LABORATORY ASSESSMENT OF HEPATIC HEMODYNAMICS

PORTAL HYPERTENSION

1089-3261/01 $15.00

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LABORATORY ASSESSMENT OF HEPATIC HEMODYNAMICS Nelson Garcia, Jr, MD, and Arun J. Sanyal, MBBS, MD

The liver is a unique organ because it has a dual blood supply from the portal vein and the hepatic artery. These vessels perfuse the hepatic lobules through a sinusoidal network of vascular channels which drain into central veins which in turn drain into the hepatic veins. It is now being increasingly understood that the flow of blood through the liver is closely regulated, and the study of the regulation of the hepatic microcirculation has become an important area of investigation. This burst of interest has been fueled, in part, by a multitude of technologic advances which have made study the hepatic microcirculation feasible. This article discusses the current methodologies available for the laboratory evaluation of the hepatic and portal circulation. The key aspects of the anatomy and physiology of the hepatoportal circulation which are germane to understanding the utility of specific methods are reviewed and the individual methods are discussed. ANATOMIC CONSIDERATIONS Portal Vein

The portal venous system contributes approximately 70% of the blood supplied to the liver and is formed by the confluence of the superior mesenteric vein and splenic vein. The portal venous system also receives contributions from the left gastric, gastroepiploic, and pan-

From the Department of Internal Medicine, Division of Gastroenterology, Hepatology and Nutrition, Medical College of Virginia, Virginia Commonwealth University

CLINICS IN LIVER DISEASE

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creaticoduodenal veins. The tributaries of the portal vein normally communicate with tributaries of veins draining directly into the systemic circulation. The watershed zones where such communications exist include (1)the gastroesophageal junction, (2) the anal canal (confluence of the superior and middle hemorrhoidal veins), (3) the falciform ligament (paraumbilical veins), (4) the splenic venous bed and the left renal vein, and (5) the retroperitoneum. Under normal circumstances, very little shunting of portal venous blood into the systemic circulation occurs. In the presence of portal hypertension, however, the veins in these regions dilate, allowing a substantial, yet variable, amount of shunting from the portal vein to the systemic circulation. Clinically, the most significant of these shunting collaterals are gastroesophageal varices, which are a major cause of the morbidity and mortality associated with portal hypertension and ~ i r r h o s i s . ~ ~

Hepatic Artery

The hepatic artery normally supplies approximately 30% of the hepatic blood With the combined blood flow from the hepatic artery and the portal vein, the liver receives approximately 25% of the cardiac Recent studiesz0indicate that the hepatic artery primarily perfuses the intrahepatic biliary ductules, portal tract interstitium, portal vein vasa vasorum, Glisson’s capsule, and the vasa vasorum of the central, sublobular, and hepatic veins (Fig. 1).The venous drainage from these regions drains into the hepatic sinusoids, thus forming several hepatic artery-derived portal systems within the liver. The teleologic benefit of this vascular arrangement is not fully understood.

Hepatic Sinusoidal Circulatory Bed

The hepatic sinusoids are perfused by branches of the portal vein, the hepatic artery, and the hepatic artery-derived portal system.zoThe sinsusoids perfusing a given hepatic lobule drain into a central vein. The hepatocytes are organized along the sinusoidal walls and form plates of cells separated by individual sinusoids. The liver parenchyma draining into a common central vein defines a hepatic lobule.79In the central regions of the lobule (Rappaport zone 3), the sinusoids radiate from the central vein, whereas in the peripheral regions of the lobule they form a network of anastamosing channels. Individual sinusoids are lined by a fenestrated endothelium which normally lacks a basement membrane. The sinusoidal endothelium is separated from the adjacent hepatocytes by the space of Disse. In recent years, hepatic stellate cells, which reside in the space of Disse, have been shown to be active in regulating sinusoidal blood The complexity of the hepatic microcirculatory anatomy has implications for the study of the physio-

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Figure 1. In the portal tract (lower right), the artery feeds the bile duct (6)as the peribiliary vascular plexus, the portal tract interstium including the nerve (N), and the wall of the portal vein (P). Drainage of these vascular beds is collected as a hepatic artery-derived portal system (APS), which joins the vein (1) in the tract or at the inlet venule on entering the lobule. The hepatic artery therefore can resume the portal flow by way of the APS. Outside the portal tract, the artery dissociates itself to supply Glisson’s capule, which drains into subcapsular lobules, and the walls of the hepatic tract system, including the central (C), sublobular (S), and hepatic (H) veins. The latter is the pathway by which arterial blood can bypass the hepatic parenchyma into the hepatic vein (2). (From Ekataksin W, Kaneda K: Liver microvasculararchitecture: An insight into the pathophysiologyof portal hypertension. Semin Liver Dis 19:359-382, 1999; with permission.)

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logic regulation of hepatic blood flow and limits the utility of specific methods in unique ways, as discussed later. HEMODYNAMIC AND PHYSIOLOGIC CONSIDERATIONS

The pressure in any vessel is defined by Ohm’s law:

P=QxR Dl where P is the pressure, Q the flow, and R the resistance to flow through the vessel. The resistance can be further measured using Poiseulle’s formula: R = 8nZ/d 121 where 1 is the length of the vessel, r is the radius of the vessel, and n is Reynold’s coefficient. These fundamental relationships form the basis for measurement and analysis of hernodynamics in the various regions of the hepatic and splanchnic circulation.

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MEASUREMENT OF HEPATIC AND PORTAL HEMODYNAMICS: HEMODYNAMIC EVALUATION OF PORTAL VEIN AND HEPATIC ARTERY Measurement of Pressure In the laboratory, rats are one of the animals most commonly studied for evaluation of portal hemodynamics. In rats, direct measurement of the portal pressure is typically performed by cannulation of the portal vein or one of its tributaries such as the ileocolic vein. Cannulation can be readily performed using of polyethylene tubing (i.e., PE-10) or a small-gauge catheter (i.e., 16-22 gauge).25,75 The catheter can be maintained in place with adequate hemostasis by the application of cyanocrylate gluez4or by tying off a terminal branch of the ileocolic vein without significantly altering portal hemodynamics (Fig. 2).25 The fluid-filled radiopaque catheter or polyethylene tubing is then attached to a pressure transducer that is calibrated to the level of the right atrium. The ability to measure portal pressure directly in the animal models provides a distinct advantage over human studies which usually involve primarily indirect measurements of portal pressure. Hepatic arterial pressure closely correlates with systemic arterial pressure. For this reason, systemic arterial pressures obtained by the cannulation of either the femoral artery or carotid artery are often used to represent the hepatic arterial pressure. There may, however, be small quantitative differences in the pressure obtained in different systemic Therefore, to minimize the error associated with measurement of intra-arterial pressures, in a given series of experiments, the same artery should be used to represent the hepatic arterial pressure.

Figure 2. Cannulation of the ileocolic vein in the rat. (See also Color Plate 1, Fig. 1.)

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Measurement of Flow Electromagnetic Flowmetry

Principle and Methodology. An electromotive force is induced in the blood as it-flows through a magnetic field, and the electromagnetic force which is induced is linearly proportional to the to the velocity of the flow. The technique of electromagnetic f l ~ w m e t r ymeasures ~~ this electromagnetic force by placing two electrodes perpendicular to the direction of flow in the vessel being studied. The voltage generated between these electrodes is directly proportional to the blood flow. Because the voltage depends on the angle between the probe and the blood vessel, the probe must be appropriately fitted over the vessel to minimize alteration of this angle.17 A secure and proper placement can be achieved in both acute and chronic preparations. Applications. For much of the preceding century, electromagnetic flowmetry served as the criterion standard in the evaluation of vascular flow. It has been widely used in vascular research including the study of hepatic hemodynamics. Electromagnetic flowmetry has been found to correlate well with flow measurements obtained through the microphere method, clearance methods, and the inert gas washout method.6,94 Electromagnetic flowmetry has been used to assess relative changes in hepatic arterial and portal venous blood flows following experimental tran~plantation.~~ Furthermore, the use of electromagnetic flowmetry has contributed to the current understanding of the hepatic arterial buffer response as described by L a ~ t t Recently, .~~ electromagnetic flowmetry has been used to assess vascular integrity at the time human orthotopic liver tran~plantation.8~ The use of electromagnetic flowmetry has, however, declined because it has been supplanted by other methods discussed in this article. Advantages and Disadvantages. Electromagnetic flowmetry can be used in both acute and chronic preparations and on both cannulated and noncannulated vessels. The disadvantages of this method include artifacts related to the movement of the probe and changes in flow and turbulence produced by constriction of the vessel by tight-fitting probes. The latter disadvantage is particularly important, because to maintain a snug fit the diameter of the probe used is usually only 5% to 10% less than that of the vessel being studied. Electromagnetic flowmeters also require frequent calibration because of zero-point drift. Some authors have described difficulties with the use of electromagnetic flowmeters in veins.57 Ultrasonic Transit-time Volume Flowmetry

History. Ultrasonic transit-time flowmetry (TTF) was first validated in large animals in 1979.18Subsequent studies have validated the use of TTF in vessels of smaller animals including rat mesenteric artery,15 rat renal artery,'O'P lo2 and rat portal vein.63Thus, TTF is currently able to

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study multiple vessels of varying sizes in a single animal through the use of a multichannel flowmeter. Principle and Methods. Transit-time flowmetry uses the effect of fluid motion in a vessel on the transit time of an ultrasound wave passed through the vessel. An appropriately sized probe is placed around the vessel of interest. The probe consists of two transducers that, under electrical excitation, produce an ultrasound wave that transects the vessel under study. The wave is then reflected by an acoustic reflector and is ultimately received by its complementary transducer (Fig. 3). The receiving transducer, in turn, creates an electrical signal that is interpreted by the flowmeter as the transit time, that is, the time required for the ultrasound wave to travel from one transducer to the other. One of the transducers emits a wave that travels along the direction of blood flow; the other produces a wave that travels against the direction of flow. The transit time decreases when the wave travels in the direction of flow and increases when it travels against the direction of flow. The difference between the two measurements provides a measure of flow volume. Applications. Transit-time flowmetry has been validated in vivo by the administration of known volumes of fluid over a specified time period through a T-cannula proximal to an implanted flowprobe.18This validation study revealed a highly linear relationship between observed and measured flow. Also, transit-time flowmetry has been validated against electromagnetic flowmetry and in microsphere flow In one study utilizing TTF to determine cardiac output in rhesus monkeys, TTF was more accurate than electromagnetic flowmetry, using thermodilution as the criterion standard.52 Transit-time flowmetry can be used in both acute and chronic preparations involving the hepatic artery and portal vein. Chronic implantation of a flowprobe on the hepatic artery may be technically difficult in

Figure 3. Ultrasonic transit-time flowmetry. A transit-time flowprobe that demonstrates two paired transducers with an acoustic reflector. Three flowprobes are demonstrated with cotton-tipped applicator as a visual reference. Flowprobe A is for vessels 1 .O-1.5 mm O.D. (rat portal vein), B for 0.5-1.0 mm O.D. (mouse ascending aorta), C 0.35-0.7mm O.D. (Rat renal artery).

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smaller animals such as the rat, but the device has been successfully implanted in pigs.19 On the other hand, chronic implantation of a flowprobe on the rat portal vein has been well d e ~ c r i b e dThe . ~ ~ placement of flowprobes permits continuous monitoring of changes in flow through the vessel of interest during various interventions such as drug administration and mechanical obstruction of flow. Given the dual blood supply of the liver, transit-time flowmetry allows an investigator to measure the effects of a particular intervention on total hepatic blood flow as well as on each of its components. Transit-time flowmetry has been used to assess the changes in hepatic arterial and portal venous blood flow after the administration of vasoactive compounds such as terlipre~sin.~~ The simultaneous measurement of hepatic arterial and portal venous inflow allows an accurate determination of their relative contributions to the changes in total hepatic blood flow. Transit-time flowmetry has also been used to assess the hepatic arterial buffer response through graded constriction of either the hepatic arterial or the portal venous inflow in cirrhotic animals.81 Another recent application of TTF has been the study of portal venous blood flow in models of laparoscopic surgery. Through placement of a flowprobe, a decrease in portal venous inflow has been noted as intraabdominal pressure increases with CO, gas insufflation.3O Advantages and Limitations. In addition to its possibly greater accuracy, TTF provides several other advantages over electromagnetic flowmetry. First, TTF is not dependent on the hematocrit of the animal being studied. This point is of particular importance to investigators studying hepatic circulation in the setting of systemic hemodynamic compromise caused by hemorrhage. Second, TTF has excellent zeropoint stability which does not require vessel occlusion to obtain a true zero. Third, there is no electrical interference from other instruments used in the preparation. Last, flowprobes can be placed on small vessels, including arteries and veins measuring from 250 p,m to 36 mm in outside diameter, without altering flow dynamics. One disadvantage of TTF is its inability to measure hepatic blood flow at the microcirculatory level. Although blood flow through the hepatic artery and portal vein can be measured simultaneously, the flow at the level of the sinusoids may be dramatically different, depending on the degree of intrahepatic shunting. A second pitfall in the use of TTF is that flow volumes are underestimated when flowprobes of an inappropriate size are used.2,1 5 r 8 0 For example, the use of an inappropriately large flowprobe may yield a large underestimation of actual flow. Theoretically, the relationship of the flowprobe to the vessel must be similar from one experiment to the next to minimize artifacts related to the angle between the probe and the vessel. In practice, however such artifacts can be obviated by using a correction factor.77The placement of flowprobes is typically not a major limiting factor in acute experiments where the flowprobe can be easily manipulated but may be a more significant factor in chronically implanted flowprobe studies. Further,

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placement of portal venous flowprobes can be difficult, if not impossible, when cirrhosis is induced by administration of intraperitoneal CC1, or by common bile duct ligation resulting from the formation of adhesions at the level of the porta hepatitis (Fig. 4). For this reason, When TTF is used to study portal hemodynamics in cirrhotic livers, other methods of inducing cirrhosis (i.e., CC1, through gavage) are re~ommended.~~, 78, 95 Endoscopic Ultrasound Flowmetry

History. The azygos vein receives a substantial amount of blood that is shunted by gastroesophageal collaterals once portal hypertension develops. Although azygos venous blood flow has been previously studied through therm~dilution~ and angiography,46the development of endoscopic ultrasound (EUS) has provided a noninvasive means for the evaluation of gastroesophageal varices and azygos blood flow. The first study using EUS was published in 1988.92Subsequent human studies performed in patients with cirrhosis and gastroesophageal varices have revealed an increased diameter of the azygos vein and an increased maximal velocity of flow through this vessel.s5 Furthermore, EUS has been shown to be useful in determining changes in azygos blood flow following the administration of vasoactive agents such as terlipressin and somatostatin.60 Principle and Methods. Endoscopic ultrasound flowmetry has been used in only a small number of animal studies to date. Because of its

Figure 4. Adhesions secondary to the administration of intraperitoneal CCI,. This complication can be prevented through the use of various other methods of cirrhosis induction. (See also Color Plate 1, Fig. 2)

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size, the use of EUS is limited to large-animal models (pigs or dogs). In these models, standard endosonographic techniques have been employed to delineate normal anatomy and to perform interventions such as fine-needle aspiration, celiac block, and coagulation therapy?, 5, 98 In a study performed by Hansen et al, portal venous flow was determined by EUS and compared with flow measurements obtained through the use of TTF flow probe^.^^ In this study, there was good correlation (R = 0.92) of flow rates obtained by EUS flowmetry and by TTF even following the administration of terlipressin. Endoscopic ultrasound flowmetry is unique among the methodologies discussed here in that it can specifically evaluate the presence of gastroesophageal varices, a major source of morbidity and mortality in cirrhosis. Furthermore, in conjunction with pressure data, the determination of variceal flow and diameter permit the determination of variceal wall tension through a modification of Laplace’s law? Wall tension

=

Portal pressure x Variceal radius

PI

Thus, EUS flowmetry provides a noninvasive method of measuring portal venous and collateral flows in large animal models. Applications. The use of EUS in basic research has been quite limited to date. Clearly EUS can fill a niche in portal hypertension research. Specifically, it can provide insight into the changes in variceal flow and wall tension in animals with portal hypertension. Second, information obtained through the use of EUS flowmetry can be readily confirmed in the clinical setting given the widespread availability of this technology. The utility of EUS flowmetry and its ability to enhance the knowledge of hepatic and splanchnic hemodynamics remains to be determined. Advantages and Disadvantages. The attractiveness of EUS flowmetry lies in its noninvasive nature. Furthermore, many clinical gastroenterologists and hepatologists are familiar with the technology associated with this method. Unfortunately, the use of EUS in basic science research is limited by its expense and by the need for a large-animal model. Also, simultaneous portal pressure measurements, which are often desired in experiments evaluating hepatic hemodynamics, generally require an invasive procedure, thereby eliminating the main benefit of EUS.

Study of the Hepatic Microcirculation lntravital Microscopy

History. The first detailed description of intravital microscopy (IVM) dates from the mid-nineteenth century, when Waller described the passage of leukocytes through the frog tongue.66The first dynamic investigations of hepatic microcirculation were performed in 1936 by Knisely who described a method for illuminating living structures for microscopic

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studies.49Modern optical and recording technologies have' dramatically advanced the study of living structures with IVM. Principle and Methods. Intravital microscopy permits the visualization of living tissue at the microscopic level by illumination and magnification of the tissue of interest. Illumination can be performed by one of several methods. In transillumination the tissue of interest is interposed between the light source and the microscope. In epi-illumination, the light source and microscopy are on the same side of the tissue. The capabilities of IVM can also be augmented by causing specific structures within the tissue to emit light by phosphorescence. In the study of hepatic microcirculation, the use of epi-illumination is generally preferred over transillumination because epi-illumination permits the study of central areas of the lobe rather than simply the edges which are more susceptible to injury and alterations in The microscopic pictures are recorded on videotape by means of a low-light-level silicon-intensified target (SIT) or charge-coupled device (CCD) video cameras and can then be analyzed using various computer-assisted image analysis systems (Fig. 5).66 The microcirculation of each liver lobule should be recorded for at least 30 to 60 seconds to minimize variations in microvascular flow caused by epi-ill~rnination.~~ The use of IVM permits determination of the percentage of perfused sinusoids, sinusoidal diameters, and sinusoidal red blood cell (RBC) velocity. Red blood cell velocity is measured by determining the length of the path of a labeled RBC by video playback and dividing this distance by the elapsed time. Sinusoidal resistance can also be calculated using Poiseuille's law, as discussed previously.

Figure 5. lntravital microscopy. An illuminated, exteriorized liver segment is visualized though the use of microscopy. The images are captured by a video camera and are then analyzed using various computer-assisted image analysis systems that allow the determination of blood flow.

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When using IVM for the study of hepatic microcirculation, the left hepatic lobe is exteriorized after being freed from the diaphragm by dissection of the falciform ligament. This dissociation minimizes movement related to respiration. The exposed hepatic tissue is covered with plastic film to minimize fluid losses during the period of investigation. The hepatic tissue is then placed under the objective lens of the microscope, and the images are captured for subsequent analysis. Applications. The use of different fluorescent markers has increased the possible applications of IVM. Red and white blood cell velocity and flux, loss of endothelial integrity, and various aspects of cell-cell interaction can now be observed.66, 67, 90 For example, polymorphonuclear leukocytes can be fluorescently labeled, and their trafficking in the microcirculo7 Also, necrosis that is associated with loss of lation can be cell membrane integrity can be studied because injured cell membranes are unable to exclude vital dyes, such as propidium iodide, and are thus readily differentiated from viable cells. These applications and the ability to visualize the hepatic sinusoids directly differentiate IVM from other methods used to study microcirculation. Orthogonal polarization spectral (OPS) imaging is a newly described technique that provides images similar to those of IVM.28Orthogonal polarization spectral imaging has recently been applied to the evaluation of ischemia-reperfusion injury in a rat Using epiillumination, an OPS imaging probe can visualize a superficial segment of hepatic tissue when positioned on the liver capsule. To date, only preliminary studies of hepatic blood flow have been performed using OPS imaging. Orthogonal polarization spectral imaging can determine the number of perfused sinusoids and red blood cell velocity more reliably than IVM.56 Although OPS imaging is a promising method for studying hepatic microcirculation,69its true potential remains to be realized. Advantages and Limitations. Intravital microscopy provides the ability to examine the hepatic microcirculation in vivo, but, because of its microvascular focus, it is unable to differentiate directly between the arterial and venous contributions to hepatic perfusion. Furthermore, the direct visualization of flow through a relatively small and superficial hepatic segment may not necessarily represent the changes that are occurring in the overall hepatic circulation. Despite these limitations, the utility of IVM has expanded with the addition of fluorescence video microscopy. Laser Doppler Flowmetry

History. The initial studies utilizing laser Doppler flowmetry (LDF) were performed in the late 1 9 8 0 ~ .Laser ~ , ~ ~Doppler Flowmetry permits serial evaluation of various hepatic regions during a period of study without requiring direct contact with the liver or its vascular structures. Laser Doppler Flowmetry has been used extensively in transplantation

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procedures, primarily to assess microcirculatory changes associated with revascularization.104 Principle and Methods. Laser Doppler Flowmetry combines laser technology with the Doppler effect caused by the movement of RBCs in the microcirculation to estimate RBC flux. Red blood cell flux is defined as the product of the mean RBC speed and RBC concentration within the measured volume. A 2-mW helium neon laser beam is transmitted into the tissue of interest, and a photodetector is used to acquire backscattered light from the tissue (Fig. 6 ) . This back-scattered light consists of a portion of light from static tissue and a spectrally broadened portion resulting from interactions with moving RBCs (Doppler shift). The electrical signal produced by the photodetector is transformed into an output voltage that varies linearly with the RBC flux. The results of the LDF signal can be presented visually in real time by data-acquisition software. Applications. Laser Doppler flowmetry has recently been used as a means to monitor hepatic microcirculation noninvasively in the posttransplantation period. Hepatic arterial flow, as measured with an ultrasonic flowprobe, can be correlated with LDF measurements. This combination of methods has been studied in a dog model by the postoperative, transcutaneous implantation of a single-fiber laser Doppler microprobe in the liver through a Chiba needle.54Using this method, hepatic arterial insufficiency can be detected. Other recent studies have used LDF to assess the effects of various agents on reperfusion injury at the microcirculatory level." Laser Doppler flowmetry has also been used to assess the microcirculation of donor livers before transplantation.88 In this study, microcirculation was diminished in steatotic livers when compared with normal livers.

BE Transmitter

Receiver

Figure 6. Laser Doppler flowmetry. A 2-mW helium neon laser is transmitted into the tissue of interest, and a photodetector is used in order to acquire back-scattered light from the tissue. This back-scattered light consists of both static tissue as well as a spectrally broadened portion resulting from red blood cell movement (Doppler shift).

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Advantages and Limitations. Although LDF provides a novel approach for evaluating the hepatic microcirculation, this method has several limitations. Laser Doppler flowmetry does not permit the determination of absolute perfusion values (i.e., in mL/min/100 g tissue); instead, measurements are expressed as arbitrary perfusion units (PUS). This inability to obtain absolute perfusion values prevents correlation with other methods or instruments used to assess the microcirculation. Quantitative comparisons can be performed, however, by using the same calibrated instrument. As previously mentioned, RBC flux is related to hematocrit. In fact, previous investigations have demonstrated a linear increase in PUS with various hematocrit measurements (2.5%-20%)at a fixed flow rate.' Given the alterations that occur with varying hematocrits, LDF is not adequate for the study of animal models with variable hematocrits (e.g., in a hemorrhagic shock investigation).lo3As long as the hematocrit remains stable, however, LDF measurements can be expected to correlate directly with RBC velocity within the microcirculation. Another difficulty with LDF arises in its inability to evaluate the entirely of the hepatic microcirculation, as can be done with the clearance and indicator dilution methods discussed later. This limitation is of particular importance because of the inhomogenous nature of the hepatic microcirc~lation.~~ This inhomogeneity of the hepatic microcirculation is clearly important when interpreting results obtained with LDF because LDF evaluates only a small fraction of the total hepatic microcirculation. Of significant importance is that the surface of the liver is largely supplied by arterial blood.3 Despite its drawbacks, LDF remains an useful tool in the evaluation of hepatic microcirculation because of its ability to monitor flow continuously without altering the region of interest. Also, LDF is more easily used than other methods for evaluating the hepatic microcirculation. Clearance Methods

History. The use of clearance methods to determine hepatic blood flow in humans was first described in 1945 by Bradley et a1.8 Clearance methods are based on the premise that certain substances, when introduced into the bloodstream, are cleared by the liver and this rate of disappearance is used to estimate hepatic blood flow. Indocyanine green (ICG) is the prototype indicator used to determine hepatic blood flow by clearance methods. Indocyanine green is a nontoxic dye with a high hepatic extraction ratio (70Y0-96%).~'It is excreted unchanged in bile and, unlike other indicators such as sulfbromopthalein sodium, does not undergo enterohepatic Principle and Methods. Clearance methods rely on the Fick principle to determine hepatic blood flow. According to this principle, under conditions of constant flow, the volume moving through an organ ( F ) can be calculated by determining the amount of indicator extracted ( R ) over that time and the concentration difference of the indicator entering

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(Ci) and leaving (C,) the organ. This principle is expressed by the following equation: F

=

R/(Ci - C,)

[41

Under experimental, steady-state conditions, the intravenous infusion rate of an indicator removed efficiently by the liver (i.e., ICG) equals the rate of hepatic removal (R). Ci can readily be determined by peripheral venous or arterial blood sampling, and C, is obtained through sampling of the hepatic venous effluent. In clinical settings, where hepatic vein catheterization is impractical, the clearance rate of ICG is assumed to be 100°/~,and the C, is set at zero. This assumption, however, is inaccurate, and ICG inevitably underestimates hepatic blood flow.6In the laboratory, the cannulation of the hepatic vein, although difficult in small-animal models, can be performed, and a true C, can be determined. Most indicators currently used are distributed only in plasma. Therefore, to determine hepatic blood flow, plasma flow needs to be converted to blood flow. If the hematocrit is known, blood flow can be calculated using the equation: Blood flow

Plasma flow x 1/(1- hematocrit)

[51 The use of multiple indicators to assess the various vascular compartments within the liver was first described by Goresky in 1963.26Labeled erythrocytes, which in the liver are normally confined to the vascular space, serve as the vascular reference. On the other hand, plasmadissolved substances such as albumin and sucrose readily gain access to the extravascular space (i.e., hepatocytes, space of Disse) through sinusoidal endothelial fenestrations. This diffusion into the extravascular space delays their appearance in the hepatic vein. This delay is inversely proportional to their molecular weights. Diffusion of water is dictated by the osmotic gradients in the perisinusoidal mileu. The difference in diffusion rates between the labeled RBC and other markers can be analyzed using a flow-limited linear two-compartment model as described by Goresky.26Hence, this method provides estimates of sinusoidal blood volume and of the extravascular volume. Applications. Clearance methods have been recently used to determine the effects on hepatic blood flow of therapeutic interventions such as high-volume plasmapheresis in patients with liver failure,13 73 and the creation of a pneumoperadministration of vasoactive drugs,lO, itoneum with laparoscopic surgery.84,96 Clearance methods have also been successfully used to determine hepatic blood flow in specific patient pop~lations.~~, lo9 Clearance methods can be used in combination with other methods such as ultrasound to compare functional hepatic flow with total hepatic flow. The difference between these two flow rates permits an estimation of intrahepatic shunting which is known to occur Io8 in the presence of ~irrhosis.~~, A recently published study reports the use of ICG measurement obtained by placing near-infrared spectroscopy probes on the liver sur=

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face. In this study, cirrhotic animals demonstrated a significant reduction in hepatic ICG uptake which correlated with a decreased hepatic blood flow." Clearly, the use of a spectroscopy probe can simplify clearance methods by eliminating the need for repetitive hepatic venous blood sampling. Advantages and Limitations. Although ICG provides a good approximation of hepatic blood flow under normal physiologic states, it is not reliable in the presence of cirrhosis because its hepatic extraction is d e c r e a ~ e dOther . ~ ~ indicators with a higher and more predictable extraction ratio have been recently used to assess hepatic blood flow in the presence of ~ i r r h o s i s Even . ~ ~ these indicators, however, show variable extraction ratios in the presence of cir~hosis.'~ Another limitation of clearance methods in the laboratory is that the accurate determination of hepatic blood flow requires cannulation of the hepatic vein. This cannulation can be extremely difficult in small animals because of the short length of the infradiaphragmatic hepatic veins. Furthermore, backflow of blood from the inferior vena cava can occur during sampling, leading to dilution of C044leading to an underestimation of hepatic blood flow. Unlike some of the previously described methods, clearance methods do not permit the distinction between hepatic arterial and portal venous flows. Furthermore, real-time flow data cannot be obtained because the concentration of the indicators must be determined to estimate blood flow. Inert Gas Washout

History. The inert gas clearance technique for measurement of blood flow within an organ was first employed in 1945 for the study of cerebral blood At that time, the Fick principle was being employed to study hepatic circulation through the use of clearance methods with sulfbromophthalein sodium used as the indicator. The use of inert gases soon led to another method of determining hepatic blood flow. Principle and Methods. Radioactive gases such as Xenon-133 (133Xe) and Krypton-85 (s5Kr), which are small, chemically inert atoms, are used in the inert gas clearance technique. The entry and subsequent exit of these inert gases from a tissue depend solely on their diffusion and solubility. They instantaneously obtain equilibrium between tissue and blood, with 95% equilibrium obtained within 1 second. Only 1%to 5% of either gas remains in the blood after a single passage through the lungs, leaving very little isotope to recirculate to the hepatic tissue under investigation. Although the inert gases may be directly injected into the liver parenchyma,3l hepatic or splenic the most widely studied method involves injection through the portal vein. The beta emissions of s5Kr have an average range in tissue of only 0.7 mm. Therefore, detection of this radionuclide requires placement of a Geiger-Muller tube in close proximity to the hepatic surface. Xenon-133 decays with the emission of more penetrating gamma photons. A gamma camera or

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scintillation crystal can be used to detect the radionuclide throughout the liver. The clearance curves obtained from the beta emissions of s5Kr are monoexponential and reflect the flow in the superficial liver tissue. On the other hand, the clearance curves obtained from the gamma emissions of 133Xeare biexponential, having a fast component and a slow component. The slow component represents extrahepatic activity from other organs such as the Therefore, the true hepatic clearance rate is determined by subtracting the slow component from the clearance curve to derive the fast component. The blood flow calculated from the inert gas clearance is proportional to the rate of clearance and also to its partition coefficient, A. Because inert gases are lipophilic in nature, their solubility in hepatic tissue is primarily determined by the hepatic lipid content. The solubility of the inert gas in the blood is determined by the hematocrit because these gases have a high affinity for hemoglobin. Although partition coefficients between blood and tissue have been determined for various animal models, there is some variability from among studies, as is to be expected. The issue of hepatic fat content is important in chronic studies where hepatic lipid content may be more variable. It is of less significance in acute studies because the A error will be the same in all quantitative measurements made during the particular acute experiment. Applications. The use of inert gas clearance to study hepatic circulation has decreased because investigators prefer other, simplified methods. In the recent past, however, inert gas clearance has been used to determine blood flow in hepatic tumors.68Furthermore, 133Xewashout has been used to determine changes in hepatic flow following microemThus, although other bolization of hepatic tumors in an animal methods are currently more widely used, inert gas clearance provides an alternative means to determine total hepatic blood flow. Advantages and Limitations. Hepatic blood flows obtained with inert gas clearance have correlated well with those obtained with other 55 methods such as electromagneticflowmetry and clearance techniq~es.~~, Intrahepatic perfusion, however, may not be homogenous in the diseased liver, and this in homogenity may result in a complex isotope clearance curve for which analysis is not as straightforward as described previously. Furthermore, this method does not allow the separate physiologic contributions of the hepatic artery and portal vein to the total hepatic blood flow to be determined. Finally, real-time determination of hepatic blood flow cannot be obtained with this technique. Before using this method, the investigator should be familiar with its limitations and with the basic principles involved in clearance curve analysis. Thermodilution and Thermodiffusion

History. The use of heat exchange techniques to determine hepatic blood flow in acute experiments involving dogs and sheep was first 23 Subsequent experiments exdescribed by Fegler in the late 1950~.~*,

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tended the use of thermodilution to chronic animal preparations.82At that time, clearance techniques were being widely applied to the study of hepatic hemodynamics. In the presence of altered hepatic function, however, the utility of these methods was greatly limited. Thermodilution arose as an alternative method for the study of hepatic hemodynamics in animals with experimentally induced liver disease. Principle and Methods. Thermodilution, similar to clearance techniques, is also based on the Fick principle. Basically, the blood flow rate is inversely proportional to the temperature change as measured by an intravascular thermistor catheter. Hepatic blood flow can be determined indirectly by calculating the difference in flows through the thoracic inferior vena cava (cephalic to the hepatic veins but inferior to the right atrium) and through the intra-abdominal inferior vena cava (below the hepatic veins). The total hepatic blood flow rate is expressed by the following equation: Total hepatic blood

=

Thoracic inferior vena cava flow [61 - Intra-abdominal inferior vena cava flow

To determine each of these flow rates, two thermistor catheters are required. Various surgical techniques for placement of thermistor catheters have been described, and one of these is discussed in detail later.9,82,86 The first thermistor is placed through the internal jugular vein and is advanced to the thoracic inferior vena cava (approximately 1-2 cm cephalic to the hepatic veins in dogs). The second thermistor catheter can be advanced through the right ovarian vein (in dogs) into the intraabdominal inferior vena cava. The room-temperature injectate can be injected through a polyethylene catheter placed in the renal vein through the left ovarian vein (Fig. 7). Accuracy of catheter location is ascertained fluoroscopically or through direct palpation during surgery. For chronic preparations, the catheter can be exteriorized as described by Roberts and Plaa.82 Applications. A recent advance in the use of thermal energy for quantification of hepatic blood flow has been the application of thermodiffusion (TD). In this method, a TD probe is introduced directly into the liver parenchyma. In principle, this method consists of a self-heated thermal transducer that is heated to a temperature of 2" C above the surrounding tissue. The heating power required to maintain this temperature differential is proportional to the dissipation of heat caused by local perfusion. The TD probes can be placed chronically and permit nearly continuous sampling. Thermodiffusion has been studied primarily in the porcine model and has been validated against clearance and microsphere methods.47, This technique has also been used to evaluate graft blood flow following porcine liver transplantation.100There has also been limited experience with TD in the clinical posttransplantation setting. Here, continuous hepatic blood flow monitoring for up to 7 days postoperatively revealed diminished hepatic perfusion in association with graft failure or rejection.48In summary, thermal methods for the @

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Figure 7. Thermodilution method. Thermistor catheters are placed in the thoracic inferior vena cava (cephalad to the hepatic veins) and in the intra-abdominal aorta (caudal to the hepatic veins). Room temperature injectate administered by way of the left renal vein permits blood flow determination through the application of Fick's principle. Total hepatic blood flow is calculated by the following formula: Total hepatic blood flow = Thoracic IVC flow - Intra-abdominal inferior vena cava flow.

measurement of hepatic blood flow are continuing to evolve. Thermodiffusion methodology has not yet been applied to smaller animal models. Advantages and Limitations. Thermodilution can can provide realtime data on hepatic flow, but it is invasive and is more readily accomplished in larger animal models. Several sources of error must be considered when utilizing thermodilution. An inadequate temperature exchange between the injectate and the blood may lead to erroneous values detected by the thermistor catheters. Because the total hepatic flow is derived from the difference of the flow rates at two levels of the inferior vena cava, random error between actual and calculated values can be large. For example, an error in a value from one thermistor can be magnified by an error in the flow value obtained from the second thermistor. Another frequent source of error is inaccurate injectate temperature or volume, but this source of error can be minimized by meticulous experimental technique. Unfortunately, the use of thermodilution does not permit the distinction between hepatic arterial and portal venous blood flow. Colored and Radioactive MicrosphereslFractional Distribution Method

History. Traditionally, radioactive microspheres have been used to assess cardiac output and regional organ flow. Fluorescent and dyelabeled microspheres have now been developed to circumvent the diffi-

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culties encountered with radioactive isotopes, including health risks, special requirements for the laboratory, and expensive radioactive waste management pr0cedures.9~ Principle and Methods. Latex microspheres, measuring approximately 15 pm in diameter, are injected into the left ventricle without a significant alteration in cardiovascular parameters.5I Because of their size, the microspheres lodge in the terminal arterioles during their first pass through the microcirculation. As many as three consecutive determinations of flow rates in the same animal can be performed through the use of microspheres of dyes possessing different photometric absorpt i o n ~After . ~ ~ completion of an experiment, the tissues of interest and a reference blood sample are digested with a solution of Potassium Hydroxide (KOH) and Tween 80 (Sigma, St. Louis, MO). The resulting solution is then filtered under vacuum suction through an 8 km pore membrane filter. The dye is recovered from the micropheres through the addition of dimethylf0rmamide.9~Finally, the photometric absorption of each dye is measured using a spectrophotometer. Tissue blood flow is calculated through the use of the following equation: Blood flow

=

number of microspheres in target organ x reference blood withdrawal rate/number of microspheres in reference blood

[7]

The reference blood sample is obtained through the placement of an intra-arterial catheter in the descending aorta; blood collection is performed at a constant rate from the time immediately preceding microsphere injection to the time at which arterial microsphere concentration is zero (i.e., 2 minutes).62 Applications. The microsphere method has been applied to studies interested in quantifying the amount of portosystemic shunting present in various animal models of liver disease.29,39 After intraventricular injection of microspheres, a portosystemic shunt percentage can be readily calculated by determining the percentage of microspheres which have bypassed the splanchnic and hepatic circulations (i.e., have become lodged in the pulmonary vas~ulature).~~ A recent study by Makin et a1 evaluated the hemodynamic changes occurring in a D-galactosamine model of acute liver injury.61 This study outlined the course of the hemodynamic derangements occurring in acute liver injury over a 72hour period. Microspheres have also been widely used in studies evaluating the hemodynamics of lo6 and response to pharmacologic intervention^.^^ In these studies, the use microspheres has permitted a determination of distributional blood flow to various organs. Advantages and Limitations. The use of microspheres to study hepatic microcirculation is limited by the inability to investigate portal venous flow. The injected microspheres are trapped in the microcirculation of the intestines without the ability to proceed to the liver itself. Although this limitation can be a drawback for investigators interested in both hepatic arterial and portal venous flows, it can be an asset be allowing the two sources of hepatic flow to be distinguished. Another

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limitation of micropheres involves the inability to study hepatic flow in real time. The use of multiple dyes allows three different hepatic arterial flows in one animal to be obtained during a single experiment. The number of obtainable flow samples can be increased by combining colored microspheres with radioactive micropheres, but doing so clearly increases the complexity of an experiment. Yet another limitation is that microspheres can be used only in experiments in which the animal is sacrificed, because an anatomic specimen is needed to measure the photometric absorption of the tissue of interest.

SUMMARY

Although the study of hepatic circulation is complicated by the dual blood supply and complex anatomy of the liver, many distinct methods are available to facilitate its study. Before embarking on an investigation of hepatic hemodynamics, the investigator must be familiar with the available methods and their applications. All methods have their own attributes and limitations. No one method is superior to the others, but, depending on the aspect of hepatic hemodynamics to be investigated, a particular methodology may yield distinct advantages.

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Address reprint requests to Nelson Garcia, Jr., MD Division of Gastroenterology and Hepatology Medical College of Virginia Virginia Commonwealth University P.O. Box 980711 Richmond, VA 232984711 e-rnail:[email protected]