thirty-two anesthetized Wistar rats randomly received 1 of 4 treatments: pretreatment with infusion of C1-INH in a concentration of 7.5 U.kg-1 body weight (C1-INH-7.5 group, n = 8) or in a concentration of 15 U.kg-1 body weight (C1-INH-15 group, n = 8) followed by continuous infusion of Escherichia coli lipopolysaccharide (LPS). The LPS group (n = 8) was pretreated with saline solution 30 minutes before LPS infusion. The control group (n = 8) received equivalent amounts of saline infusion. Leukocyte adherence, red blood cell velocity, and vessel diameters in postcapillary venules of rat mesentery were determined every 60 minutes during a period of 120 minutes using in vivo videomicroscopy. Vascular permeability was determined by measuring the extravasation of fluorescence-labeled albumin. Venular wall shear rate was calculated from mean red blood cell velocity and vessel diameter. RESULTS: LPS infusion induced a decrease in venular wall shear rate and an increase in leukocyte adherence and vascular permeability in postcapillary venules of rat mesentery. All microcirculatory disturbances were attenuated by pretreatment with C1-INH, showing no significant difference between the 2 concentrations. CONCLUSIONS: Pretreatment with C1-INH attenuates endotoxin-induced leukocyte adherence and macromolecular leakage in postcapillary venules of rat mesentery, indicating that complement inhibition might be a therapeutic tool in the treatment of sepsis.

Influence of N-acetylcysteine treatment on endotoxin-induced microcirculatory disturbances.

OBJECTIVES: To determine the influence of N-acetylcysteine (NAC) in a treatment model, its effects on endotoxin-induced leukocyte-endothelial cell adhesion, vascular leakage, and venular microhemodynamics in postcapillary venules of rat mesentery. DESIGN: Prospective, randomized, controlled, experimental study. SETTING: Animal research laboratory. SUBJECTS: 40 male Wistar rats. INTERVENTIONS: The rats randomly received one of four treatments: infusion of saline (SAL) or Escherichia coli lipopolysaccharides (LPS) followed by treatment with saline (SAL) or NAC (150 mg.kg-1 body weight) 30 min after induction of endotoxemia. MEASUREMENTS AND MAIN RESULTS: Leukocyte adherence, red blood cell velocity, and vessel diameters in postcapillary venules of rat mesentery were evaluated every 30 min over a period of 120 min using in vivo videomicroscopy. Vascular permeability was determined by measuring the extravasation of fluorescence-labeled albumin. Venular wall shear rate was calculated from red cell velocity, and vessel diameter. NAC in rats without endotoxemia (SAL + NAC group) compared to the control group (SAL + SAL) did not change microcirculatory parameters in postcapillary venules of rat mesentery. In both LPS-treated groups (LPS + SAL and LPS + NAC), leukocyte adherence increased after just 30 min. NAC treatment prevented a further increase in leukocyte adherence and attenuated the extravasation of fluorescence-labeled albumin during endotoxemia. Venular diameters remained unchanged, while erythrocyte velocity decreased in the LPS + SAL group. This led to a lower venular wall shear rate in this group. CONCLUSIONS: Treatment with NAC attenuates endotoxin-induced leukocyte adherence and macromolecular leakage in postcapillary venules of rat mesentery, showing that NAC is also effective after the onset of endotoxemia.

Effect of the 21-aminosteroid tirilazad mesylate on leukocyte adhesion and macromolecular leakage during endotoxemia.

BACKGROUND: Interstitial accumulation of leukocytes has been related to the development of multiple organ failure after sepsis. Oxygen radicals are involved in the process of leukocyte adherence to the microvascular wall. This study investigates the effects of the oxygen radical scavenger tirilazad mesylate on leukocyte-endothelial interactions, macromolecular leakage, and microhemodynamics in mesenteric venules during endotoxemia. METHODS: Male Wistar rats were randomly allocated to receive tirilazad mesylate (group A, n = 10), its vehicle (group B, n = 10), or saline 0.9% (group C, n = 10) before a 120-minute infusion of endotoxin (2 mg/kg/hr). Furthermore, a control group without receiving endotoxin (group D, n = 10) was investigated. Leukocyte adherence, emigration of leukocytes, and macromolecular leakage were determined in postcapillary venules of the mesentery by using intravital videomicroscopy. RESULTS: During the administration of endotoxin the number of adherent leukocytes per square millimeter of vessel surface progressively increased in group B (baseline, 431 +/- 35 cells/mm2; 120 minutes, 1121 +/- 71 cells/mm2) and group C (baseline, 398 +/- 44 cells/mm2; 120 minutes, 1290 +/- 116 cells/mm2). In group A no increase in leukocyte adherence was observed after 120 minutes (baseline, 415 +/- 81 cells/mm2; 120 minutes, 638 +/- 87 cells/mm2). In control animals the leukocyte adherence remained unchanged (baseline, 347 +/- 41 cells/mm2; 120 minutes, 507 +/- 75 cells/mm2). After 120 minutes, tirilazad mesylate prevented the increase in leukocyte emigration observed in group B and C. Increased macromolecular leakage during endotoxemia (groups B and C) was not influenced by pretreatment with tirilazad. Tirilazad did not affect the decrease in red cell velocity, volumetric blood flow, and venular shear rate observed during endotoxemia. CONCLUSIONS: This study demonstrates inhibitory effects of tirilazad on endotoxin-induced leukocyte adherence and emigration, suggesting a potential therapeutic role for this substance in the prevention of sepsis-induced multiple organ failure.

Influence of steroids on microvascular perfusion injury of the bowel induced by extracorporeal circulation.

BACKGROUND: Extracorporeal circulation is associated with gastrointestinal complications. By means of intravital microscopic methods, we investigated whether preoperative treatment with steroids can attenuate the impairment of the bowel microcirculation. METHODS: In 20 pigs, a partial left heart bypass (pLHB) was established. A loop of the terminal ileum was exteriorized for intravital-microscopic observation. Seven sham-operated animals served as controls. In 13 animals, pLHB was established for 2 hours with a flow rate of 2,000 mL per minute; 7 of the animals received 20 mg/kg body weight prednisolone preoperatively. The microcirculatory network was analyzed before, during pLHB, and 2 hours after bypass. RESULTS: Despite unchanged macro-hemodynamics, pLHB resulted in a significant microvascular perfusion injury of the small bowel. Arteriolar vasoconstriction and a reduction of perfused capillaries per unit area (functional capillary density) to 30% of prebypass values could be found. Blood cell velocities were reduced in submucuous collecting venules. In the steroid-treated animals, the functional capillary density remained normal. In addition, arteriolar vasoconstriction could be prevented. CONCLUSIONS: Treatment with prednisolone largely prevents the microcirculatory alterations in the small bowel induced by extracorporeal circulation.

Cigarette smoke enhances ethanol-induced pancreatic injury.

Alcohol induces pancreatic ischemia, but the mechanisms promoting pancreatic inflammation are unclear. We investigated whether cigarette smoke inhalation is a cofactor in the development of ethanol-induced pancreatic injury. Cigarette smoke was administered to anesthetized rats alone or in combination with intravenous ethanol infusion. Control animals received either saline or ethanol alone. Pancreatic capillary blood flow and leukocyte-endothelium interaction in postcapillary venules were evaluated by intravital microscopy. Leukocyte sequestration was assessed by measurement of myeloperoxidase activity in pancreatic tissue, and pancreatic injury evaluated by histology. Ethanol decreased pancreatic blood flow progressively over 90 minutes (p < 0.001 vs. baseline), but neither leukocyte-endothelium interaction nor leukocyte sequestration was altered. Cigarette smoke alone reduced pancreatic blood flow temporarily (p < 0.01 vs. baseline) and increased leukocyte-endothelium interaction (roller p < 0.001, sticker p < 0.01 vs. baseline). Cigarette smoke potentiated the impairment of pancreatic capillary perfusion caused by ethanol, and both the number of rolling leukocytes and myeloperoxidase activity levels were increased compared to ethanol or nicotine administration alone (p < or = 0.05 and p < or = 0.01, respectively). This study demonstrates that ethanol induces pancreatic ischemia and that cigarette smoke leads to both temporary pancreatic ischemia and minimal leukocyte sequestration. Cigarette smoke potentiates the amount of pancreatic injury generated by ethanol alone. Smoking therefore seems to be a contributing factor in the development of alcohol-induced pancreatitis in the rat model.