Review
B. van der Hoven, D. Gommers, and J. Bakker
Introduction
One of the most important goals of therapy in critically ill patients is restoring and maintaining adequate perfusion and oxygenation of vital organs in the recovery from a variety of disruptive processes, such as circulatory failure in myocardial infarction, sepsis, and trauma. The gastrointestinal tract is generally regarded as sig- nificant in the development of shock and multiple organ failure (MOF) as a conse- quence of loss of its barrier function against luminal bacteria and bacterial prod- ucts, such as endotoxin in hypoxic conditions. Insufficient blood flow to the splanchnic organs is believed to be the essential mechanism [1]. Translocation of bacteria and endotoxin to the lymphatic and portal system is a first step towards distant organ damage. The gut and liver macrophages (Kupffer cells) are important as a first barrier against spread of translocated bacteria and endotoxins to the bloodstream.
The autoregulation of microcirculatory blood flow distribution in the splanchnic area is compromised under septic or shock conditions. Regional blood flow distri- bution is marred and changes under normal circumstances are not reflected in con- ditions of sepsis and septic shock. The flow distribution to the mucosa is decreased in sepsis while the general splanchnic flow remains largely intact [2]. Blood flow dis- tribution and regional perfusion has to adapt to local metabolic demand. Because the diffusion of oxygen in tissues is limited, nutritional delivery has to be through a dense, finely distributed network of capillaries. This capillary network is generally regarded as vessels smaller than 300 µm in diameter and comprises the largest endo- thelial surface area ( 8 0.5 km2).
Hiltebrand et al showed that the microcirculatory blood flow in the splanchnic organs is remarkably heterogeneous both in early, hypodynamic shock as well as later in hyperdynamic shock and cannot be predicted based upon general changes in systemic or even regional blood flow [3]. So, monitoring the microcirculation seems to be the answer to the question of how we could identify patients at risk of developing further organ system failure and how to evaluate our therapeutic inter- ventions [4].
Unlike other organs, the microcirculation of the liver receives blood from two types of afferent vessels: The portal system and the hepatic artery. Both portal venous and hepatic arterial systems are obviously not routinely accessible for flow or other forms of direct measurement except at laparotomy. The aim of this chapter is to highlight several aspects of the hepatic microcirculation and oxygenation, and what changes occur during sepsis or other critical illness conditions.
Characteristics of Hepatic Anatomy
Together with the small bile ductuli culminating in the common bile duct, the termi- nal hepatic venules and the terminal hepatic arterioles form a triad, the portal tract, upon which the base of the microscopic structure of the liver rests. The hepatocytes are organized in the Kiernan’s or classic lobules around central hepatic venules, which form the hepatic vein, in a hexagonal outline. The portal tracts are situated in the ‘cor- ners’ of the hepatic lobules [5, 6]. Rappaport, based on intravital microscopy of the microcirculation, proposed a further refinement with the ‘simple liver acinus’ as the functional and structural basic liver unit, consisting of the smallest portal tract including the terminal portal venules and hepatic arterioles as the afferent vessels together with the bile ducts, lymphatic vessels and nerves at the center, and the termi- nal hepatic venule as the efferent vessel in the periphery. The zone closest to the affer- ent vessel is referred to as ‘zone 1’, the area surrounding the peripheral efferent hepatic venule as ‘zone 3’. ‘Zone 2’ is located between these two areas (Fig. 1). This morphologic and functional structure provides a rational way to study the heteroge- neous hepatic metabolism in each zone [7]. For instance, there is an oxygen gradient from zone 1 to zone 3, making zone 3 cells the most vulnerable to oxygen deprivation.
The portal venous system, terminating in the portal venules, and the hepatic arte- rial system derived from the celiac trunk, forming terminal hepatic arterioles, con- fluence into the liver capillary bed, called the sinusoids. These have characteristic structures, different from normal capillaries, e.g., they have sinusoidal endothelial fenestrae and the basement membrane beneath the sinusoidal endothelial cells is absent (Fig. 2) [5, 8]. The hepatic arterial blood flows indirectly into the sinusoids via an anastomosis between the terminal hepatic arteriole and the portal venule, but also directly into the sinusoids [8, 9]. Regulation of the blood flow into the sinusoids is maintained by the relaxation and contraction of a precapillary sphincter at the end of the terminal hepatic arteriole and also by the coordinated contraction and dilatation of the sinusoidal endothelial fenestrae around the portal tract zone and by regulators in the portal venous system [9]. Furthermore, the stellate or Ito cells have an important regulatory role to be discussed hereafter.
Fig. 1. A schematic drawing of ‘simple liver aci- nus” grouped around the portal tract (PT) with the zonal arrangements of the hepatocytes. The arterial and portal blood flow congregates at the central venule (CV).
Fig. 2. Relationship between sinusoid, sinusoid lining cells, and hepatocyte. The sketch illustrates the vari- ous hepatocyte organelles. Note Kupffer cells of which cytoplasmic processes are anchored in endothelial fenestrae. From [8] with permission.
Current Physiology of the Hepatic Microcirculation
The terminal hepatic arteriole supplies oxygen enriched blood to the bile ducts, the walls of the portal venules as ‘vasa vasorum’, the nerves, and the connective tissue [5, 9]. The portal system carries nutrients from the digestive tract to the sinusoids and hepatocytes. Blood flow in the sinusoids, examined by intravital video enhanced contrast microscopy, was found to be significantly slower (400 – 450 µm/sec) than in normal or ‘true’ capillaries (500 – 1000 µm/sec), thus facilitating the metabolic
exchange of sinusoidal blood with the hepatocytes. The diameter of the sinusoids was smaller and the blood flow velocity was slower in zone 1 than in zone 3, indicat- ing a distinction in metabolic exchange.
Endothelins as vasoconstrictors and nitric oxide (NO) as a vasodilator are derived from the endothelial cells and are important for the local regulation of the microcirculation in the liver [5, 6, 10]. Endothelin infusion results in a constriction of the portal venules and terminal portal venules, comparable to the effects of nor- epinephrine: the sinusoidal blood flow slows down, leading to stasis and final col- lapse of the sinusoids. The sieving plates or fenestrae of the sinusoids contract, as well as the terminal hepatic arterioles directly connected to the sinusoids, contribut- ing to an overall elevation of portal pressure [9]. The role of NO will be discussed later.
The role of the autonomic nervous system has not been clarified entirely. Para- sympathetic branches from the vagus nerve innervate the liver, as well as sympa- thetic branches derived from the celiac ganglion. Although the sympathetic and parasympathetic influences on liver blood flow have not been established, excitation of the sympathetic nerves leads to a general flow reduction [6]. Complete denerva- tion, such as in liver transplants, results in an almost complete loss of epinephrine and norepinephrine concentrations in the liver, but effects on hepatic macro- and microcirculation are contradictory. Although in humans, total liver blood flow is increased after orthotopic liver transplantation, experimental animal models of liver denervation showed a variable picture of decreased hepatic microcirculation, increased hepatic artery blood flow, stable hepatic venous flow, or no significant changes whatsoever [11].
About 25 % of the cardiac output is distributed to the liver, with a portal to hepatic arterial ratio of about 3:1 to 4:1, with a compensatory mechanism between both, i.e., when the portal blood flow decreases, the hepatic arterial flow increases and vice versa [12 – 14]. One of the proposed regulatory mechanisms in reduced por- tal flow is the ‘adenosine washout hypothesis’: Adenosine is released into the space of Mall (surrounding the hepatic arterioles and portal venules) and washed out by both systems, maintaining a constant concentration. When the ‘wash-out’ is reduced by a reduction in flow of one of the systems, the adenosine concentration increases and dilates the hepatic artery resulting in increased arterial blood flow. This theory, however, is speculative and subject to debate [15].
Cellular function in the Hepatic Microcirculation
Endothelial cells that enclose Disse’s space underneath the fenestrated cell processes, line hepatic sinusoids with an average diameter of about 10 µm. The hepatocyte sur- face delineates the other border of this space, in which the stellate or Ito cells are sit- uated, their long cytoplasmic processes surrounding the sinusoids (Fig. 2). The endothelial basement membrane is typically absent in the sinusoids. In liver cirrho- sis, the hepatic sinusoidal endothelial fenestrae are decreased in diameter and num- ber and formation of a basal membrane is found beneath the sinusoidal endothe- lium, influencing normal vascular tone and exchange of nutrients. This change is induced by endothelin-1 and contributes to the development of portal hypertension [16].
Hepatic stellate cells represent 5 – 8 % of all liver cells in humans. On the luminal side of the endothelium lie the phagocytic Kupfer cells and liver-associated lym-
phatic cells [8]. Smooth muscle cells in portal venules and hepatic venules regulate the pre- and post sinusoidal vascular resistance. In the sinusoids the endothelial and stellate cells are the resistance regulators.
Several substances influence the stellate cell tonus in vitro, e.g., endothelins, angiotensin II, prostaglandin F2[ , and vasopressin are constrictive; dilatation was shown with NO, carbon monoxide and prostaglandin E2[6, 17]. In addition to their influence on vascular tone, in the normal liver the predominant function of the stel- late cells is storage of vitamin A, but when stimulated, such as in sepsis, they go through myofibroblastic transformation with loss of vitamin A and release of pro- inflammatory and pro-fibrinogenic mediators [17].
The role of arginine metabolism and nitric oxide
The arginine-NO pathway plays an important role in infection, inflammation and organ failure. NO is derived from arginine by normally active endogenous, and inducible NO synthases (NOS). These are located in different cell types, but mainly in endothelium and hepatocytes from which the inducible form was first found in humans [18]. NO acts as a strong vasodilator; it precludes leukocyte adhesion to endothelium and inhibits thrombocyte aggregation. It regulates immunologic cyto- toxicity and lymphocyte function.
The physiological role of NO in the regulation of the hepatic microcirculation remains complex. Endogenous NOS (eNOS) produces NO ‘on demand’.
eNOS in the liver is expressed by sinusoidal endothelial cells only and is upregu- lated, e.g., in endotoxemia, in an attempt to maintain adequate sinusoid perfusion.
In several conditions, such as ischemia/reperfusion (discussed below in detail), hemorrhagic shock, and endotoxemia, eNOS-produced NO overall exerted a benefi- cial and cell-protective effect [18]. Li et al. disclosed in an in vivo animal model that augmentation of endogenous NO production by L-arginine and 3-morpholinosyd- nonimine, an NO donor, increased portal blood flow. Inhibition of endogenous NO production had no effect on portal hemodynamics [19].
All liver cells have been reported to express inducible NOS (iNOS) [18, 20]. iNOS once expressed continually produces NO and, in contrast to endogenous NO-syn- thase (eNOS), is independent of intracellular Ca2+levels. The role of iNOS was more difficult to establish than for eNOS. For instance, in cold ischemia/reperfusion injury, iNOS had a cytoprotective, anti-apoptotic effect, but selective NO blocking in hemorrhagic shock experiments showed a general deleterious effect of iNOS [18].
Recently, in a rabbit model of hemorrhagic shock, Lhuillier et al showed a rapid and continuous increase in hepatic NO, that could only partly be blocked by an NOS inhibitor, suggesting enzyme-independent NO sources, e.g., tissue-stored S-nitroso- compounds, nitrosyl-hemoglobin, and nitrite ions [21].
Arginine, an NO donor, and arginine metabolism and its role in the regulation of microcirculatory flow have been investigated with great interest in recent years.
Asymmetric dimethylarginine (ADMA), a derivative of arginine metabolism, inhib- its the actions of NO and acts as a natural compensatory feedback mechanism for NO production. Symmetric dimethylarginine (SDMA), another arginine metabolite, has no NOS inhibiting properties, but competitively inhibits arginine uptake by the cell through an action on the so-called y+-pump.
In a rat model of endotoxic shock, a significant role of the liver in extracting ADMA from the circulation through dimethylarginine dimethylaminohydrolase
(DDAH) was disclosed [22]. Nijveldt et al. confirmed the role of the kidney and the liver in the elimination of ADMA by DDAH. In careful organ balance studies in a rat model, they could establish a predominant elimination of ADMA in the liver, thereby regulating NO synthesis. Blocking of DDAH, which has been found in oxida- tive stress and inflammation, could lead to elevated ADMA levels and consequently blocking of hepatic NO production, leading to decreased sinusoid blood flow and further damage to hepatocytes and ultimately liver failure [23].
In critically ill patients, plasma ADMA concentration was found to be indepen- dently related to the presence of hepatic failure, to lactic acid and bilirubin concen- tration as markers of hepatic function and was a better predictor of outcome than traditional scoring systems [24, 25]. Significantly higher ADMA levels were found in non-survivors.
Recently, a relationship between arginine-NO metabolism and insulin was revealed. The infusion of L-arginine together with insulin caused a dose-dependent vasodilator effect of L-arginine. A vasodilatory effect of L-arginine infusion in kid- ney and ocular vasculature was enhanced by insulin, possibly by facilitated L-argi- nine membrane transport, but enhanced intracellular NO production or increased NO bioavailability were also proposed [26]. Evidence for a correlation between ADMA and intensive insulin therapy in the critically ill and the liver was established with a significant increase in plasma ADMA levels in traditionally treated patients as opposed to stable levels in the intensive insulin group. Modification of ADMA con- centrations by insulin, with improved NO mediated microcirculation and perfusion, may in part explain the beneficial role of one of the most discussed therapeutic strategies in critically ill patients developed in recent years [27].
Ischemia/reperfusion Injury of the Liver
Ischemia-reperfusion injury of the liver occurs in several clinically relevant condi- tions, such as hemorrhagic shock, liver transplantation, liver resection, and sepsis.
Interruption of blood flow followed by reperfusion in liver transplants leads to sig- nificant cellular damage. Experimental evidence suggests that activation of Kupffer cells and T cells mediates the activation of polymorphonuclear cells (PMN), sinusoi- dal endothelial cells and the activation of reactive oxygen species (ROS) [18, 28]. In response to ischemia/reperfusion, sinusoidal endothelial cells become activated and on reperfusion express an array of adhesion molecules and MHC antigens. This results in further PMN interactions and disruption of sinusoidal blood flow. PMN- induced hepatocyte injury results from adhesion of the two cell types, release by the PMNs of toxic enzymes like elastase, and induction of oxygen free radical species [28, 29].
Mitogen activated protein kinases (MAPKs) play a pivotal role in regulating cyto- kine production in ischemia/reperfusion injury [30]. Activation of p38 MAPK by tumor necrosis factor (TNF)-[ exposure stimulates further production of TNF- [ , creating a vicious circle. Inhibition of TNF-[ by pentoxifylline resulted in improved sinusoidal blood flow [30].
Carbon monoxide acts as a regulatory molecule similar to NO and is released from heme in ischemia/reperfusion. It activates soluble guanylate cyclase (sGC) leading to smooth muscle relaxation and endothelial vasodilation, positively influ- encing the sinusoidal blood flow. It also inhibits platelet aggregation and suppresses iNOS in the hepatocytes, which has been shown to be detrimental in warm ische-
mia/reperfusion injury in the liver [18, 28]. The role of NOS in ischemia/reperfusion remains controversial though: Experiments in eNOS knockout mice demonstrated an increase in hepatotoxicity in ischemia/reperfusion liver damage, while iNOS was shown to contribute to warm ischemia/reperfusion injury, but to beneficially influ- ence apoptosis in cold ischemia/reperfusion injury as seen in organ transplantation [18]. Thus the mechanisms by which NO exerts an effect depends on the type of ischemia/reperfusion and the source of the NO produced.
Ischemia/reperfusion also causes extensive DNA damage, with overactivation of poly(ADP-ribose)-polymerase (PARP). PARP consumes large quantities of oxidized NAD and adenosine triphosphate (ATP), depleting cellular energy and leading to cell death. A reduction of hepatic microvascular damage was demonstrated by PARP inhibition after warm ischemia of the liver and after hemorrhagic shock [31, 32].
This is a promising novel approach in reducing hepatic microcirculatory damage in ischemia/reperfusion injury.
Conclusion
Impairment of the microcirculation with altered tissue oxygenation is central in crit- ical illness and therapeutically correcting and improving this correlates with out- come. Knowledge of the microcirculatory alterations in sepsis and other clinically relevant situations is expanding and will be of crucial importance in the develop- ment of new treatment strategies. Evaluating therapeutic interventions with moni- toring of the hepatosplanchnic microcirculation seems feasible, but is, unfortunately, not routinely available in everyday ICU practice.
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