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Clinical Science (1985) 68,23-28 23 Effect of two models of portal hypertension on splanchnic organ blood flow in the rat D . LEBREC AND L. BLANCHET Unite' de Recherches de Physiopathologie He'patique (INSERMJ, H6pital Beaujon, Clichy, France (Received 9 February/3 June 1984; accepted 3 July 1984) Summary 1. Splanchnic organ blood flow and cardiac output were measured by the microsphere method in fasted rats with prehepatic portal hypertension due to portal vein stenosis, in rats with intrahepatic portal hypertension due to bile duct ligation, and in unoperated normal rats. 2 . Portal venous pressure was higher in both groups of portal hypertensive rats than in normal rats. Cardiac output was significantly higher in portal hypertensive rats than in normal rats. 3. In rats with portal vein stenosis, splanchnic blood flow was higher than in controls. This increase was caused by increased perfusion of all organs drained by the portal vein, and by increased hepatic arterial blood flow. In rats with bile duct ligation, splanchnic blood flow was not significantly higher than in normal rats: haemoperfusion of all organs contributing to the portal circulation decreased, whereas hepatic arterial blood flow increased. As cardiac output rose similarly, the differences observed between the two types of portal hypertension depend mainly on the difference in distribution of flow within the splanchnic bed. Key words: blood flow, liver, portal hypertension. Introduction An increase in portal venous pressure is associated with changes in systemic haemodynamics [l-31. In patients with portal hypertension, no method is currently available to measure blood flow in splanchnic organs individually. The purpose of this Correspondence: Dr D. Lebrec, INSERM U 24, Hapita1 Beaujon, 921 18 Clichy, France. investigation was to study changes in splanchnic blood flow, by using the microsphere method, in rats with prehepatic portal hypertension due to portal vein stenosis and intrahepatic portal hypertension due to bile duct ligation. Methods Animals Three groups of adult male rats (Charles River, Saint-Aubin-L&Elbeuf, France), initially weighing 200-250 g, were studied: ten with portal hypertension due to portal vein stenosis, ten with portal hypertension due to bile duct ligation and ten control animals. Portal vein stenosis To induce prehepatic portal hypertension, partial portal vein ligation was performed according to the method of Halvorsen & Myking [4]. Under ether anaesthesia, the portal vein was exposed by median laparotomy. A polyethylene catheter of 0.96 mm outside diameter (Biotrol Pharma, Paris, France) was placed beside the portal vein. A ligature between the bifurcation of the portal vein and the junction of splenic and mesenteric veins was tied around both the catheter and the portal vein; the catheter was immediately removed and the abdominal incision closed. Bile duct ligation To induce intrahepatic portal hypertension, ligature of the common bile duct was performed according to the method of Franco et al. [S]. Under ether anaesthesia, the common bile duct was exposed by median laparotomy and occluded by double ligature with a non-resorbable suture 24 D, Lebrec and L. Blanchet (7-0 silk). The first tie was below the junction of the hepatic ducts, and the second was above the entrance of the pancreatic ducts. The common bile duct was then resected between the two ligatures and the abdominal incision closed. Two to 4 weeks after bile duct ligation, marked jaundice and mild ascites developed. Secondary biliary cirrhosis due to chronic bile duct ligation [5] was histologically confirmed in these rats. Haemodynamic measurements Haemodynamic studies were performed 3 weeks after partial portal vein ligation and 4 weeks after bile duct ligation. All rats then weighed 300350 g (after ligation of the bile duct, rats slightly decreased their weight and then return to normal). Rats were fasted 18 h before the haemodynamic study but were given water ad libitum. Haemodynamic studies were performed under pentobarbital anaesthesia (5 mg/100 g body wt. intraperitoneally); rats were fixed in a supine position; rectal temperature was maintained at 38'C. Splanchnic organ blood flow was measured by using the microsphere method peviously described [6-91. Under pentobarbital anaesthesia, the right common carotid artery was cannulated with a catheter of 0.70 mm outside diameter (William Cook Europe, ApS, Soborg, Denmark). The catheter was advanced into the left ventricle; its position was demonstrated by a left ventricular pressure curve and verified at autopsy. The left common carotid artery was then cannulated with a catheter of 0.96 mm outside diameter (Biotrol Pharma, Paris, France). This catheter was used for monitoring heart rate and arterial pressure (Telco, Gentilly, France) and for taking the reference blood sample (see below). Each catheter was flushed with 50 i.u. of heparin. Microspheres (15 f 3 pm diameter; sp. activity, 10 mCi/g; New England Nuclear, Boston, MA, U.A.S.), labelled with 14'Ce, were suspended in 10% glucose, and dispersed in an ultrasonic disintegrator (MSE, Scientific Instruments, Crawley, U.K.) for 10 min before injection. A 0.1 ml suspension of approx. 60 000 microspheres was injected into the left ventricle over 15 s; the catheter was then flushed with 0.5 ml of saline (154 mmol of NaC1/1). Starting 5 s before microsphere injection, the reference blood sample was withdrawn through the left carotid artery catheter, at a rate of 0.8 ml/min, for a period of 60 s (Syringe infusion withdrawal pump; Sage Instruments, Cambridge, MA, U.S.A.). The number of microspheres collected in this sample ranged from 400 to 600. After portal venous pressure measurement (see below), animals were killed by injection of 25 mg of pentobarbital. The stomach, intestine, colon, mesentery-pancreas (because of the difficulty in detaching all pancreas fragments of the mesentery in the rat, these organs were not divided), and liver were dissected, washed, dried with a compress, weighed, and placed in counting tubes. The amount of injected radioactivity was calculated from the difference between initial and residual radioactivity in the syringe. Radioactivity of the syringe, reference blood sample and removed organs was counted (c.p,m.) by gamma scintillation (Picker Nuclear Intertech, U.S.A.). The number of microspheres collected in the organs ranged from 400 to 12000. Distribution of cardiac output to various organs was calculated from the ratio of organ radioactivity to injected radioactivity. Organ perfusion was calculated according to the following formula: organ blood flow (ml/min) = (organ radioactivity/injected radioactivity x cardiac output) (ml/min). Cardiac output was calculated from the following formula: cardiac output (ml/min) = (injected radioactivity/reference blood sample radioactivity) x 0.8 (ml/min). Microsphere mixing was considered adequate because differences between right and left renal blood flow measurements [ 101 were insignificant. Arterial pressure was unaffected by left ventricular catheterization, by withdrawal of reference blood samples, o r by microspheres (except for a transient fall during injection). Within 5 min after microsphere injection median laparotomy was performed to measure portal venous pressure. A polyethylene catheter (0.7 mm outside diameter; William Cook Europe, ApS, Soborg, Denmark) was inserted into the junction of two small ileal veins and gently advanced into the portal vein. The correct position of the catheter was visually checked through the portal vein wall. Portal venous pressure was measured with the catheter and recorded on a square-wave electromagnetic manometer (Telco, Gentilly, France). The zero reference level was aligned with the right atrium. The adequacy of portal catheterization for pressure measurement was confirmed by rapid response of the tracing and easy aspiration of blood. Statistical analysis The values are expressed as means+ 1 SD. The Wilcoxon test for unpaired samples was used for statistical comparisons. Results The mean values for arterial pressure were not significantly different in the three groups [110+ 8 mmHg (mean ~ S D )in normal rats; 107 f 1 4 Splanchnic blood flow in portal hypertensive rats mmHg and 98 k 9 mmHg in rats with portal hypertension due to portal vein stenosis and bile duct ligation, respectively]. Mean values of blood flow to splanchnic organs are presented for all groups in Table 1 . In rats with portal hypertension due to portal vein stenosis, the total arterial flow to organs drained by the portal vein ranged from 21.12 to 29.91 ml/min (26.14 f 3.55 ml/min). Those with portal hypertension due to bile duct ligation ranged from 8.73 to 20.16 ml/min (14.65 f 3.39 ml/min). In the former group, the mean value was significantly higher than that found in normal rats, whereas in the latter group, the mean value was significantly lower than normal (Table 1). Spanchnic blood flow in the two groups ranged from 23.65 to 36.33 ml/min (29.82 f 3.91 ml/min) in rats with portal vein stenosis, and from 13.52 to 27.19 ml/min (20.73 f 4.06 ml/min) in rats with bile duct ligation; the mean value in the first group was significantly higher than that measured in normal rats, whereas, in the second group, the mean value was not significantly different from normal (Table 1). Distribution of cardiac output to various splanchnic organs in rats with portal hypertension due to portal vein stenosis or bile duct ligation, compared with normal rats, is shown in Table 2. In rats with portal hypertension due to portal vein stenosis, the percentage of cardiac output supply- 25 ing organs drained by the portal vein (stomach, intestine, colon, spleen and mesentery-pancreas) ranged from 18.87 to 32.49%,with a mean value (25.24%) significantly higher than that found in normal rats (20.77%). In those with portal hypertension due to bile duct ligation the distribution of cardiac output to the portal bed ranged from 9.22 to 22.84% with a mean value (15.10%) significantly lower than normal (Table 1). Distribution of cardiac output to splanchnic bed (portal venous bed and liver) in the two groups ranged from 22.51 t o 35.53% in rats with portal vein stenosis, and from 14.3'8 to 27.21% in rats with bile duct ligation. In the former group, the mean value was significantly higher than in normal rats, whereas, in the latter group, the mean value was not significantly different (Table 1). Splanchnic organ blood flow expressed per g tissue wt. in rats with portal hypertension due to portal vein stenosis or to bile duct ligation, and in normal rats, is summarized in Table 3. In rats with portal vein stenosis the differences with the control group of splanchnic organ blood flow expressed per g tissue wt. were similar to those expressed per ml/min except for mesentericpancreatic and hepatic blood flows (Table 3). In rats with bile duct ligation, blood flows expressed per g tissue wt. were different from those expressed per ml/min except for gastric, splenic and hepatic blood flows (Table 3). TABLE 1. Splanchnic organ blood flow, portal venous tributary blood flow and splanchnic blood flow in rats with portal hypertension due to portal vein stenosis or to bile duct ligation, and in normal rats Results are mean values f SD. Portal venous tributary blood flow is equal to the sum of gastric, intestinal, colonic, splenic and mesenteric-pancreatic blood flows. Hepatic blood flow is equal t o hepatic arterial blood flow. Splanchnic blood flow is equal to the sum of portal venous tributary blood flow and hepatic arterial blood flow. Blood flow (ml/min) Normal (n = 10) Gastric Intestinal Colonic Splenic Mesenteric-pancreatic Portal venous tributary Hepatic Splanchnic 0.592 0.06 1.67A 1.68 1.81 0.49 1.77 f 0.62 2.05 0.49 7.89* 1.70 1.99k0.52 19.88 k 2.02 * * Portal vein stenosis (n = 10) Bile duct ligation (n = 10) 0.84 2 0.36 *t 17.64*4.03$§ 2.12k 0.43*11 1.99* 0.43*11 3.55* 1.38**t 26.14i: 3.55tt§ 3.68i 1.78s $ $ 29.82k 3.91ttll 0.66f 0.24* 7.96* 2.44: 1.34* 0.55 1 . 0.53n ~ 3.57* 1.18$ 14.65i3.39n 6 . 0 8 2.09tt ~ 20.73f4.06* ~~ *Not significantly different from normal; 1- not significantly different from bile duct ligation; $significantlydifferentfrom normal (P< 0.01); § significantly different from bile duct ligation (P< 0.001); II significantly different from bile duct ligation (P< 0.01); 1[ significantly different from from normal (P< 0.001); $ $ significantly different from bile duct ligation (P< 0.02). D. Lebrec and L . Blanchet 26 TABLE 2 . Percentage of distribution of cardiac output to splanchnic organs, portal venous tributary, and splanchnic tributary in rats with portal hypertension due to portal vein stenosis or to bile duct ligation, and in normal rats Results are mean values f SD.Portal venous tributary is equal to the sum of percentages of cardiac output of the stomach, intestine, colon, spleen and mesentery-pancreas. Liver is equal to the percentage of cardiac output of the hepatic artery. Splanchnic tributary is equal to the sum of percentages of cardiac output of the portal venous tributary and the liver. Percentage of cardiac output Normal (n = 10) Stomach Intestine Colon Spleen Mesentery-uancreas Portal venobs tributary Liver Splanchnic tributary 0.69i 0.09 13.53 i 2.14 2.09i 0.56 2.09i0.66 2.37 f 0.57 20.77f 1.94 2.31 f 0.55 23.08f 1.90 Portal vein stenosis (n = 10) Bile duct ligation 0.78f0.25*t 17.17*4.60*$ 2.03*0.35*11 1.90t 0.30*11 3.36f 1.237 t 25.24+4.36**$ 3.63f 1.46711 28.87i 3.79ttll 0.71 f 0.27* 8.25 i 2.805 1.36i0.508 1.13 f 0.48 5 3.655 1.127 15.10i3.605 6.17i 1.87tt 21.27f 3.82* (n = 10) * Not significantly different from normal; t not significantly different from bile duct ligation; (P< 0.001); 5 significantly different from normal (P< 0.01); II significantly different from bile duct ligation (P< 0.01); ll significantly different from normal (P< 0.02): ** significantly different from normal (P< 0.05); tt significantly different from normal (P< 0.001). $. significantly different from bile duct ligation TABLE 3 . Splanchnic organ blood flow in rats with portal hypertension due to portal vein stenosis or to bile duct ligation, and in normal rats Results are mean values f SD. Hepatic is equal to hepatic arterial blood flow. Blood flow (ml min-' g-' tissue wt.) Normal In = 10) Gastric Intestinal Colonic Splenic Mesenteric-pancreatic Hepatic 0.41 i 0.08 2.32f 0.47 1.67* 0.54 2.63 f 0.94 0.62i 0.24 0.21 i 0.27 Portal vein stenosis In = 10) 0.66 f 0.23*t 3.65f 1.04*8 2.54f00.78*11 1.72 +0.30$11 0.97i 0.425t 0.52t0.41*t Bile duct ligation In = 10) 0.47i 0.145 1.88f 0.785 1.12f0.407 0.49t 0.32* 0.67 i 0.26$. 0.38i0.135 * Significantly different from normal (P< 0.01); t not significantly different from bile duct ligation; $not significantly different from normal; 5 significantly different from bile duct ligation (P< 0.01); II significantly different from bile duct ligation (P< 0.001); 11 significantly different from normal (P< 0.05). Mean values for cardiac output in rats with portal hypertension due to portal vein stenosis and in rats with portal hypertension due to bile duct ligation were: 104.95 5 16.80 ml/min and 97.50k 6.78 ml/min, respectively. No significant difference in cardiac output was found between these two groups of rats, whereas the mean values for cardiac output in these rats were significantly higher than in normal rats (86.75 f 6.71 ml/min) (P< 0.02 and P < 0.01, respectively). In rats with portal hypertension due t o portal vein stenosis and in those with portal hypertension due to bile duct ligation the mean values for portal venous pressure were 13.85 2.0 mmHg and 13.1 k 1.8 mmHg, respectively, and were significantly higher than those measured in normal rats (7.2 f 0.7 mmHg) (P< 0.001 in both). Discussion Methods used in the present study to induce portal hypertension in the rat were previously described [4-6, 81. The degree of portal hypertension was similar in all cases, but mechanisms and haemo- Splanchnic blood flow in portal hypertensive rats dynamic effects differed between the two groups. In rats with portal vein stenosis, portal hypertension is of so-called ‘prehepatic’ type: the liver is normal, neither liver failure nor liver fibrosis occurs, and, as shown by others, portosystemic collateral circulation is extensive [8, 111. In rats with bile duct ligation, portal hypertension is of the so-called ‘intrahepatic’ type: secondary biliary cirrhosis develops [S] associated with manifestations of liver failure (jaundice and ascites). The liver lesions are similar to those observed in rats with cirrhosis induced by repeated injections of CC14 [8]. In both types of portal hypertensive rats, it has already been demonstrated that by 3-4 weeks of the haemodynamic study, induced portal hypertension is constant, permanent and stable [4, 5, 81. In the present study, haemodynamic studies were performed in fasted rats and consequently splanchnic organ weights, mainly the intestine, as well as splanchnic organ blood flows markedly differ from fed animals [121. The two types of portal hypertension have opposite effects on both distribution of cardiac output to splanchnic organs and organ blood flow. In rats with portal hypertension due to portal vein stenosis, portal venous tributary blood flow increased, whereas in those with portal hypertension due to bile duct ligation, this tributary blood flow decreased. Such a discrepancy in results was marked with intestinal, colonic and splenic perfusion, but was minimal with gastric and mesenteric-pancreatic flow. Changes in splanchnic organ blood flow did not directly depend on changes in cardiac output, which increased in both groups, but mainly on the distribution of cardiac output. The increase in portal venous bed perfusion in rats with portal vein stenosis was the sum of an increase in both the percentage of cardiac output and the cardiac output itself. Such effects of portal hypertension on perfusion of splanchnic organs are not clearly understood; induced splanchnic resistance may differ between the two types of portal hypertension, thereby inducing various modifications in portal venous drainage. A sustained increase in hepatic arterial blood flow was observed in both types of portal hypertensive rats. This effect disappeared when hepatic arterial blood flow was expressed per g of liver tissue, and depended mainly on an increase in the fraction of cardiac output, rather than on a rise in cardiac output. Changes in hepatic arterial blood flow are related to changes in portal venous blood flow [13]. However, the correlation between hepatic arterial and portal venous blood flows probably does not directly depend upon portal 27 venous tributary blood flow, but rather upon portal liver perfusion. This perfusion is reduced in the two types of portal hypertensive rat, since a large amount of blood from the portal venous tributary directly reaches the systemic tributary through spontaneous portosystemic shunts [8,11]. The marked increase in hepatic arterial blood flow measured in rats with portal hypertension due to bile duct ligation has been previously reported in the dog [14-161. This increase is nearly twice that measured in rats with portal hypertension due to portal vein stenosis. The difference in the hepatic arterial blood flow in the two types of portal hypertensive rats is unclear. In those with bile duct ligation, total splanchnic blood flow increased slightly, because the increase in hepatic arterial blood flow compensated for the decrease in portal venous tributary blood flow. However, changes in splanchnic blood flow in these rats were less marked than those observed in rats with portal vein stenosis. The difference between the two types might be related to the degree of development of portosystemic collateral venous circulation. The haemodynamic changes common to both types of portal hypertension were a sustained elevation in portal venous pressure and an increase in cardiac output. The increase in cardiac output seems to be at least partially the consequence of the rise in portal venous pressure. This effect was recently demonstrated in rats [6, 171, and is a common response t o portal hypertension in man [3, 18-20]. The mechanism for increased cardiac output is unknown, but this study suggests two possibilities. In rats with portal vein stenosis and wide portosystemic collateral venous circulation, the increase in cardiac output may be due to the opened splanchnic shunt, as in rats with arteriovenous fistulae [21]. This relationship between the increase in cardiac output and the presence of portosystemic shunts may be compared with that observed in patients with portal hypertension due to portal vein obstruction; in these patients the liver is normal [20]. In rats with bile duct ligation, the increase seems to be due mainly to liver failure, a known effect in man with or without cirrhosis [22, 231. Acknowledgments The authors thank Dr C. Degott for histological examinations and Dr C. T. Nuzum and Miss C. Girod for their help. This work was supported by grant CRL 8 2 7 018 from the Institut National de la Sant6 et de la Recherche MBdicale. 28 D. Lebrec and L. Blanchet References 1. Bayley, T.J., Segel, N. & Bishop, J.M. 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