Brigitte Vollmar

The Hepatic Microcirculation: Mechanistic Contributions and Therapeutic Targets in Liver Injury and Repair

The complex functions of the liver in biosynthesis, metabolism, clearance, and host defense are tightly dependent on an adequate microcirculation. To guarantee hepatic homeostasis, this requires not only a sufficient nutritive perfusion and oxygen supply, but also a balanced vasomotor control and an appropriate cell-cell communication. Deteriorations of the hepatic homeostasis, as observed in ischemia/reperfusion, cold preservation and transplantation, septic organ failure, and hepatic resection-induced hyperperfusion, are associated with a high morbidity and mortality. During the last two decades, experimental studies have demonstrated that microcirculatory disorders are determinants for organ failure in these disease states. Disorders include 1) a dysregulation of the vasomotor control with a deterioration of the endothelin-nitric oxide balance, an arterial and sinusoidal constriction, and a shutdown of the microcirculation as well as 2) an overwhelming inflammatory response with microvascular leukocyte accumulation, platelet adherence, and Kupffer cell activation. Within the sequelae of events, proinflammatory mediators, such as reactive oxygen species and tumor necrosis factor-alpha, are the key players, causing the microvascular dysfunction and perfusion failure. This review covers the morphological and functional characterization of the hepatic microcirculation, the mechanistic contributions in surgical disease states, and the therapeutic targets to attenuate tissue injury and organ dysfunction. It also indicates future directions to translate the knowledge achieved from experimental studies into clinical practice. By this, the use of the recently introduced techniques to monitor the hepatic microcirculation in humans, such as near-infrared spectroscopy or orthogonal polarized spectral imaging, may allow an early initiation of treatment, which should benefit the final outcome of these critically ill patients.

Leukocytes contribute to hepatic ischemia/reperfusion injury via intercellular adhesion molecule-1-mediated venular adherence*

BACKGROUND Leukocytes are suggested to modulate ischemia/reperfusion injury via membrane receptor-controlled interaction with the microvascular endothelium. METHODS With the use of intravital fluorescence microscopy we investigated the role of the intercellular adhesion molecule-1 (ICAM-1) in a rat model of hepatic reperfusion injury with a neutralizing monoclonal antibody (anti-ICAM-1). RESULTS Sixty minutes of left lobar ischemia and reperfusion (isotype-matched immunoglobulin G1 control antibody) caused leukostasis in sinusoids (240 +/- 15 cells per liver lobule), leukocyte adherence in postsinusoidal venules (679 +/- 76 cells per mm2 endothelial surface of postsinusoidal venules), nutritive perfusion failure (15% +/- 2% nonperfused sinusoids), excretory dysfunction (bile flow, 1.2 +/- 0.3 microliters.min-1.gm-1), and loss of hepatocellular integrity (serum aspartate aminotransferase, 1353 +/- 317 units.L-1; serum alanine aminotransferase, 1055 +/- 265 units.L-1). Anti-ICAM-1 did not affect sinusoidal leukostasis; however, it effectively inhibited postischemic leukocyte adherence to the venular endothelial lining (217 +/- 38 cells/mm2, p < 0.01). Concomitantly, hepatic reperfusion injury, including sinusoidal perfusion (6% +/- 1% nonperfused sinusoids, p < 0.01), excretory function (bile flow, 1.8 +/- 0.1 microliters.min-1.gm-1, p < 0.05), and hepatocellular integrity (aspartate aminotransferase, 480 +/- 108 units.L-1; alanine aminotransferase, 447 +/- 80 units.L-1, p < 0.05), was significantly ameliorated by anti-ICAM-1. CONCLUSIONS These findings prove in vivo the pivotal role of ICAM-1 in leukocyte-dependent manifestation of postischemic liver damage.

Surgical trauma: hyperinflammation versus immunosuppression?

BackgroundExperimental and clinical studies have brought evidence that surgical trauma markedly affects the immune system, including both the specific and the non-specific immune response.Materials and methodsThis report reviews the present knowledge on the mechanisms of surgical trauma-induced immune dysfunction and outlines experimental and clinical approaches to find effective treatment strategies.ResultsMajor surgical trauma induces an early hyperinflammatory response, which is characterized by (1) pro-inflammatory tumour necrosis factor alpha (TNF), interleukin (IL)-1, and IL-6 cytokine release and (2) neutrophil activation and microvascular adherence, as well as (3) uncontrolled polymorphonuclear (PMN) and macrophage oxidative burst. The massive and continuous IL-6 release induces an acute phase response, but, more importantly, also accounts for the up-regulation of major anti-inflammatory mediators, such as prostaglandin (PG) E2, IL-10 and transforming growth factor (TGF)-ß. This results in surgical, trauma-induced, immunosuppression, as indicated by (1) monocyte deactivation, reflected by the lack of monocytic TNF- production upon lipopolysaccharide (LPS) stimulation, and (2) a shift of the Th1/Th2 ratio towards a Th2-dominated cytokine pattern. The imbalance between pro-inflammatory and anti-inflammatory cytokines and immuno-competent cells determines the phenotype of disease and should help the physician to compose the therapeutic strategy. In fact, recent clinical studies have shown that both the initial uncontrolled hyperinflammation and the continued cell-mediated immunosuppression represent primary targets to counteract post-surgery immune dysfunction. The balance between inflammatory and anti-inflammatory forces may be restored by interferon gamma (IFN-γ) to counteract monocyte deactivation; the anti-inflammatory PGE2 may be inhibited by indomethacin to attenuate immunosuppression; or the initial hyperinflammation may be targeted by administration of anti-inflammatory substances, such as granulocyte colony-stimulating factor (G-CSF), hydoxyethyl starch, or pentoxifylline.ConclusionsWhen drawing up the therapeutic regimen the physician should not consider hyperinflammation versus immunosuppression, but hyperinflammation and immunosuppression, aiming at restoring an appropriate mediator- and immune cell-associated balance.

Systemic and Regional Hemodynamics of Isoflurane and Sevoflurane in Rats

The authors studied the effects of sevoflurane and isoflurane on systemic hemodynamics and regional blood flow distribution (microsphere technique) in 15 rats during general anesthesia with intravenous chloralose and controlled ventilation. Inhaled anesthetics were applied to reduce mean arterial blood pressure (MAP) to 70 mm Hg (1.66 vol% sevoflurane and 0.96 vol% isoflurane) and 50 mm Hg (MAP 50; 3.95 vol% sevoflurane and 2.43 vol% isoflurane). Control recordings were obtained with intravenous chloralose only. At a MAP of 70 mm Hg, both anesthetics reduced heart rate, cardiac output, and systemic vascular resistance to a similar degree. Isoflurane decreased systemic vascular resistance markedly at a MAP of 50 mm Hg and thereby maintained cardiac output at higher levels than sevoflurane. The left ventricular rate-pressure product decreased comparably with both anesthetics. Cerebral blood flow increased dose-dependently with both inhaled anesthetics but to a greater degree with isoflurane. Total hepatic blood flow remained unchanged from control at a MAP of 70 mm Hg but decreased at a MAP of 50 mm Hg. This was due to reductions of hepatic arterial and portal venous tributaries. Renal blood flow was reduced with only the high concentrations of the anesthetics. Myocardial blood flow was reduced at all concentrations of volatile anesthetic; however, the decrease was less with isoflurane. This would indicate a more pronounced coronary vasodilation by isoflurane as the rate-pressure product, as a measure of the actual left ventricular oxygen demand, decreased by comparable degrees with both anesthetics. Our results indicate that sevoflurane and isoflurane (each approximately 0.7 MAC) have no dissimilar systemic and regional hemodynamic effects at a MAP of 70 mm Hg in this animal model. At higher concentrations (approximately 1.7 MAC), cerebral blood flow was more with isoflurane than with sevoflurane and was associated with a more pronounced vasodilation in the myocardium.

Is the intravascular administration of mesenchymal stem cells safe?: Mesenchymal stem cells and intravital microscopy

We investigated the kinetics of human mesenchymal stem cells (MSCs) after intravascular administration into SCID mouse cremaster vasculature by intravital microscopy. MSCs were injected into abdominal aorta through left femoral artery at two different concentrations (1 x 10(6) or 0.2 x 10(6) cell). Arterial blood velocity decrease by 60 and 18% 1 min after high/low dose MSCs injection respectively. The blood microcirculation was interrupted after 174+/-71 and 485+/-81 s. Intravital microscopy observation and histopathologic analysis of cremaster muscles indicated MSCs were entrapped in capillaries in both groups. 40 and 25% animals died of pulmonary embolism respectively in both high and low MSCs dose groups, which was detected by histopathologic analysis of the lungs. Intraarterial MSCs administration may lead to occlusion in the distal vasculature due to their relatively large cell size. Pulmonary sequestration may cause death in small laboratory animals. MSCs should be used cautiously for intravascular transplantation.

Microhemodynamic and cellular mechanisms of activated protein C action during endotoxemia.

ObjectiveTo characterize microcirculatory actions of activated protein C in an endotoxemia rodent model that allows in vivo studies of microvascular inflammation and perfusion dysfunction. DesignAnimal study using intravital microscopy. SettingAnimal research facility. SubjectsMale Syrian golden hamsters, 6–8 wks old with a body weight of 60–80 g. InterventionsIn skinfold preparations, endotoxemia was induced by intravenous administration of 2 mg/kg endotoxin (lipopolysaccharide, Escherichia coli). Intravital microscopy allowed quantitative analysis of arteriolar and venular leukocyte adhesion and functional capillary density (cm−1) that served as a measure of microvascular perfusion failure. Activated protein C (APC group, n = 8, 24 &mgr;g/kg intravenously) was substituted continuously during 8 hrs after lipopolysaccharide, whereas endotoxemic buffer-treated animals (control, n = 7) served as controls. Measurements and Main ResultsLipopolysaccharide increased leukocyte adhesion and decreased functional capillary density to 50% of baseline values (p < .01 vs. baseline). Activated protein C treatment inhibited (p < .05) lipopolysaccharide-mediated leukocytic response and attenuated (p < .05) endotoxic perfusion failure in nutritive capillaries. ConclusionsActivated protein C-induced protection from lipopolysaccharide-mediated microcirculatory dysfunction was characterized in vivo for the first time. The impressive modification of leukocyte cross-talk indicates systemic anti-inflammatory activated protein C effects on leukocytes and the endothelium, subsequently improving capillary perfusion. These actions could represent the in vivo mechanism of activated protein C interactions observed in patients with severe sepsis.

Viewing the Microcirculation through the Window: Some Twenty Years Experience with the Hamster Dorsal Skinfold Chamber

Intravital microscopy represents a sophisticated technique to study the microcirculation in health and disease. While most preparations used for those studies are acute in nature, the use of chamber preparations in the skinfold bear the advantage to allow for chronic studies with repeated analysis of the microcirculation over a prolonged period of time. The skinfold chamber model for microcirculatory analysis has been adapted to mice, rats and hamsters. Although the use of rats and, in particular, the use of mice has the advantage of the availability of species-specific tools, the use of the hamster as the experimental animal may be preferred due to anatomical reasons, which facilitate the microsurgical preparation and improve the quality of microscopic imaging. The use of the hamster dorsal skinfold chamber, firstly described by Endrich and coworkers in 1980, has brought out during the last two decades a considerable number of experimental studies within the fields of microcirculation physiology, inflammation and sepsis, ischemia-reperfusion, angiogenesis, and transplantation, indicating that the model has to be considered a versatile tool to study the microcirculation in health and disease.

Capillary dysfunction in striated muscle ischemia/reperfusion: on the mechanisms of capillary "no-reflow".

The major dysfunction of capillaries after prolonged periods of ischemia is the lack of re-establishment of nutritive blood flow upon onset of reperfusion, i.e., capillary no-reflow. Several mechanisms have been proposed to cause capillary no-reflow, including intravascular hemoconcentration and thrombosis, leukocyte plugging, endothelial cell swelling, vasomotor dysfunction, and interstitial edema formation. Electron microscopic studies suggest that thrombus formation and intravascular clotting are not significant mechanisms. Moreover, intravital microscopic studies have demonstrated that plugging of capillaries by leukocytes is not a primary cause for the manifestation of no-reflow in postischemic striated muscle. In contrast, both in vivo studies and histological examinations support the concept that ischemia/reperfusion induces the disruption of the endothelial integrity with loss of fluid to endothelial cells and the interstitial space. As a consequence, these pathological sequelae are associated with intravascular hemoconcentration, endothelial cell swelling and interstitial edema formation, which contribute to capillary lumenal narrowing, increase of hydraulic resistance, and, thus, impairment of perfusion. Whether the postischemic diameter response with dilation of reperfused capillaries and lumenal narrowing of no-reflow capillaries involves endothelin/nitric oxide-triggered capillary pericyte function remains to be determined.

Intestinal ischemia/reperfusion: microcirculatory pathology and functional consequences

BackgroundIntestinal ischemia and reperfusion (I/R) is a challenging and life-threatening clinical problem with diverse causes. The delay in diagnosis and treatment contributes to the continued high in-hospital mortality rate.ResultsExperimental research during the last decades could demonstrate that microcirculatory dysfunctions are determinants for the manifestation and propagation of intestinal I/R injury. Key features are nutritive perfusion failure, inflammatory cell response, mediator surge and breakdown of the epithelial barrier function with bacterial translocation, and development of a systemic inflammatory response. This review provides novel insight into the basic mechanisms of damaged intestinal microcirculation and covers therapeutic targets to attenuate intestinal I/R injury.ConclusionThe opportunity now exists to apply this insight into the translation of experimental data to clinical trial-based research. Understanding the basic events triggered by intestinal I/R may offer new diagnostic and therapeutic options in order to achieve improved outcome of patients with intestinal I/R injury.

Hydroxyethyl starch (130 kD), but not crystalloid volume support, improves microcirculation during normotensive endotoxemia.

Background Increased leukocyte–endothelial cell interaction (LE) and deterioration of capillary perfusion represent key mechanisms of septic organ dysfunction. The type of volume support, however, which may be used during septic disorders, remains controversial. Using intravital microscopy, the authors studied the effect of different regimens of clinically relevant volume support on endotoxin-induced microcirculatory disorders, including the synthetic colloid hydroxyethyl starch (HES, 130 kD) and a crystalloid regimen with isotonic saline solution (NaCl). Methods In Syrian Golden hamsters, normotensive endotoxemia was induced by intravenous application of Escherichia coli lipopolysaccharide (LPS, 2 mg/kg). The microcirculation was analyzed in striated muscle of skinfold preparations. HES 130 kD (Voluven®, 16 ml/kg, n = 7) or isotonic saline (NaCl, 66 ml/kg, n = 6) were infused 3 h after LPS exposure over a 1-h period (posttreatment mode). Animals receiving LPS without volume therapy served as control subjects (n = 8, control). LE, functional capillary density (FCD), and macromolecular leakage were repeatedly analyzed in the awake animals during a 24-h period using intravital fluorescence microscopy. Results HES 130 kD significantly reduced LPS-induced arteriolar and venular leukocyte adherence (P < 0.05), whereas NaCl resuscitation had no effect when compared with nontreated control animals. The LPS-induced decrease in FCD and increase in macromolecular leakage were also significantly attenuated by HES 130 kD but not by NaCl. Improvement of LPS-induced microcirculatory disorders by HES was unlikely the result of macro- and microhemodynamic changes because arterial blood pressure, heart rate, and venular wall shear rate did not differ between HES- and NaCl-treated animals. Conclusions Thus, our study provides microhemodynamic and cellular mechanisms of HES 130 kD-mediated protection on microcirculation during endotoxemia, even when used in a clinically relevant posttreatment mode during normotensive conditions.