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Effects of reserpine administration in two models of portal hypertension in rats
Journal ofHepatology, 1993; 19:413-417
413
© 1993ElsevierScientificPublishers Ireland Ltd. All rights reserved. 0168-8278/93/$06.00 HEPAT 01454
Effects of reserpine administration in two models of portal hypertension in rats
H a n - C h i e h L i n a, P i - C h i n Y u b, S h o u - D o n g L e e a, Y a n g - T e Tsai c, J o n - S o n K u o d and May C.-M. Yang b "Division of Gastroenterology. Department of Medicine and hDepartment of Medical Research. Taipei Veterans General Hospital. 'Department of Medicine and dDepartment of Medical Research, Taichung Veterans General Hospital. Republic of ChhTa
(Received 5 November 1992)
The effects of reserpine were investigated in two models of portal hypertension in rats. Twenty-four hours after 1 mg/kg of reserpine was administered intraperitoneally to normal and portal vein stenosed rats, the cardiac index, mean arterial pressure, heart rate, and portal pressure were significantly decreased compared with normal and portal vein stenosed rats receiving placebo.'ln addition, the portal tributary blood flow was significantly decreased in portal vein stenosed rats receiving reserpine, but was unchanged in normal rats. In cirrhotic rats receiving a single dose of reserpine, 0.1 mg/kg intraperitoneally for 24 h, there were significant decreases in cardiac index, mean arterial pressure and heart rate compared with cirrhotic rats receiving placebo, while the portal pressure and portal tributary blood flow followed a decreasing trend after reserpine administration. The degree of hemodynamic change was similar in the groups of rats receiving reserpine, even though cirrhotic rats received lower doses than either normal or portal vein stenosed rats. This study suggests enhanced sympathetic nervous activity observed in cirrhotic rats. Key words: Hemodynamics; Cirrhosis; Sympathetic nervous activity
Portal hypertension is associated with marked hemodynamic changes characterized by increased cardiac ~utput and splanchnic blood flow, and decreased systemic vascular resistance and portal territory vascular resistance (1-4). The mechanisms underlying these hemodynamic changes are poorly understood. Recently, it has been suggested that sympathetic nervous activity might play a role in the pathogenesis of the hyperdynamic circulation associated with portal hypertension, since inhibition of central sympathetic outflow by clonidine reduced the severity of portal hypertension and hyperdynamic circulation in both portal hypertensive animals and cirrhotic patients (5-8). Moreover, it has been shown that sympathetic nervous activity is increased in patients with cirrhosis (9,10). However, the role of sympathetic nervous activity in the pathogenesis
of portal hypertension is not clearly established. Reserpine is known to reduce or deplete the catecholamine content in various tissues, and to exert its pharmacological action on circulation in this way. This mechanism is different from that of clonidine (11). The aim of this study was to investigate the effects of decreased sympathetic nervous activity following reserpine administration on systemic and splanchnic hemodynamics in two models of portal hypertension in rats. Methods Anhnals
Adult male Sprague-Dawley rats weighing between 300 and 350 g were used in the study. Two models of portal hypertension were produced by portal vein
Correspomlence to: May C.-M. Yang, Ph.D., Department of Medical Rcsearch. Taipei Veterans General Hospital. 201. Sec. 2. Shih-Pai Road, Taipei 112,Taiwan.
414 stenosis and by carbon tetrachloride-induced cirrhosis as previously described (12,13). In brief, under ether anesthesia, portal vein stenosis was induced using a 3-0 silk to ligate both the portal vein and a PE-50 polyethylene catheter. The catheter was then removed and a calibrated stenosis of the portal vein was produced (12). The abdomen was closed with catgut. Cirrhosis was induced by carbon tetrachloride. After induction with phenobarbital 0.33 mg/ml in the drinking water for 10 days, carbon tetrachloride was administered once a week by intragastric gavage for 10 consecutive weeks and phenobarbital was given continuously in the drinking water. Both treatments were discontinued 1 week before the experiments (13). All rats were caged at 24°C, with a 12-h light-dark cycle and allowed free access to food and water.
Study protocol Three groups of 14 rats each were studied. The groups consisted of normal (control) rats, rats with portal hypertension induced by portal vein stenosis and rats with cirrhosis induced by carbon tetrachloride. Reserpine is rapidly absorbed after either oral, subcutaneous or intraperitoneal administration (14), and i.p. injection has been commonly used in rats to study sympathetic nervous system and function (15-17). Withrington and Zaimis suggested that doses of at least 1 mg/kg of reserpine be used to 'reserpinize' animals (11). In addition, the hemodynamic effects of reserpine in rats has been evaluated at the dose of 2 mg/kg intraperitoneally (15). In a preliminary study, following i.p. injection of reserpine I mg/kg, marked reductions in heart rate and mean arterial pressure were observed in both normal and portal vein stenosed rats. Therefore, in this study, a single i.p. injection of reserpine I mg/kg (n = 7) and of placebo (n = 7) was administered to all rats in the group of normal and portal vein stenosed rats. On the other hand, in the preliminary study cirrhotic rats showed severe bradycardia and hypotension after an i.p. injection of 1 mg/kg of reserpine and could not tolerate the experimental procedure. Severe bradycardia and hypotension were still noted in cirrhotic rats when the dose of reserpine was reduced to 0.3 mg/kg. Finally, 0.1 mg/kg of reserpine administration in cirrhotic rats resulted in similar changes in heart rate and mean arterial pressure compared with normal and portal vein stenosed rats receiving reserpine 1 mg/kg. Therefore, in the present study, a single i.p. injection of 0.1 mg/kg of reserpine was administered to cirrhotic rats (n = 7) and the results were compared with cirrhotic rats receiving placebo (n = 7). Twenty-four hours following reserpine administra-
H.-C. LINet al. tion, hemodynamic parameters were measured for comparison between rats treated with reserpine and placebo in each group. Administration of reserpine or placebo was made 13 days after the induction of portal vein stenosis and 10 weeks after induction of cirrhosis by carbon tetrachloride.
Hemodynamic measurements All rats were fasted 18 h before the hemodynamic studies and had free access to water. Under pentobarbital sodium anesthesia (50 mg/kg, i.p.), a tracheostomy was performed to keep the airway patent. A catheter was inserted into the left ventricle via the right carotid artery for radioactive microspheres injection. Correct positioning of the catheter was confirmed by blood pressure tracing. A femoral artery catheter was also inserted to monitor the arterial pressure and heart rate, and to withdraw the reference blood sample. The abdomen was then opened via a midline incision, and the portal vein was cannulated via a small ileal vein for measurement of portal pressure. The rectal temperature was maintained at 37°C by a heating lamp. All pressures were measured and recorded by a multi-channel recorder (model RS 3400, Gould Inc., Cupertino, CA, USA). After the hemodynamic values were stabilized, cardiac output and regional organ blood flows were measured using the radioactive microsphere technique with the reference sample method as previously described (12). In brief, the reference sample was withdrawn from the femoral artery into a syringe for 75 s at a rate of 0.8 ml/min using a Harvard pump (Harvard Apparatus, Millis, MA, USA). Ten seconds after the withdrawal of the reference sample, approximately 60 000 57Co-labeled microspheres of 15 /.tin diameter (New England Nuclear, Boston, MA, USA) were injected into the left ventricle over 25-30 s. The catheter was then flushed with 0.5 ml of 0.9% saline. After hemodynamic measurements, the animals were killed with a bolus of saturated KCI, and the individual organs were dissected, The radioactivity of each organ and the reference blood sample were counted in a zscintillation counter (Auto Gamma 5000, Packard, Downers Grove, IL, USA). Adequate mixing of microspheres was assumed when the difference of radioactivity between the left and right kidney was below 10%. Cardiac output (CO) was calculated by the following formula: CO (ml/min) = radioactivity injected (counts/min) reference sample radioactivity (counts/rain)
x 0.8 (ml/min)
415
RESERPINE AND PORTAL HYPERTENSION
Cardiac index (CI) was derived from the following formula:
temic vascular resistance (SVR) was calculated according to the following formula:
CO
CI ( m l . m i n -~. 100 g-t b o d y wt.) =
mean arterial pressure (MAP, mmHg)
SVR =
100 g b o d y wt.
x 80
CI
Regional organ blood flows were calculated according to the following formula:
Portal territory vascular calculated as follows: MAP-
organ b l o o d flow ( m l / m i n ) =
resistance
(PTVR)
was
portal pressure ( m m H g )
PTVR-
x 80 PTBF
o r g a n radioactivity ( c o u n t s / m i n ) x CO
V a s c u l a r r e s i s t a n c e s w e r e e x p r e s s e d as d y n . s . c m -5 x
radioactivity injected
Hepatic arterial blood flow was expressed as ml.min -I. 100 g-t body weight Portal tributary blood flow (PTBF, expressed in ml.min -I • 100 g-i body wt.) was taken as the sum of spleen, stomach, small bowel, colon, and mesentery with pancreas blood flows. Sys-
10 3. 100 g - I
body wt.
Statistical analysis Results were expressed as mean ± S.E.M. In each group of rats, the unpaired Student's t-test was used for
tABLE I Hemodynamic values in rat receiving reserine and placebo Normal rats receiving
Portal vein stenosed rats receiving
Cirrhotic rats receiving
placebo (n = 7)
reserpine I mg/kg (n = 7)
placebo (n = 7)
reserpine 1 mg/kg (n = 7)
placebo (n = 7)
reserpine 0.1 mg/kg (n = 7)
22.0 .4- 1.6'
35.7 .4- 1.0"***
27.7 .4- 1.1"*
33.8 ± 0.9****
26.4 .4- 2.1"
Cardiac index (ml-min -~27.1 .4- 1.5 100 g-J body wt.) Mean arterial pres- 140 ± 6 sure (mmHg) Ileart rate 362 ± 9 (beats/min) Systemic vascular 130 ± I I resistance ( d y n . s . c m -5 x 103. 100 g-i body wt.) Portal pressure 6.9 .4- 0.5 (mmHg) Portal tributary 3.75 .4- 0.34 blood flow (ml. min -j . 100 -I body wt.) Hepatic arterial 1.22 .4- 0.19 blood flow (ml. min -I . 100 -I body wt) Portal territory 914.4- 78 vascular resistance (dyn. s. cm-5 x 103 • 100 g-I body wt.)
105 .4- 2**
125 ± 5
95 .4- 2**
266 .4- 16"*
347 ± 13
281 ± 16"*
115 .4- 8
116 .4- 7****
II0 ± 7
5.1 ± 0.6*
14.3 .4- 0.4****
11.8 .4- 0.6**.*****
14.7 .4- 0.5****
12.7 ± 0.9*****
3.52 .4- 0.47
6.07 .4- 0.54***
4.43 ± 0.47*
5.49 .4- 0.70***
3.55 ± 0.79
0.83 .4- 0.14
1.69 4- 0.22***
1.08 ± 0.26
0.86 .4- 0.19
1.66 ± 0.50
738 ± 93
653 .4- 97****
641 .4- 85
Results were expressed as mean .4- S.E.M. Reserpine was administered by intraperitoneal injection. *P < 0.05 vs. rats receiving placebo. **P < 0.01 vs. rats receiving placebo. ***P < 0.05 vs. normal rats receiving placebo. ****P < 0.01 vs. normal rats receiving placebo. *****P < 0.01 vs. normal rats receiving reserpine.
134 ± 6
101 .4- 4**
369 .4- 18
265 .4- 12"*
88 .4- 6****
518 .4- 60****
84 .4- 10
686 .4- 154
416 comparison between rats receiving reserpine and those receiving placebo. One way analysis of variance with Scheffe's correction was used for statistical analysis in three groups of rats receiving reserpine and in three groups of rats receiving placebo, respectively. P-values < 0.05 were considered statistically significant.
Results
The hemodynamic values are shown in Table 1. In rats receiving placebo, the portal pressure, portal tributary blood flow, and cardiac index were significantly higher in portal vein-stenosed and cirrhotic rats than in normal rats. In contrast, the systemic vascular resistance and portal territory vascular resistance were significantly lower in both models of portal hypertensive rats than in normal rats. On the other hand, in normal and portal vein-stenosed rats that received I mg/kg of reserpine and in cirrhotic rats receiving 0.1 mg/kg of reserpine, there were no significant differences in hemodynamic values except portal pressure which was significantly higher in portal vein-stenosed and cirrhotic rats than in normal rats. In addition, following reserpine administration, the degree of reduction in the mean value of mean arterial pressure was nearly identical in normal (-25%), portal vein-stenosed (-24%) and cirrhotic (-25%) rats. Similarly, the degree of change in the mean value of cardiac index was nearly the same in normal ( - 19%), portal vein-stenosed (-22%) and cirrhotic (-22%) rats after reserpine administration, In normal rats, administration of I mg/kg of reserpine resulted in significant decreases in the cardiac index, heart rate, mean arterial pressure, and portal pressure compared with rats receiving placebo. However, there were no significant differences in the systemic vascular resistance, portal territory vascular resistance, hepatic arterial blood flow, and portal tributary blood flow between rats receiving reserpine and placebo. In portal vein-stenosed rats, the cardiac index, heart rate, mean arterial pressure, portal pressure, and portal tributary blood flow were significantly decreased in rats receiving 1 mg/kg of reserpine compared with those receiving placebo. In cirrhotic rats, the cardiac index, heart rate and mean arterial pressure were significantly decreased in rats receiving 0.1 mg/kg of reserpine compared with those receiving placebo. The portal pressure and portal tributary blood flow were lower (P = 0.07, respectively) in cirrhotic rats receiving reserpine than in those receiving placebo. As in the normal rats, the systemic vascular resistance, portal territory vascular resistance and hepatic arterial blood flow were not
H.-C. LIN et
al.
significantly altered in either model of portal hypertensive rat following reserpine administration.
Discussion
In the present study, significant reductions in heart rate, mean arterial pressure and cardiac index were clearly demonstrated in the three groups of animals studied, thus indicating a decreased cardiovascular sympathetic tone caused by reserpine (15,18). However, the dose of reserpine given to cirrhotic rats to produce similar reductions in cardiac index, heart rate and mean arterial pressure was one-tenth of the dose given to both normal and portal vein stenosed rats. The lower dose required for cirrhotic rats could be due to decreased hepatic elimination of reserpine or enhanced sympathetic nervous activity observed in cirrhosis. Previous studies reported that hydrolytic reactions are one of the major ways reserpine is broken down (19,20). In addition, enzymes such as esterases which hydrolyze reserpine are found not only in plasma and in the liver but also in the intestinal mucosa, and in vivo administration of reserpine is probably metabolized by a number of non-hepatic esterases (14). Therefore, the smaller dose required for cirrhotic rats may not be due to a decreased hepatic elimination of the drug. Results of this study suggest that sympathetic nervous activity may be enhanced in cirrhosis. In other words, the increased sympathetic nervous activity in cirrhosis may be associated with the presence of liver disease or increase in sinusoidal pressure. In contrast, in portal hypertension ofa presinusoidal origin and normal liver function, sympathetic nervous activity is not increased. Similar observations regarding sympathetic nervous activity in portal hypertension have been demonstrated by Gaudin and coworkers (21,22). The mechanism of liver disease and sinusoidal portal hypertension in the pathogenesis of enhanced sympathetic nervous activity is not clearly established. However, Kostreva et al. suggested that a hepatic baroreceptor may play a role in the activation of sympathetic nervous activity observed in cirrhosis (23). In this study, systemic vascular resistance was unaffected despite decreased cardiovascular sympathetic tone following reserpine administration. The lack of effect of reserpine on systemic vascular resistance may be due to the decrease in both blood pressure and cardiac index, since the resistance was calculated by these two factors. Alternatively, the effect of other endogenous vasoactive substances in response to hypotension, such as the renin-angiotensin system and vasopressin may also play a role. A similar effect on systemic vascu-
417
RESERPINE AND PORTAL HYPERTENSION
lar resistance has been demonstrated in dogs receiving reserpine (18). In addition, the portal territory vascular resistance was not affected despite a significant decrease in arterial pressure following reserpine administration. The absence of reflex-mediated splanchnic vasoconstriction was mostly due to depleted catecholamines stores in various tissues which subsequently abolished this reflex (18). In conclusion, results of this study showed that the degrees of hemodynamic responses to reserpine administration were similar in the three groups of rats. At the same time, the dose required for cirrhotic rats was lower than for normal and portal vein stenosed rats thus suggesting enhanced sympathetic nervous activity in the cirrhotic animals.
Acknowledgements
7 8
9 10 II 12 13 14 15
This work was supported by Grant No. DOH82-HRC08 from the National Health Research Institutes, Taiwan, Republic of China.
16 17
References 18 I 2
3 4
5 6
Lebrec D, Blanchet L. Effect of two models of portal hypertension on splanchnic organ blood flow in rat. Clin Sci 1985: 68: 23-8. VorobioffJ, Bredfeldt JE. Groszmann RJ. Hyperdynamic circulation in portal-hypertensive rat model: a primary factor for maintenance of chronic portal hypertension. Am J Physiol 1983: 244: G52-7. VorobioffJ, Bredfeldt JE, Groszmann RJ. Increased blood flow through the portal system in cirrhotic rats. Gastroenterology 1984; 87: 1120-6. Bosch J, Enriquze R. Groszmann RJ. Storer EH. Chronic bile duct ligation in the dog: hemodynamic characterization of a portal hypertensive model. Hepatology 1983: 3: 1002-7. Roulot D, Braillon A, Gaudin C. Ozier Y. Girod C, Lcbrec D. Mechanism of a clonidine-induced decrease in portal pressure in normal and cirrhotic rats. Hepatology 1989: 10: 477-81. Lin HC, Soubrane O. Lebrec D. Prevention of portal hyperten-
19 20 21
22 23
sion and portal-systemic shunts by early chronic administration of clonidine in conscious portal vein stenosed rats. Hepatology 1991; 14: 325-30. Willet IR, Esler M, Jennings G, Dudley FJ. Sympathetic tone modulates portal venous pressure in alcoholic cirrhosis. Lancet 1986; ii: 939-43. Moreau R, Lee SS, Hadengue A, Braillon A, Lebrec D. Hemodynamic effects of a clonidine-induced decrease in sympathetic tone in patients with cirrhosis. Hepatology 1987; 7: 149-54. Bichet DG, Van Putten VJ, Schrier RW. Potential role of increased sympathetic activity in impaired sodium and water excretion in cirrhosis. N Engl J Med 1982; 307: 1552-7. Wilier I, Esler M, Burke F, Leonard P, Dudley F. Total and renal sympathetic nervous system activity in alcoholic cirrhosis. J Hepatol 1985; I: 639-48. Withrington P, Zaimis E. The reserpine-treated cat. Br J Pharmacol 1961: 17: 380-91. Yang MCM, Pang PKT, Lay CS, et al. Effects of parathyroid hormone on portal pressure in portal hypertensive rats. Liver 1990: 10: 11-6. Chiang JH, Cheng HC, Yang MCM, et al. Lung deposit of lipiodol in normal and cirrhotic rats. Acta Radiol 1991; 32: 474-8. Stitzel RE. The biological fate of reserpine. Pharmacol Rev 1977: 28: 179-205. Kisin I. Yuzhakov S. Effects of reserpine, guanethidine and methyldopa on cardiac output and its distribution. Eur J Pharmacol 1976: 35: 253-60. Clarke DE. Restoration of tyramine responses by bretylium, BW392C60, bethanidine and monoamine oxidase inhibitors in reserpine-treated rats. Br J Pharmacol 1970; 38:1-1 I. Nadeau R, Champlain J. Comparative effects of 6-hydroxydopamine and of reserpine on quabain toxicity in the rat. Life Sci 1973; 13: 1753-61. Jandhyala BS, Cavero I, Adams HR. Smookler HH, Dixit BN, Buckley J P. Cardiovascular effects of chronic reserpine administration in mongrel dogs. Eur J Pharmacol 1971: 16: 261-70. Glasko A J, Dill WA, Wolf LM, Kasenko A. Studies on the riletabolism of reserpine. J Pharmacol Exp Ther 1956; 118: 377-87. Numerof P, Gordon M. Kelley J M. The metabolism or reserpine. I. Studies in the mouse with C-14 labeled reserpine. J Pharmacol Exp Ther 1955: 115: 427-31. Gaudin C, Braillon A, Poo JL, Kleber G, Moreau R, Lebrec D. Plasma catecholamines in patients with presinusoidal portal hypertension: comparison with cirrhotic patients and nonportal hypertensive subjects. Hepatology 1991; 13: 913-6. Gaudin C. Ruget G, Braillon A, Selz F, Cuche JL, Lebrec D. Portal and arterial free and conjugated noradrenalin in two models of portal hypertension in rats. Life Sci 1989: 45: 1333-9. Kostreva DR, Castaner A, Kampine JP. Reflex effects of hepatic baroreceptors on renal and cardiac sympathetic nerve activity. Am J Physiol 1980: 238: R390-4.
413
© 1993ElsevierScientificPublishers Ireland Ltd. All rights reserved. 0168-8278/93/$06.00 HEPAT 01454
Effects of reserpine administration in two models of portal hypertension in rats
H a n - C h i e h L i n a, P i - C h i n Y u b, S h o u - D o n g L e e a, Y a n g - T e Tsai c, J o n - S o n K u o d and May C.-M. Yang b "Division of Gastroenterology. Department of Medicine and hDepartment of Medical Research. Taipei Veterans General Hospital. 'Department of Medicine and dDepartment of Medical Research, Taichung Veterans General Hospital. Republic of ChhTa
(Received 5 November 1992)
The effects of reserpine were investigated in two models of portal hypertension in rats. Twenty-four hours after 1 mg/kg of reserpine was administered intraperitoneally to normal and portal vein stenosed rats, the cardiac index, mean arterial pressure, heart rate, and portal pressure were significantly decreased compared with normal and portal vein stenosed rats receiving placebo.'ln addition, the portal tributary blood flow was significantly decreased in portal vein stenosed rats receiving reserpine, but was unchanged in normal rats. In cirrhotic rats receiving a single dose of reserpine, 0.1 mg/kg intraperitoneally for 24 h, there were significant decreases in cardiac index, mean arterial pressure and heart rate compared with cirrhotic rats receiving placebo, while the portal pressure and portal tributary blood flow followed a decreasing trend after reserpine administration. The degree of hemodynamic change was similar in the groups of rats receiving reserpine, even though cirrhotic rats received lower doses than either normal or portal vein stenosed rats. This study suggests enhanced sympathetic nervous activity observed in cirrhotic rats. Key words: Hemodynamics; Cirrhosis; Sympathetic nervous activity
Portal hypertension is associated with marked hemodynamic changes characterized by increased cardiac ~utput and splanchnic blood flow, and decreased systemic vascular resistance and portal territory vascular resistance (1-4). The mechanisms underlying these hemodynamic changes are poorly understood. Recently, it has been suggested that sympathetic nervous activity might play a role in the pathogenesis of the hyperdynamic circulation associated with portal hypertension, since inhibition of central sympathetic outflow by clonidine reduced the severity of portal hypertension and hyperdynamic circulation in both portal hypertensive animals and cirrhotic patients (5-8). Moreover, it has been shown that sympathetic nervous activity is increased in patients with cirrhosis (9,10). However, the role of sympathetic nervous activity in the pathogenesis
of portal hypertension is not clearly established. Reserpine is known to reduce or deplete the catecholamine content in various tissues, and to exert its pharmacological action on circulation in this way. This mechanism is different from that of clonidine (11). The aim of this study was to investigate the effects of decreased sympathetic nervous activity following reserpine administration on systemic and splanchnic hemodynamics in two models of portal hypertension in rats. Methods Anhnals
Adult male Sprague-Dawley rats weighing between 300 and 350 g were used in the study. Two models of portal hypertension were produced by portal vein
Correspomlence to: May C.-M. Yang, Ph.D., Department of Medical Rcsearch. Taipei Veterans General Hospital. 201. Sec. 2. Shih-Pai Road, Taipei 112,Taiwan.
414 stenosis and by carbon tetrachloride-induced cirrhosis as previously described (12,13). In brief, under ether anesthesia, portal vein stenosis was induced using a 3-0 silk to ligate both the portal vein and a PE-50 polyethylene catheter. The catheter was then removed and a calibrated stenosis of the portal vein was produced (12). The abdomen was closed with catgut. Cirrhosis was induced by carbon tetrachloride. After induction with phenobarbital 0.33 mg/ml in the drinking water for 10 days, carbon tetrachloride was administered once a week by intragastric gavage for 10 consecutive weeks and phenobarbital was given continuously in the drinking water. Both treatments were discontinued 1 week before the experiments (13). All rats were caged at 24°C, with a 12-h light-dark cycle and allowed free access to food and water.
Study protocol Three groups of 14 rats each were studied. The groups consisted of normal (control) rats, rats with portal hypertension induced by portal vein stenosis and rats with cirrhosis induced by carbon tetrachloride. Reserpine is rapidly absorbed after either oral, subcutaneous or intraperitoneal administration (14), and i.p. injection has been commonly used in rats to study sympathetic nervous system and function (15-17). Withrington and Zaimis suggested that doses of at least 1 mg/kg of reserpine be used to 'reserpinize' animals (11). In addition, the hemodynamic effects of reserpine in rats has been evaluated at the dose of 2 mg/kg intraperitoneally (15). In a preliminary study, following i.p. injection of reserpine I mg/kg, marked reductions in heart rate and mean arterial pressure were observed in both normal and portal vein stenosed rats. Therefore, in this study, a single i.p. injection of reserpine I mg/kg (n = 7) and of placebo (n = 7) was administered to all rats in the group of normal and portal vein stenosed rats. On the other hand, in the preliminary study cirrhotic rats showed severe bradycardia and hypotension after an i.p. injection of 1 mg/kg of reserpine and could not tolerate the experimental procedure. Severe bradycardia and hypotension were still noted in cirrhotic rats when the dose of reserpine was reduced to 0.3 mg/kg. Finally, 0.1 mg/kg of reserpine administration in cirrhotic rats resulted in similar changes in heart rate and mean arterial pressure compared with normal and portal vein stenosed rats receiving reserpine 1 mg/kg. Therefore, in the present study, a single i.p. injection of 0.1 mg/kg of reserpine was administered to cirrhotic rats (n = 7) and the results were compared with cirrhotic rats receiving placebo (n = 7). Twenty-four hours following reserpine administra-
H.-C. LINet al. tion, hemodynamic parameters were measured for comparison between rats treated with reserpine and placebo in each group. Administration of reserpine or placebo was made 13 days after the induction of portal vein stenosis and 10 weeks after induction of cirrhosis by carbon tetrachloride.
Hemodynamic measurements All rats were fasted 18 h before the hemodynamic studies and had free access to water. Under pentobarbital sodium anesthesia (50 mg/kg, i.p.), a tracheostomy was performed to keep the airway patent. A catheter was inserted into the left ventricle via the right carotid artery for radioactive microspheres injection. Correct positioning of the catheter was confirmed by blood pressure tracing. A femoral artery catheter was also inserted to monitor the arterial pressure and heart rate, and to withdraw the reference blood sample. The abdomen was then opened via a midline incision, and the portal vein was cannulated via a small ileal vein for measurement of portal pressure. The rectal temperature was maintained at 37°C by a heating lamp. All pressures were measured and recorded by a multi-channel recorder (model RS 3400, Gould Inc., Cupertino, CA, USA). After the hemodynamic values were stabilized, cardiac output and regional organ blood flows were measured using the radioactive microsphere technique with the reference sample method as previously described (12). In brief, the reference sample was withdrawn from the femoral artery into a syringe for 75 s at a rate of 0.8 ml/min using a Harvard pump (Harvard Apparatus, Millis, MA, USA). Ten seconds after the withdrawal of the reference sample, approximately 60 000 57Co-labeled microspheres of 15 /.tin diameter (New England Nuclear, Boston, MA, USA) were injected into the left ventricle over 25-30 s. The catheter was then flushed with 0.5 ml of 0.9% saline. After hemodynamic measurements, the animals were killed with a bolus of saturated KCI, and the individual organs were dissected, The radioactivity of each organ and the reference blood sample were counted in a zscintillation counter (Auto Gamma 5000, Packard, Downers Grove, IL, USA). Adequate mixing of microspheres was assumed when the difference of radioactivity between the left and right kidney was below 10%. Cardiac output (CO) was calculated by the following formula: CO (ml/min) = radioactivity injected (counts/min) reference sample radioactivity (counts/rain)
x 0.8 (ml/min)
415
RESERPINE AND PORTAL HYPERTENSION
Cardiac index (CI) was derived from the following formula:
temic vascular resistance (SVR) was calculated according to the following formula:
CO
CI ( m l . m i n -~. 100 g-t b o d y wt.) =
mean arterial pressure (MAP, mmHg)
SVR =
100 g b o d y wt.
x 80
CI
Regional organ blood flows were calculated according to the following formula:
Portal territory vascular calculated as follows: MAP-
organ b l o o d flow ( m l / m i n ) =
resistance
(PTVR)
was
portal pressure ( m m H g )
PTVR-
x 80 PTBF
o r g a n radioactivity ( c o u n t s / m i n ) x CO
V a s c u l a r r e s i s t a n c e s w e r e e x p r e s s e d as d y n . s . c m -5 x
radioactivity injected
Hepatic arterial blood flow was expressed as ml.min -I. 100 g-t body weight Portal tributary blood flow (PTBF, expressed in ml.min -I • 100 g-i body wt.) was taken as the sum of spleen, stomach, small bowel, colon, and mesentery with pancreas blood flows. Sys-
10 3. 100 g - I
body wt.
Statistical analysis Results were expressed as mean ± S.E.M. In each group of rats, the unpaired Student's t-test was used for
tABLE I Hemodynamic values in rat receiving reserine and placebo Normal rats receiving
Portal vein stenosed rats receiving
Cirrhotic rats receiving
placebo (n = 7)
reserpine I mg/kg (n = 7)
placebo (n = 7)
reserpine 1 mg/kg (n = 7)
placebo (n = 7)
reserpine 0.1 mg/kg (n = 7)
22.0 .4- 1.6'
35.7 .4- 1.0"***
27.7 .4- 1.1"*
33.8 ± 0.9****
26.4 .4- 2.1"
Cardiac index (ml-min -~27.1 .4- 1.5 100 g-J body wt.) Mean arterial pres- 140 ± 6 sure (mmHg) Ileart rate 362 ± 9 (beats/min) Systemic vascular 130 ± I I resistance ( d y n . s . c m -5 x 103. 100 g-i body wt.) Portal pressure 6.9 .4- 0.5 (mmHg) Portal tributary 3.75 .4- 0.34 blood flow (ml. min -j . 100 -I body wt.) Hepatic arterial 1.22 .4- 0.19 blood flow (ml. min -I . 100 -I body wt) Portal territory 914.4- 78 vascular resistance (dyn. s. cm-5 x 103 • 100 g-I body wt.)
105 .4- 2**
125 ± 5
95 .4- 2**
266 .4- 16"*
347 ± 13
281 ± 16"*
115 .4- 8
116 .4- 7****
II0 ± 7
5.1 ± 0.6*
14.3 .4- 0.4****
11.8 .4- 0.6**.*****
14.7 .4- 0.5****
12.7 ± 0.9*****
3.52 .4- 0.47
6.07 .4- 0.54***
4.43 ± 0.47*
5.49 .4- 0.70***
3.55 ± 0.79
0.83 .4- 0.14
1.69 4- 0.22***
1.08 ± 0.26
0.86 .4- 0.19
1.66 ± 0.50
738 ± 93
653 .4- 97****
641 .4- 85
Results were expressed as mean .4- S.E.M. Reserpine was administered by intraperitoneal injection. *P < 0.05 vs. rats receiving placebo. **P < 0.01 vs. rats receiving placebo. ***P < 0.05 vs. normal rats receiving placebo. ****P < 0.01 vs. normal rats receiving placebo. *****P < 0.01 vs. normal rats receiving reserpine.
134 ± 6
101 .4- 4**
369 .4- 18
265 .4- 12"*
88 .4- 6****
518 .4- 60****
84 .4- 10
686 .4- 154
416 comparison between rats receiving reserpine and those receiving placebo. One way analysis of variance with Scheffe's correction was used for statistical analysis in three groups of rats receiving reserpine and in three groups of rats receiving placebo, respectively. P-values < 0.05 were considered statistically significant.
Results
The hemodynamic values are shown in Table 1. In rats receiving placebo, the portal pressure, portal tributary blood flow, and cardiac index were significantly higher in portal vein-stenosed and cirrhotic rats than in normal rats. In contrast, the systemic vascular resistance and portal territory vascular resistance were significantly lower in both models of portal hypertensive rats than in normal rats. On the other hand, in normal and portal vein-stenosed rats that received I mg/kg of reserpine and in cirrhotic rats receiving 0.1 mg/kg of reserpine, there were no significant differences in hemodynamic values except portal pressure which was significantly higher in portal vein-stenosed and cirrhotic rats than in normal rats. In addition, following reserpine administration, the degree of reduction in the mean value of mean arterial pressure was nearly identical in normal (-25%), portal vein-stenosed (-24%) and cirrhotic (-25%) rats. Similarly, the degree of change in the mean value of cardiac index was nearly the same in normal ( - 19%), portal vein-stenosed (-22%) and cirrhotic (-22%) rats after reserpine administration, In normal rats, administration of I mg/kg of reserpine resulted in significant decreases in the cardiac index, heart rate, mean arterial pressure, and portal pressure compared with rats receiving placebo. However, there were no significant differences in the systemic vascular resistance, portal territory vascular resistance, hepatic arterial blood flow, and portal tributary blood flow between rats receiving reserpine and placebo. In portal vein-stenosed rats, the cardiac index, heart rate, mean arterial pressure, portal pressure, and portal tributary blood flow were significantly decreased in rats receiving 1 mg/kg of reserpine compared with those receiving placebo. In cirrhotic rats, the cardiac index, heart rate and mean arterial pressure were significantly decreased in rats receiving 0.1 mg/kg of reserpine compared with those receiving placebo. The portal pressure and portal tributary blood flow were lower (P = 0.07, respectively) in cirrhotic rats receiving reserpine than in those receiving placebo. As in the normal rats, the systemic vascular resistance, portal territory vascular resistance and hepatic arterial blood flow were not
H.-C. LIN et
al.
significantly altered in either model of portal hypertensive rat following reserpine administration.
Discussion
In the present study, significant reductions in heart rate, mean arterial pressure and cardiac index were clearly demonstrated in the three groups of animals studied, thus indicating a decreased cardiovascular sympathetic tone caused by reserpine (15,18). However, the dose of reserpine given to cirrhotic rats to produce similar reductions in cardiac index, heart rate and mean arterial pressure was one-tenth of the dose given to both normal and portal vein stenosed rats. The lower dose required for cirrhotic rats could be due to decreased hepatic elimination of reserpine or enhanced sympathetic nervous activity observed in cirrhosis. Previous studies reported that hydrolytic reactions are one of the major ways reserpine is broken down (19,20). In addition, enzymes such as esterases which hydrolyze reserpine are found not only in plasma and in the liver but also in the intestinal mucosa, and in vivo administration of reserpine is probably metabolized by a number of non-hepatic esterases (14). Therefore, the smaller dose required for cirrhotic rats may not be due to a decreased hepatic elimination of the drug. Results of this study suggest that sympathetic nervous activity may be enhanced in cirrhosis. In other words, the increased sympathetic nervous activity in cirrhosis may be associated with the presence of liver disease or increase in sinusoidal pressure. In contrast, in portal hypertension ofa presinusoidal origin and normal liver function, sympathetic nervous activity is not increased. Similar observations regarding sympathetic nervous activity in portal hypertension have been demonstrated by Gaudin and coworkers (21,22). The mechanism of liver disease and sinusoidal portal hypertension in the pathogenesis of enhanced sympathetic nervous activity is not clearly established. However, Kostreva et al. suggested that a hepatic baroreceptor may play a role in the activation of sympathetic nervous activity observed in cirrhosis (23). In this study, systemic vascular resistance was unaffected despite decreased cardiovascular sympathetic tone following reserpine administration. The lack of effect of reserpine on systemic vascular resistance may be due to the decrease in both blood pressure and cardiac index, since the resistance was calculated by these two factors. Alternatively, the effect of other endogenous vasoactive substances in response to hypotension, such as the renin-angiotensin system and vasopressin may also play a role. A similar effect on systemic vascu-
417
RESERPINE AND PORTAL HYPERTENSION
lar resistance has been demonstrated in dogs receiving reserpine (18). In addition, the portal territory vascular resistance was not affected despite a significant decrease in arterial pressure following reserpine administration. The absence of reflex-mediated splanchnic vasoconstriction was mostly due to depleted catecholamines stores in various tissues which subsequently abolished this reflex (18). In conclusion, results of this study showed that the degrees of hemodynamic responses to reserpine administration were similar in the three groups of rats. At the same time, the dose required for cirrhotic rats was lower than for normal and portal vein stenosed rats thus suggesting enhanced sympathetic nervous activity in the cirrhotic animals.
Acknowledgements
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9 10 II 12 13 14 15
This work was supported by Grant No. DOH82-HRC08 from the National Health Research Institutes, Taiwan, Republic of China.
16 17
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