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Intravital microscopy and FITC-labeled erythrocytes were used to investigate villous perfusion in the rat small intestine in a model of inflammatory bowel disease. Inflammation was induced with s.c. application of Indomethacin. It has previously been demonstrated that systemic Indomethacin leads to an increase in villous blood flow in the small intestine of the rat. In order to determine whether mast cell activation may contribute to the increase in villous perfusion, Ketotifen was used to inhibit mast cell degranulation. We found that Ketotifen significantly reduced villous perfusion in the inflamed intestine, but had no effect in the control group. We conclude that mast cell activation is one of the mechanisms leading to hyperemia in the mucosa of the small intestine in this animal model. Further studies are required to determine whether mast cell stabilizers may be beneficial in the treatment of inflammatory bowel disease in man.

Objective: To validate the safety of gadolinium-diethylenetriamine pentaacetic acid (GD-DTPA) by measuring its effect on pancreatic capillary perfusion and acinar injury in acute pancreatitis., Background: Contrast-enhanced computed tomography (CECT) is proposed as a gold standard for early evaluation of acute necrotizing pancreatitis. However, iodinated contrast media used for CECT have been shown in these circumstances to reduce pancreatic capillary flow and increase necrosis and mortality. Recent reports suggest that post-GD MRI provides images comparable to CECT in the assessment of severe acute pancreatitis., Methods: Necrotizing pancreatitis was induced in 14 Wistar rats by intraductal glycodeoxycholic acid (10 mM/L) and intravenous caerulein (5 microg/kg/h) over 6 hours. Intravital microscopic quantitation of pancreatic capillary blood flow was performed using fluorescein isothiocyanate-labeled erythrocytes after induction of pancreatitis and 30 and 60 minutes after an intravenous bolus of either Ringer's solution or GD-DTPA (0.2 mL/kg)., Results: The two study groups were comparable with regard to mean arterial pressure, heart rate, arterial blood gases, hematocrit, amylase, lipase, and trypsinogen activation peptide production throughout the experiment. GD-DTPA did not reduce capillary flow (1.93 +/- 0.05 nL/capillary/min) compared to animals infused with Ringer's solution (1.90 +/- 0.06 nL/capillary/min)., Conclusions: Intravenous injection of GD-DTPA does not further impair pancreatic microcirculation or increase acinar injury in acute necrotizing pancreatitis. Because of this advantage over CT contrast medium, further development of MRI as a staging tool in acute pancreatitis seems desirable.

Unlabelled: The interaction between immunocompetent cells and tumor-endothelium is essential for effective immunologic recognition. In the present study we evaluated resting and CD11b/CD18-mediated leukocyte adhesion on tumor-endothelium of experimental pancreatic carcinoma and in healthy pancreatic venules., Methods: 22 male Lewis rats (120-140 g) were anesthetized. Duct-like pancreatic carcinoma (DSL6A, Am. J. Pathol. 1993; 143:292) was induced by intrapancreatic implantation of tumor fragments between inert polymethylmetacrylat plates. After 4 wks the tumor-bearing pancreas was exposed and the microcirculation studied. Parameters in tumor vessels (15-40 microns) and healthy pancreatic collecting venules (20-40 microns) included: Erythrocyte velocity, Leukocyte adhesion, Vessel diameter and wall shear rate. Measurements were obtained before and 5 min after adding f-MLP (100 mM) or PAF (50 mM), two CD11b/CD18 agonists of different potency to the immersion chamber., Results: [table: see text], Conclusion: In experimental pancreatic carcinoma leukocyte adhesion of low affinity is reduced despite comparable wall shear rates. The CD11b/Cd18-mediated adhesion of high affinity, which is inducible by f-MLP and PAF in healthy pancreatic venules, is absent in tumor vessels. This may be a mechanism by which malignant tumors escape immune control.

In this study we investigated the effect of donor and recipient conditioning with N-acetylcysteine on the ischemia/reperfusion injury after experimental pancreas-transplantation. We performed standardized pancreaticoduodenal transplantation in male lewis rats. The pancreas was perfused with UW-solution, harvested and conserved at 4 degrees C. Cold ischemia time was 1.5 hours and 16 hours respectively. The microcirculation in the transplanted organ was quantified by means of intravital microscopy 1.5 hours after implantation and reperfusion in the recipient. After 16 hours of cold ischemia we found a significant reduction in capillary erythrocyte velocity and a significantly enhanced leucocyte/endothelium interaction. The treatment with N-acetylcysteine resulted in a significant improvement of these microcirculatory disorders after prolonged cold ischemia.

To determine if disturbances of the liver microcirculation may be of pathophysiological relevance for liver damage during acute biliary obstruction, we studied the effects of bile duct ligation (BDL) on hepatic microhemodynamics and leukocyte adhesion in rat liver in vivo. Male Wistar rats were subjected to BDL for 3 days and 7 days, respectively. Sham-operated controls underwent laparotomy without BDL. After 3 days, intravital fluorescence microscopy (IVM) and hydrogen gas (H2) clearance were performed to study hepatic microvascular perfusion. Furthermore, leukocyte-endothelial cell interactions were assessed by IVM. Intercellular adhesion molecule 1 (ICAM-1) protein expression was studied by Western blot analysis and tissue immunofluorescence after 3 and 7 days, respectively. Analysis of microvascular perfusion by IVM revealed a marked impairment of sinusoidal perfusion after 3 days. Assessment of H2 clearance confirmed that overall hepatic microvascular perfusion was decreased. In addition, increased leukocyte adhesion in sinusoids and venules could be observed. A concomitant increase of ICAM-1 expression in liver tissue was also noted within the first week after BDL. Our results show that BDL is followed by a marked depression of the hepatic microcirculation and increased leukocyte adhesion in vivo within 3 to 7 days. Together, these findings suggest that deficits in microvascular perfusion and increased neutrophil infiltration may represent a potential source of liver injury during acute biliary obstruction.

Background: Endotoxin activates leukocyte-endothelial cell adhesion, vascular leakage, and changes in vascular micro-hemodynamics. The aim of this study was to determine whether lidocaine, which inhibits the activation of leukocytes, could attenuate microcirculatory disturbances during endotoxemia., Methods: Thirty anesthetized male rats were randomly assigned to receive one of three treatments (n = 10 for each group): infusion of saline (control group), infusion of Escherichia coli endotoxin (LPS group: 2 mg x kg(-1) x h(-1) lipopolysaccharides) without lidocaine treatment, or infusion of endotoxin with lidocaine pretreatment 30 min before baseline measurements (lidocaine group: intravenous bolus of 2 mg/kg and continuous infusion of 2 mg x kg(-1) x h(-1)). Leukocyte adherence, erythrocyte velocity (V(RBC), and vessel diameters (Dv) were determined at baseline and at 60 and 120 min in mesenteric postcapillary venules using in vivo videomicroscopy. Macromolecular leakage was determined by measuring the extravasation of fluorescence-labeled albumin. Venular wall shear rate (tau) was calculated according to the equation tau = 8 x V(RBC) x Dv(-1)., Results: Lidocaine significantly attenuated the increase of leukocyte adherence during endotoxemia. There were no significant differences of tau within or between the groups. Macromolecular leakage exhibited the greatest increase in the LPS group. In the lidocaine group, it was significantly decreased but still increased compared with the control group., Conclusions: These results show that lidocaine attenuates endotoxin-induced alterations in leukocyte-endothelial cell adhesion and macromolecular leakage, which suggests that lidocaine may have a therapeutic role in preventing endothelial damage in sepsis.

Objective: To determine the influence of N-acetylcysteine on endotoxin-induced leukocyte-endothelial cell adhesion, vascular leakage, and venular microhemodynamics., Design: Randomized, blinded, controlled trial., Setting: Experimental laboratory., Subjects: Thirty male Wistar rats., Interventions: After pretreatment with N-acetylcysteine (150 mg/kg; n = 40; group A) or 0.9% saline solution (n = 10; group B) animals were given an intravenous infusion of endotoxin (Escherichia coli lipopolysaccharide 026:B6; 2 mg/kg/hr) over 120 mins. Animals in the control group (n = 10; group C) received a volume-equivalent infusion of 0.9% saline solution., Measurements and Main Results: Leukocyte adherence, red cell velocity (VRBC), vessel diameters, venular wall shear rate, and macromolecular leakage were determined in mesenteric postcapillary venules using in vivo videomicroscopy at baseline and at 30, 50, 90, and 120 mins after the start of the endotoxin challenge. Endotoxin exposure induced a marked increase in adherent leukocytes (group B: baseline, 391 +/- 24 cells/mm2; 120 mins, 1268 +/- 131 cells/mm2; p < .01). N-acetylcysteine pretreatment attenuated the adherence of leukocytes during endotoxemia (baseline, 366 +/- 28 cells/mm2; 120 mins, 636 +/- 49 cells/mm2; p < .01 vs. baseline; p < .01 vs. group B). Leukocyte adherence in control animals (group C) did not increase significantly. Administration of N-acetylcysteine did not influence the decrease in VRBC observed during endotoxemia. In group B1 VRBC decreased during the infusion of endotoxin from 2.0 +/- 0.2 mm/sec at baseline to 1.1 +/- 0.2 mm/ sec after 120 mins (p < .01 vs. baseline; p < .05 vs. group C), and in group A from 2.2 +/- 0.2 mm/sec to 1.1 +/- 0.1 mm/sec after 120 mins (p < .01 vs. baseline; p < .05 vs. group C). In group C, VRBC remained unchanged (baseline, 1.7 +/- 0.2 mm/sec; at 120 mins, 1.5 +/- 0.2 mm/sec). The venular diameters remained unchanged in all groups during the entire study period. After 120 mins, the venular wall shear rate decreased from 502 +/- 62 secs-1 at baseline to 272 +/- 46 sec-1 in group B (p < .01), and from 563 +/- 45 secs-1 at baseline to 283 +/- 31 secs-1 in group A (p < .01). No differences in venular wall shear rate were observed between these groups. In group C, the venular wall shear rate remained unchanged (baseline, 457 +/- 54 secs-1; at 120 mins, 409 +/- 51 secs-1). Macromolecular leakage, expressed as perivenular/intravenular fluorescence intensity after injection of fluorescence-labeled albumin, increased from 0.29 +/- 0.03 to 0.58 +/- 0.03 (p < .01) during the infusion of endotoxin in group B. In contrast, pretreatment with N-acetylcysteine diminished the extravasation of albumin (baseline, 0.27 +/- 0.01; at 120 mins, 0.37 +/- 0.02; p < .01 vs. baseline; p < .01 vs. group B)., Conclusion: These results demonstrate that N-acetylcysteine attenuates endotoxin-induced alterations in leukocyte-endothelial cell adhesion and macromolecular leakage, suggesting N-acetylcysteine might be therapeutic in the prevention of endothelial damage in sepsis.

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.

A strain of Yucatan micropigs is known to have heritable ventricular septal defects (VSDs) and thus may develop overflow pulmonary hypertension. Since inhaled nitric oxide (NO) selectively dilates pulmonary vessels, we determined its hemodynamic and co-agulatory effects in this new animal model. Eight Yucatan micropigs were anesthetized with midazolam, piritramide (a synthetic opioid) and vecuronium bromide. The presence and the size of the VSD were determined by using transesophageal color flow Doppler echocardiography. Four animals showed VSDs of 1-2 mm size. Inhaled NO was then administered with increasing inspired concentrations of 0, 5, 10, 20, 40, 80 and again 0 ppm NO for 10-min periods. NO inhalation did not affect heart rate, right cardiac output, mean arterial pressure, pulmonary arterial wedge pressure, or central venous pressure. Inhaled NO in animals with proven VSDs decreased pulmonary artery pressure (PAP) in a dose dependent manner; 5 ppm NO reduced mean PAP from 25 +/- 2.3 mm Hg to 18 +/- 0.8 mm Hg (p < 0.05), while pulmonary vascular resistance (PVR) decreased from 954 +/- 143 dyn.cm. s-5 to 661 +/- 88 dyn.cm.s-5 (p < 0.01) at the same dose. The maximum reduction in mean PAP and PVR occurred when 80 ppm NO was inhaled. Yucatan micropigs without VSDs did not respond hemodynamically to NO inhalation. Methemoglobin levels remained unchanged during the entire study. Platelet function was assessed according to the method of BREDDIN and BORN (BORN 1962). Initial aggregation and slope were affected when NO inhalation commenced. Yucatan micropigs with VSDs may represent a suitable model for further research of the in vivo effects of inhaled NO.

In this study we investigated the influence of N-acetylcysteine (NAC) on the hepatic microcirculation after warm ischemia by intravital fluorescence microscopy. Clamping of the left liver lobe was performed in 20 male Wistar rats for 70 min. The treatment group (n = 10) received 400 mg NAC/kg body weight 20 min prior to clamping. After reperfusion, acinar and sinusoidal perfusions were observed as well as the leukocyte-endothelium interaction. Phagocytic activity was assessed after application of latex beads. NAC reduced the number of nonperfused sinusoids in all acinar zones. A reduction in zone 1 (portal) was achieved from 15.5 to 7.1% (p < 0.0001), in zone 2 (midzonal) from 14.6 to 6.1% (p < 0.0001) and in zone 3 (central) from 11.9 to 2.9% (p < 0.0001). There were no significant differences in leukocyte adherence as well as in phagocytic activity detectable. We conclude that NAC improves hepatic microcirculation after warm ischemia by increasing sinusoidal blood flow.

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.

Intestinal mucosal hypoperfusion with subsequent ischemia during endotoxemia might cause a breakdown of the gut barrier with translocation of bacteria and their toxins into the systemic circulation, thus maintaining a "gut-derived" septic state. The aim of this study was to investigate the influence of endotoxin on the microcirculation of intestinal villi, which represent the most vulnerable part of the mucosa. The changes in blood flow and in the diameters of the central villus arterioles located in the distal ileum were monitored in control rats without lipopolysaccharide (LPS) exposure (n=7), and in rats receiving 1.5 mg/kg b.w. LPS (n=7) or 15 mg/kg b.w. LPS (n=7) over 60 min. The blood flow and the arteriolar diameters were determined using in vivo videomicroscopy at baseline, and 60 min and 120 min later. In control animals, no change in blood flow and arteriolar diameters were observed during the entire experiment. Administration of 1.5 mg/kg b.w. LPS reduced the blood flow to 69.5 +/- 9.0% of the baseline value at the end of the study period. This decrease in blood flow was associated with a decrease in the villus arteriolar diameters by 17.4 +/- 2.5% from the baseline values. In animals exposed to 15 mg/kg b.w. LPS, the decrease in villus blood flow at 60 min was 64.8 +/- 10.9% of baseline, and at 120 min 66.9 +/- 12.6% of baseline. The diameters of the villus arterioles were reduced by 11.5 +/- 2.4% and 15.1 +/- 1.7%, respectively. In the control group and in the 1.5-mg/kg LPS group, the mean arterial blood pressure did not change during the entire study period. In the 15-mg/kg LPS group, the mean arterial pressure tended to decrease after 60 min. These data suggest a reduction of villus blood flow due to vasoconstriction in the central villus arterioles during normotensive endotoxmia, which might represent the mechanism for the mucosal ischemia observed in critically ill patients.

The aim of this study was to investigate the influence of the platelet-activating factor (PAF) antagonist BN52021 on endotoxin-induced leukocyte-endothelium interactions and on venular microcirculation. The rates of leukocyte adherence and changes in red cell velocity, volumetric blood flow, vessel diameter, and venular shear rate were monitored in rat mesenteric venules following intravenous lipopolysaccharide (LPS) exposure (15 mg/kg body wt). The experiments were performed in LPS-treated control animals and in animals pretreated with the PAF receptor antagonist BN52021. LPS exposure induced a marked increase in adherent leukocytes in the control group compared to the BN52021-pretreated group. Thirty minutes after LPS injection, 6.1 +/- 0.8 leukocytes were adherent to 100 microns of venule in the control group compared to 2.5 +/- 0.5 in BN52021-pretreated animals (P < 0.01). The increased leukocyte adherence in the control group was accompanied by leukocytopenia. Red cell velocity, volumetric blood flow, vessel diameter, and venular shear rate did not differ between groups, indicating that increased leukocyte adherence in the control group was not due to diminished hydrodynamic dispersal forces. The attenuation of leukocyte adherence by the PAF receptor antagonist BN52021 suggests that PAF is involved in the mediation of the initial process of leukocyte sticking to the endothelium after LPS stimulation.

Objectives: To determine the influence of ketamine on endotoxin-induced leukocyte adherence and venular microhemodynamics., Design: Randomized, controlled trial., Setting: Experimental laboratory., Subjects: Thirty male Wistar rats., Interventions: The rats were pretreated with ketamine (10 mg/kg iv) or 0.9% saline, and both groups were given endotoxin (Escherichia coli lipopolysaccharide; 5 mg/kg iv). The control group received two doses of 0.9% saline., Measurements and Main Results: The rates of leukocyte adherence and changes in microhemodynamics were monitored in rat mesenteric venules, using in vivo video microscopy. The number of adherent leukocytes was determined on-line in 10-min intervals from 60 mins before until 2 hrs after endotoxin administration. Venular diameters, red blood cell velocity, volumetric blood flow, and the venular wall shear rate were monitored before and at 10, 30, and 60 mins after endotoxin exposure. A 6.3-fold increase in the number of adherent leukocytes was observed 10 mins after administration of endotoxin when compared with control animals (5.87 +/- 0.69 vs. 0.93 +/- 0.21 adherent cells/100 microns; p < .001). This increase remained unchanged for 120 mins. In ketamine-pretreated rats, a 2.6-fold increase in leukocyte adherence occurred during the first 20 mins after endotoxin exposure (2.40 +/- 0.46 vs. 0.93 +/- 0.21 adherent cells/100 microns; p < .01). However, no difference in the number of adherent leukocytes between ketamine-pretreated and control animals was found after this 20-min period. In animals of the control group, no increase in leukocyte adherence occurred during the entire observation time. Diameters of mesenteric venules did not change after endotoxin exposure in any of the groups. Red blood cell velocity and venular blood flow in the endotoxin-treated groups decreased 10 mins after the injection of endotoxin when compared with controls, but these values did not show any difference when they were compared between ketamine and saline-pretreated animals. Similarly, venular wall shear rate in the endotoxin-treated groups decreased 10 and 30 mins after injection of endotoxin. However, no significant difference occurred between ketamine and saline-pretreated animals., Conclusions: Pretreatment with ketamine attenuates endotoxin-induced leukocyte adherence by a shear rate-independent mechanism, suggesting reduced expression of adhesion molecules. These results indicate that ketamine exerts an anti-inflammatory effect, which might be beneficial in septic patients.

Characterization of pancreatic capillary blood flow by in vivo microscopy has been limited by technical shortcomings associated with the use of plasma tracers and the lack of quantitative data. Therefore, fluorescent-labeled erythrocytes were evaluated for quantitation of pancreatic capillary blood flow in rats in physiological state and under defined conditions of increased and impaired pancreatic blood flow. Physiological blood flow was 1.21 +/- 0.06 nl/min per capillary with stable capillary perfusion pattern. Reduction of pancreatic blood flow by decreasing systemic arterial pressure to 60 mmHg through controlled hemorrhage produced a profound decrease of volumetric flow to 0.21 +/- 0.05 nl/min (P < 0.05). The number of perfused capillaries was reduced to 52 +/- 8% of baseline (P < 0.001) and the intermittent perfusion pattern was altered in 26 +/- 5% (P < 0.001) of observed capillaries. Stimulation of pancreatic perfusion with intravenous secretin (5 CU/kg/hr) induced a transient decline to 0.94 nl/min (P < 0.05) followed by a continuous increase to 2.39 nl/min (P < 0.001). The intermittent flow pattern was modified in 15 +/- 3% of capillaries (P < 0.05). Fluorescent-labeled erythrocytes provide unique and reliable qualitative and quantitative data about pancreatic microcirculatory changes. Confinement of the fluorescent tracer to erythrocytes prevents extravasation and minimizes phototoxicity, thereby improving intravital analysis of blood flow in pancreatic exchange capillaries.

Aim: Usually, in-situ preparation of the hepatic hilar structures is performed prior to the perfusion with preservation solution. Aim of this study was to investigate mechanical effects of liver preparation on the hepatic microcirculation., Methods: 16 pigs (German landrace) were randomized in two groups. In both groups, laparotomy was performed after intratracheal intubation. Subsequently, a thermal diffusion probe was implanted into the medial left liver lobe for quantification of microperfusion. In group A (n = 8), bile duct, hepatic artery, and portal vein were exposed and the lesser omentum transsected thereafter. Ultrasound-volume-probes were placed around the hepatic artery and portal vein. Simultaneous measurement of hepatic microperfusion and total liver blood flow was performed five minutes after the end of liver preparation. In group B (n = 8) hepatic microperfusion was quantified 45 minutes after laparotomy without further manipulations., Results: By the preparation, liver perfusion was significantly reduced in group A from 78 +/- 13 ml/100g/min to 61 +/- 16 ml/100g/min. After preparation a total liver blood flow of 137 +/- 46 ml/100g/min was recorded indicating a shunt fraction of 51 +/- 21%. In contrast, hepatic microperfusion in group B remained at baseline during the whole observation period (79 +/- 3 ml/100g/min vs. 78 +/- 5 ml/100g/min)., Conclusion: In-situ liver preparation induces a relevant disturbance of hepatic microcirculation. Preservation perfusion shortly after surgical manipulation could become ineffective because of an increase in shunt flow. If the regeneration period is too short, e.g. lack of heart explantation, the quality of the liver graft could be limited.

Over a 30-month period, 60 patients (30 in each group) suffering from end-stage liver disease or primary hepatic malignancy and scheduled for liver transplantation were enrolled in a prospective, randomized study to compare two methods of liver preservation: histidine-tryptophan-ketoglutarate (HTK) solution versus University of Wisconsin (UW) solution. Entry criteria for both groups were: age (18-65 years), elective surgery (transplantable or urgent category of the recipients), first transplantations and harvesting procedure performed by the same team. The parameters under investigation were the clinical and laboratory data pre- and post-transplantation, as well as follow-up data such as complications and survival. There were no significant differences in the two groups as far as the evaluation criteria were concerned, even when cold ischemia time was more than 15 h (n = 7). A slight, yet not significant, increase in late complications of the biliary anastomoses could be seen in the UW group. Hepatocellular injury (SGOT, SGPT, GLDH, lactate) appeared to be more marked in the HTK group. These results suggest that both HTK and UW solutions are appropriate for clinical use in liver transplantation, even if cold ischemia time is more than 15 h.

This study was undertaken in order to obtain information on the mode of reaction of the contractile apparatus after different forms of cardiac arrest, global ischemia and reperfusion, as well as on possible correlations between the contraction state of myofibrils and biochemical parameters. During the survival time, before the level of 3 mumol/gww creatine phosphate (CP) is reached, the contraction state shows only minor changes. During the revival time in which ATP tissue concentrations decay to 4 mumol/gww, the contribution of ATP, lactate, anorganic phosphate (Pa) and acidosis to the degree of relaxation depends on the method of cardiac arrest. At defined biochemical values, the degree of relaxation is comparable after aortic cross clamping (ACC) and St. Thomas perfusion, but significantly different compared to HTK perfusion. Thus, during the revival time, the relaxation of sarcomeres depends predominantly on the composition of the solutions used for cardiac arrest. The re-entry of contraction below 3 mumol/gww ATP is correlated with the ATP concentration, independent of the form of cardiac arrest. Reperfusion after HTK or St. Thomas cardioplegia and reversible ischemia leads to the focal formation of contraction bands, which do not occur during ischemia. This contraction state is significantly more pronounced after reperfusion of St. Thomas arrested hearts. Thus, the contraction state of myofibrils is influenced not only by alterations in metabolite concentrations, but also by the composition of cardioplegic solutions and by the characteristic conditions (sufficient energy, oxygen and Calcium) during reperfusion.

In 22 human donor livers the measurement of the non-invasive bioelectrical impedance was performed prospectively to evaluate the degree of tissue damage sustained during cold ischemia. The results of the measurement were correlated with liver function, the method of organ preservation and the period of ischemia. The impedance was measured in vivo as 620 ohm (at 192 Hz), the phase angle as -7.4 degrees (at 5 kHz). The results were compared with the data obtained from 72 patients who underwent elective laparotomies. The 22 donor livers were studied further during ischemia. The method was found to be a reliable way of detecting severe damage to the hepatocytes during the cold ischemia.

Ischemia causes changes in organ tissue (e.g. during operation or transplantation) which may finally lead to irreversible injury, so that the organ can no longer be resuscitated. To the extent that these changes affect the electrical properties of the tissue they are manifested in the impedance spectrum. As an example, the course of impedance of a HTK-protected porcine liver is presented in the frequency range of 0.1 Hz to 10 MHz, which includes two dispersion--alpha- and beta-dispersion. Using a suitable electrical equivalent circuit analogue to the structure of the liver, the behavior of the alpha- and beta-dispersion is explained on the basis of gap junction closure and narrowing of the extracellular space due to cell swelling.

The influence of glycogen content, temperature, and Euro Collins (EC) solution on membrane potential (Vm) and intracellular sodium activity (aNai) were measured in cells of superfused porcine liver slices by means of double-barrelled ion-sensitive microelectrodes. Vm was -26.1mV in fasted pigs and -20.6mV after glucose feeding, when measured in HEPES-buffered solution (P less than 0.0001). aNai was not measurably affected by glucose feeding. During superfusion with Tyrode solution, lowering the temperature from 35.5 degrees C to 15.5 degrees C led to a fast Vm decrease of roughly 2mV followed by an increase of 1-3mV. At the same time, aNai increased from 12.8 to 18.2mM within 10 min. Superfusion with EC solution for 10 min caused comparable changes in fed and fasted pigs. Vm depolarized at either temperature by about 16mV. At 35.5 degrees C the initial aNai of 17.5mM was roughly halved, whereas at 15.5 degrees C it decreased from 21.0 to 14.3mM. The results suggest that the nutritional state markedly affects the electric properties of liver. However, the effect on membrane potential of high-potassium organ-protective solutions seems to be distinctly more pronounced. Moreover, cellular Na+ activity decreases in consequence of an extracellular Na+ reduction with protective solutions, which might be balanced to some extent by a simultaneous temperature decrease.

During open heart surgery, reperfusion-induced arrhythmias arising after short periods of ischaemia may originate from subendocardial Purkinje fibres. We investigated the ultrastructure of these fibres during 30 min of global ischaemia at 25 degrees C. The effects both with myocardial protection (HTK cardioplegia) and without it (pure ischaemia) were compared qualitatively and morphometrically. After 30 min pure ischaemia overcontraction of sarcomeres, hypercontraction and contraction bands, together with considerable changes in organelles, predominate over cellular oedema. In Purkinje fibres, both cellular and mitochondrial swelling were significantly increased within this 30-min time period from the onset of pure ischaemia. In contrast, following HTK cardioplegia and 30 min ischaemia, cellular and mitochondrial swelling remain moderate and over-contractions are almost entirely lacking. This means that despite remarkable differences between pure ischaemia and HTK cardioplegia in the degree of protection attained it is clear that, compared with the working myocardium, subendocardial Purkinje fibres do not display a higher resistance to early global ischaemia. Further investigations of this sensitivity of Purkinje fibres to global ischaemia and certain drugs may bring about new insights into myocardial protection and pharmacotherapy of arrhythmias.

The pathophysiological fundamentals of the tolerance of the heart to the ischemic condition are discussed, with special reference to three contributions in Issue 1/1990 of this journal. The relationship 'duration of/damage done by' ischemia is of sigmoidal form. The time needed to recover from the damage caused is dependent on the extent of the damage and not on the duration of ischemia. This time is thus a measure of the damage caused. The two main means of reducing this damage are cardioplegia and hypothermia. The fundamental differences in the various cardioplegic methods, and the factors on which their effectiveness depends, are explained. The background to the use of hypothermia and the limits in its application are presented. The dependence on hypothermia to extend the useful duration of ischemia demands, however, a careful consideration of the physiological thermodynamics involved, when estimating the probable extent of damage reached at any time.

The cardiac conduction system is considered to be particularly resistant to ischaemia. Nevertheless, following open heart surgery with short periods of ischaemia disturbances in AV conduction or ventricular arrhythmia have been reported. We compared the ultrastructure of AV node and working myocardium following 30 min global ischaemia at 25 degrees C, during pure ischaemia and with HTK cardioplegia qualitatively and morphometrically. After 30 min of pure ischaemia, interstitial and intracellular oedema together with considerable changes in organelles in AV nodes predominate over mainly cellular oedema in working myocardium. Sometimes irregular overcontractions of sarcomeres occur in the AV node, though very seldom in working myocardium. In pure ischaemia, mitochondrial swelling is comparable in both types of tissue. Following HTK cardioplegia and 30 min ischaemia, cellular oedema and mitochondrial swelling are significantly reduced in AV nodal cells and working myocardium, but remain more extensive in the AV nodes. Irregularities in the contractile state of sarcomeres are not observed. The extent of the ultrastructural alterations corresponds to the degree of metabolic change in the working myocardium. Thus, despite considerable differences during pure ischaemia and HTK cardioplegia, ultrastructurally the AV nodal cells do not display a greater resistance to ischaemia than working myocardium.

Cellular changes occurring in the left ventricular myocardium during ischaemia after different methods of cardiac arrest have been evaluated by morphological and morphometric parameters: volume densities of mitochondria (VVMi), sarcoplasm (VVSp), myofibrils (VVMf), surface densities of mitochondria (SVMi). The surface to volume ratio of mitochondria (SVratioMi) has been used as an independent parameter of mitochondrial swelling. Since ischaemic swelling of myocardial cells increases the volume of the reference space and ischaemic swelling of mitochondria decreases the free sarcoplasm, VVMi and VVSp cannot be considered as reliable indicators of the degree of oedema. SVMi/VVMf remains nearly constant after different forms of cardiac arrest, demonstrating the integrity of mitochondrial outer membranes. The inverse linear ratio between SVratioMi and the mean mitochondrial volume indicates that the increase in mitochondrial volume is achieved by surface smoothing. Loss of matrix structure and fragmentation of cristae occur at an SVratioMi of about 5.8, cristolysis at 5.5 to 5.6 and amorphous matrix densities at an SVratioMi of less than 5.5 micron2/micron3. The SVratioMi is a suitable parameter for evaluating mitochondrial swelling both at the onset and during global myocardial ischaemia, independent of the method of cardiac arrest used. It serves as an indicator of the state of structural preservation of mitochondria during ischaemia.

In this study, the interstitial space and myocytes were investigated qualitatively and morphometrically in samples from beating, fibrillating as well as from cardioplegically HTK-arrested hearts fixed by immersion or perfusion. The size of tissue clefts separating bundles of myocytes and that of the interstitial space within bundles of myocytes depend on the functional state and on the kind of fixation. Cellular preservation is significantly better in HTK-arrested hearts compared to beating or fibrillating hearts. Thus, for the structural evaluation of myocytes and interstitium, the pretreatment constitutes a highly significant factor.

Contraction bands usually occur in the intramural working myocardium following post-ischaemic reperfusion. In the subendocardium, however, they are found during ischaemia. Thus, we ascertained the contraction states of Purkinje fibres, transitional cells, subendocardial and intramural parts of the working myocardium during 30 min global ischaemia at 25 degrees C. The effects with and without myocardial protection were compared. At the onset of pure ischaemia contraction bands are completely lacking in all cell types. During pure ischaemia contraction bands are found in all subendocardial cell types but not in the intramural working myocardium. A peak of pathological contraction states is found in the intramural working myocardium at the onset (0 min), in the subendocardial working myocardium at 10 min, in the transitional cells and Purkinje fibres at 30 min of pure ischaemia. Histidine-, tryptophan-, ketoglutarate-enriched (HTK) cardioplegia prevents contraction bands completely at the onset of ischaemia and prevents both contraction bands and pathological contraction states during ischaemia almost completely. Striking differences in the physiological contraction states are seen only in the working myocardium: HTK cardioplegia brings about dominance of relaxation during ischaemia. These findings may be due mainly to the effects of global ischaemia on the one hand and to catecholamines, calcium and oxygen on the other.

The influence of pre-ischemic treatment with propranolol (0.8 mg/kg bw) or with verapamil (0.1 mg/kg bw) on myocardial O2 consumption and on coronary resistance after cardioplegic arrest (300 minutes at 22 degrees - 24 degrees C) was investigated, using a Langendorff technique in open chest experiments (canine hearts). For myocardial protection histidine buffered Bretschneider solution was used, while for standardization all hearts were postischemically reperfused with a modified Tyrode solution at 35 degrees C. Both drugs were given intravenously in two fractions, 60 and 30 minutes before the onset of cardioplegic perfusion; a third group was not pretreated. Although the verapamil group had the lowest O2 consumption post-ischemically, the most impressive results were found in coronary resistance. Propranolol significantly diminished coronary resistance (p less than 0.05), while verapamil distinctly enhanced it compared to the non treated group. The "membrane labilizing" effect of verapamil combined with calcium-free cardioplegic solution was confirmed by a massive postischemic myocardial sodium uptake.

The effects of the cardioplegic solution HTK on membrane potential (EM) and intracellular K and Na activities (aiK, aiNa) were studied in sheep cardiac Purkinje fibres by means of conventional and ion-selective microelectrodes. HTK contains (mM): Na 15, K 10, Ca 0, Mg 4, histidine 180. (1) In control conditions EM was -74.3 +/- 3.3 mV (n = 25), aiK was 116.4 +/- 4.1 mM (n = 7) and aiNa was 8.2 +/- 1.4 mM (n = 15). (2) Exposure to HTK led to a depolarization to -59.7 +/- 3.6 mV (n = 25) which exceeded by about 5-7 mV that induced in a Tyrode solution of 10 mM K and in a modified HTK solution supplemented by 2 mM Ca (n = 6). (3) Addition of 0.5 mM barium eliminated the difference in the steady-state depolarization. (4) HTK superfusion increased aiK to 120.1 +/- 4.4 mM (n = 7) and decreased aiNa to 3.9 +/- 0.9 mM (n = 15). (5) The decrease in aiNa was insensitive to amiloride (1 mM) and to external alkalization but was slightly increased by addition of 2 mM calcium. (6) When the calcium in Tyrode solution was lowered from 2.0 mM to 0.05 mM, aiNa hardly decreased during subsequent exposure to unmodified HTK and it increased in the presence of 0.1 mM dihydroouabain. We propose the hypothesis (1) that the difference in membrane depolarization between HTK and a 10 mM K-Tyrode is caused by a decrease in K conductance by the HTK solution and (2) that the aiNa decline mainly results from a coupled Ca influx via Na-Ca exchange due to a delayed washout of external calcium.

Unlabelled: During myocardial ischemia the phase angle phi of the complex electric impedance of myocardial tissue at 5 kHz AC exhibits a characteristic behaviour, the progress of which depends on the cardioplegic method applied. By extending the frequency range to 200 Hz and 10 MHz and by analyzing in addition to phase and magnitude also real and imaginary part of the impedance it was possible to elucidate which ischemic changes in the myocardium are responsible for the course of phi (5 kHz). This method we call impedance spectroscopy. Canine hearts were cardioplegically perfused with either the standard solution HTK[4] or the solution HTK[4] + 50 mumol/l Ca++. During the following ischemia at 25 degrees C energy-rich phosphate level, the ultrastructure, the real part, imaginary part and phase angle of the impedance between 200 Hz and 10 MHz were analyzed., Results: phi (5 kHz) displays very similar characteristics during the ischemic period to those of the real part of the impedance at 200 Hz, Re (200 Hz). Re (200 Hz) increases, when--according to electron microscopic findings--an intracellular myocardial edema begins to develop. The changes of Re(200 Hz) are always smaller, however, than those of phi (5 kHz). This indicates that phi (5 kHz) increases in the course of ischemia not only as a consequence of confinement of the extracellular space by myocardial cellular edema but also because of changes of passive electrical characteristics of the myocardial cell membranes.(ABSTRACT TRUNCATED AT 250 WORDS)