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Effect of propranolol on surgical bleeding and hemostasis: An in vivo study in the rat
JOURNAL
OF SURGICAL
36,89-9 1 ( 1984)
RESEARCH
Effect of Propranolol on Surgical Bleeding and Hemostasis: An in Vivo Study in the Rat J. J. B. VAN LANSCHOT,
M.D.,
R. G. M. TEN
BERG,PH.D.,
AND H. A. BRUINING,
Laboratory for Experimental Surgery, Erasmus University, Dr. Molenaterpleim The Netherlands
M.D.
50, 3015 GE, Rotterdam.
Submitted for publication November 10, 1982 The influence of propranolol on surgical bleeding and hemostasis was studied in two different models in the normotensive rat. Propranolol was administered for 6 days subcutaneously and for 28 days orally. In both the tail and the muscle bleeding test the blood loss after the short-term as well as the long-term administration of propranolol was diminished as compared to control values. Highly significant difkrences were reached in the tail bleeding test. Previous studies have described the effect of propranolol on several constituents of the highly complex hemostatic process. Some of these effects enhance, others inhibit hemostasis. It is concluded that in the normotensive rat these hemostasis-enhancing effectsare of greater importance than the inhibiting ones.
to the water intake in such a way, that the average dose was about 10 mg/kg/day (n = 15). In both experiments nontreated rats were used as control in equal numbers (n = 10 and 15, respectively). In all rats, two bleeding tests were performed under pentobarbitone anesthesia (Nembutal, 60 mg/kg ip). in the muscle bleeding test [9] the skin above the side of each hind paw was opened and the M. biceps femoris was cut through longitudinally with a razor blade (length = 11 mm) without damaging the underlying structures. Blood was absorbed from the wounds by gauzes during 20 min and then extracted in 20 cc of water. Subsequently the tail bleeding test was carried out [ 11. The rats were placed into a cage of a constant temperature (27 f 1°C) with the tail in vertical hanging position. After 10 min the tail was transected at a diameter of 2 mm. The bleeding tail tip was placed in 20 cc of water of the same temperature during 10 min. Hemoglobin concentrations were measured by grating spectrophotometry (Beckman DB6) and the amounts of lost hemoglobin per paw and per tail were calculated. The values of the muscle bleeding were averaged per rat. Statistical analysis was performed with Student’s t test.
INTRODUCTION
Beta-adrenergic receptor blocking agents have been described as influencing directly and indirectly various parts of the hemostatic process (reaction of vessels, thrombocyte function, blood coagulation, and fibrinolysis) [3-7, 10, 141. Some of these influences accelerate hemostasis, others inhibit it. This study was to investigate the summarized effect of propranolol on surgical bleeding in the normotensive rat. MATERIALS
AND
METHODS
Male rats of a WR strain (inbred, University Animal Breeding Centre), weighing 200-300 g at the beginning of the experiment, were used. Rats had free access to food (Hope Farms, Woerden, The Netherlands) and to tap water. They were housed in a temperaturecontrolled room (20-22”(Z), illuminated intermittently. Two experiments were carried out. In the first experiment propranolol was administered subcutaneously during 6 days (5 mg/kg/day) via an osmotic minipump (Alzet, model 200 1) (n = 10). In the second experiment propran0101 (Inderal) was given during 28 days via the drinking water. The concentration of propranolol in the drinking water was adjusted 89
0022-4804/84 $1 SO Copyright 8 1984 by Academic Res. Inc. All ri&!bU of reproduction in any form rem’wd.
90
JOURNAL
OF SURGICAL
RESEARCH:
VOL.
36, NO.
1, JANUARY
1984
the direct force expelling the blood. However, in normotensive rats propranolol has no effect The results are given in Table 1. In both on blood pressure [ 121. tests a decreased blood loss after both shortBeta-adrenergic receptor blockade leads to term and long-term administration of pro- increase of bleeding via inhibition of the rise pranolol was found. In the long-term muscle of factor VIII immediately after physical extest the decrease was statistically significant ertion or stress [4, 61. Propranolol inhibits ag(P < 0.05); in the short-term as well as the gregation, adhesion, and clot retraction of the long-term tail test it was highly significant (P thrombocytes [3, 141. Propranolol lowers < 0.01). blood pressure partly by decreasing cardiac Comparing the tail bleeding tests of both output secondary to a venous pooling due to control groups, significantly more blood was venodilatation [7]. This venodilatation enlost in the long-term model (P < 0.01). No hances bleeding. Plasma renin activity (PRA) significant difference was found between the is suppressed by propranolol [lo]. two control groups in the muscle bleeding test. Rats have frequently been used to study the mechanism of beta-adrenergic receptor blockDISCUSSION ade on the cardiovascular system [8, 1 l-131. Hemostasis depends on the following in- Dosages vary between 1 and 100 mg/kg/day. terdependent mechanisms: reaction of vessels, To study the summarized effect of beta thrombocyte function, blood coagulation, and blockade on hemostasis in the rat, two bleedfibrinolysis. Beta-adrenergic receptor blockade ing tests were used. Dejana et al. showed [2] is reported both to accelerate and to delay that reaction of vessels, thrombocyte function, these different constituents [3-7, 10, 141. The blood coagulation, and fibrinolysis all connet result depends on the importance of the tribute to the ultimate result of the tail bleeding various complex and contrasting effects. test. The circulation and hemostasis in the tail Beta-adrenergic receptor blockade leads to varies significantly with the ambient temperdecrease of bleeding via inhibition of the in- ature, therefore the test was carried out in a crease of fibrinolysis which occurs after phys- cage of 27 + l°C. ical exertion or stress and is due to a rise of The muscle bleeding test was used to test the plasminogen activator [4, 61. another superficial region not only with Furthermore, immediately after the ad- smaller vessels, but also with probably different ministration of propranolol the peripheral re- beta-adrenergic receptors. sistance increases, due to an arteriolar vasoPropranolol diminishes tail bleeding sigconstriction [5]. However, this effect disap- nificantly and decreases rather than provokes pears within 4 weeks. Beta-adrenergic receptor bleeding in the muscle model. In previous exblockade also lowers blood pressure which is periments [3-7, 10, 141 different hemostasisRESULTS
TABLE THE
AMOUNT
OF HEMOGLOBIN
Prop. 6 days (N = 10) Muscle Tail
5.4 (1.2) 28.8 (3.5)
1
(mg) COLLECTED DURING THE TAIL Control (N = 10) 1.3 (1.4) 58.9 (10.3)**
AND
MUSCLE
Prop. 28 days (N = 15) 5.4 (0.7) 43.2 (6.1)
BLEEDING
TEST
Control (N = 15) 8.0 (0.8)* 108.4 (1 I .O)**
Notes. Propranolol was given for 6 days subcutaneously (5 mgjkg day) or for 28 days via the drinking water (about 10 mg/kg day). N = Number of rats used. Values represent means (fSEM). Level of significance between propranololtreated and control rats: *P < 0.05; **P < 0.01.
VAN
LANSCHOT,
TEN
BERG,
enhancing as well as hemostasis-inhibiting effects of propranolol have been described. From our findings it can be concluded, that in the normotensive rat these enhancing effects are of greater importance than the inhibiting ones. Further studies have to be carried out before this finding can be extrapolated to the clinical situation. Significantly more blood loss occurred in the long-term tail test control group than in the short-term one. A difference in age of the two groups at the time of the performance of the tests has to be taken in account. REFERENCES 1. Dejana, E., Calhoni, A., Quintana, A., and de Gaetano, G. Bleeding time in laboratory animals. II-A comparison of different assayconditions in rats. Thromb. Res. 15: 191, 1979.
2. Dejana, E., Quintana, A., Calliono, A., and de Gaetano, G. Bleeding time in laboratory animals. IIIDo tail bleeding times in rats only measure a platelet defect? Thromb. Rex 15: 199, 1979. 3. Frishman, W. H., Weksler, B., Christodoulou, J. P., Smithen, C., and Killip, T. Reversal of abnormal platelet aggregability and change in exercise tolerance in patients with angina pectoris following oral propranolol. Circulation 50: 887, 1974. 4. Gader, A. M. A., Clarkson, A. R., and Cash, J. D. The plasminogen activator and coagulation factor VIII responses to adrenaline, noradrenaline, isoprenahne and salbutamol in man. Thromb. Res. 2: 9, 1973. 5. Hansson, L., Zweifler, A. J., Julius, S., and Hunyor, S. N. Hemodynamic effects of acute and prolonged
AND
BRUINING:
PROPRANOLOL
b&-adrenergic Acta Med.
&and.
91
blockade in essential hypertension. 196: 27, 1974.
Kral, J. G., Ablad, B., Johnsson, G., and KorsamBengtsen, K. Effects of adrenaline and alprenolol (Apti#) on blood coagulation and fibrinolysis in man. Eur. J. Clin. Pharmacol. 3: 144, 197 1. Krauss, X. H., Schalekamp, M. A. D. H., Koisters, G., Zaal, G. A., and Birkenhager, W. H. Effects of chronic beta-adrenergic blockade on systemic and renal haemodynamic response to hyperosmotic saline in hypertensive patients. Clin. Sci. 43: 385, 1972. 8. Lundgren, Y. Blood pressure and vascular design in renal hypertension in rats after prolonged propranolol treatment. Acta Physiol. Stand. 91: 409, 1974. 9. Meuleman, D. G., Hobbelen, P. M. J., Dedem, G. van, and Moelker, H. C. T. A novel anti-thrombotic heparinoid (Org 10172) devoid of bleeding inducing capacity; a survey of its pharmacological properties in experimental animal models. Thromb. Res. 27: 353, 1982.
10. Michelakis, A. M., and McAllister, R. G. The effect of chronic adrenergic receptor blockade on plasma renin activity in man. J. Clin. Endocrinol. Metab. 34: 386, 1972.
11. Smits, J. F. M. The Antihypertensive Effect of Propranolol. Thesis Maastricht, 1980. 12. Vavra, I., Tom, H., and Greselin, E. Chronic propranolol treatment in young spontaneously hypertensive and normotensive rats. Canad. J. Physiol. Pharmacol.
51: 727, 1973.
13. Weiss, L., Lundgren, Y., and Folkow, B. Effects of prolonged treatment with adrenergic beta-receptor antagonists on blood pressure, cardiovascular design and reactivity in spontaneously hypertensive rats (SHR). Acta Physiol. &and. 91: 447, 1974. 14. Weksler, B. B., Gillick, M., and Pink, J. Effect of propranolol on platelet function. Blood 49: 185, 1977.
OF SURGICAL
36,89-9 1 ( 1984)
RESEARCH
Effect of Propranolol on Surgical Bleeding and Hemostasis: An in Vivo Study in the Rat J. J. B. VAN LANSCHOT,
M.D.,
R. G. M. TEN
BERG,PH.D.,
AND H. A. BRUINING,
Laboratory for Experimental Surgery, Erasmus University, Dr. Molenaterpleim The Netherlands
M.D.
50, 3015 GE, Rotterdam.
Submitted for publication November 10, 1982 The influence of propranolol on surgical bleeding and hemostasis was studied in two different models in the normotensive rat. Propranolol was administered for 6 days subcutaneously and for 28 days orally. In both the tail and the muscle bleeding test the blood loss after the short-term as well as the long-term administration of propranolol was diminished as compared to control values. Highly significant difkrences were reached in the tail bleeding test. Previous studies have described the effect of propranolol on several constituents of the highly complex hemostatic process. Some of these effects enhance, others inhibit hemostasis. It is concluded that in the normotensive rat these hemostasis-enhancing effectsare of greater importance than the inhibiting ones.
to the water intake in such a way, that the average dose was about 10 mg/kg/day (n = 15). In both experiments nontreated rats were used as control in equal numbers (n = 10 and 15, respectively). In all rats, two bleeding tests were performed under pentobarbitone anesthesia (Nembutal, 60 mg/kg ip). in the muscle bleeding test [9] the skin above the side of each hind paw was opened and the M. biceps femoris was cut through longitudinally with a razor blade (length = 11 mm) without damaging the underlying structures. Blood was absorbed from the wounds by gauzes during 20 min and then extracted in 20 cc of water. Subsequently the tail bleeding test was carried out [ 11. The rats were placed into a cage of a constant temperature (27 f 1°C) with the tail in vertical hanging position. After 10 min the tail was transected at a diameter of 2 mm. The bleeding tail tip was placed in 20 cc of water of the same temperature during 10 min. Hemoglobin concentrations were measured by grating spectrophotometry (Beckman DB6) and the amounts of lost hemoglobin per paw and per tail were calculated. The values of the muscle bleeding were averaged per rat. Statistical analysis was performed with Student’s t test.
INTRODUCTION
Beta-adrenergic receptor blocking agents have been described as influencing directly and indirectly various parts of the hemostatic process (reaction of vessels, thrombocyte function, blood coagulation, and fibrinolysis) [3-7, 10, 141. Some of these influences accelerate hemostasis, others inhibit it. This study was to investigate the summarized effect of propranolol on surgical bleeding in the normotensive rat. MATERIALS
AND
METHODS
Male rats of a WR strain (inbred, University Animal Breeding Centre), weighing 200-300 g at the beginning of the experiment, were used. Rats had free access to food (Hope Farms, Woerden, The Netherlands) and to tap water. They were housed in a temperaturecontrolled room (20-22”(Z), illuminated intermittently. Two experiments were carried out. In the first experiment propranolol was administered subcutaneously during 6 days (5 mg/kg/day) via an osmotic minipump (Alzet, model 200 1) (n = 10). In the second experiment propran0101 (Inderal) was given during 28 days via the drinking water. The concentration of propranolol in the drinking water was adjusted 89
0022-4804/84 $1 SO Copyright 8 1984 by Academic Res. Inc. All ri&!bU of reproduction in any form rem’wd.
90
JOURNAL
OF SURGICAL
RESEARCH:
VOL.
36, NO.
1, JANUARY
1984
the direct force expelling the blood. However, in normotensive rats propranolol has no effect The results are given in Table 1. In both on blood pressure [ 121. tests a decreased blood loss after both shortBeta-adrenergic receptor blockade leads to term and long-term administration of pro- increase of bleeding via inhibition of the rise pranolol was found. In the long-term muscle of factor VIII immediately after physical extest the decrease was statistically significant ertion or stress [4, 61. Propranolol inhibits ag(P < 0.05); in the short-term as well as the gregation, adhesion, and clot retraction of the long-term tail test it was highly significant (P thrombocytes [3, 141. Propranolol lowers < 0.01). blood pressure partly by decreasing cardiac Comparing the tail bleeding tests of both output secondary to a venous pooling due to control groups, significantly more blood was venodilatation [7]. This venodilatation enlost in the long-term model (P < 0.01). No hances bleeding. Plasma renin activity (PRA) significant difference was found between the is suppressed by propranolol [lo]. two control groups in the muscle bleeding test. Rats have frequently been used to study the mechanism of beta-adrenergic receptor blockDISCUSSION ade on the cardiovascular system [8, 1 l-131. Hemostasis depends on the following in- Dosages vary between 1 and 100 mg/kg/day. terdependent mechanisms: reaction of vessels, To study the summarized effect of beta thrombocyte function, blood coagulation, and blockade on hemostasis in the rat, two bleedfibrinolysis. Beta-adrenergic receptor blockade ing tests were used. Dejana et al. showed [2] is reported both to accelerate and to delay that reaction of vessels, thrombocyte function, these different constituents [3-7, 10, 141. The blood coagulation, and fibrinolysis all connet result depends on the importance of the tribute to the ultimate result of the tail bleeding various complex and contrasting effects. test. The circulation and hemostasis in the tail Beta-adrenergic receptor blockade leads to varies significantly with the ambient temperdecrease of bleeding via inhibition of the in- ature, therefore the test was carried out in a crease of fibrinolysis which occurs after phys- cage of 27 + l°C. ical exertion or stress and is due to a rise of The muscle bleeding test was used to test the plasminogen activator [4, 61. another superficial region not only with Furthermore, immediately after the ad- smaller vessels, but also with probably different ministration of propranolol the peripheral re- beta-adrenergic receptors. sistance increases, due to an arteriolar vasoPropranolol diminishes tail bleeding sigconstriction [5]. However, this effect disap- nificantly and decreases rather than provokes pears within 4 weeks. Beta-adrenergic receptor bleeding in the muscle model. In previous exblockade also lowers blood pressure which is periments [3-7, 10, 141 different hemostasisRESULTS
TABLE THE
AMOUNT
OF HEMOGLOBIN
Prop. 6 days (N = 10) Muscle Tail
5.4 (1.2) 28.8 (3.5)
1
(mg) COLLECTED DURING THE TAIL Control (N = 10) 1.3 (1.4) 58.9 (10.3)**
AND
MUSCLE
Prop. 28 days (N = 15) 5.4 (0.7) 43.2 (6.1)
BLEEDING
TEST
Control (N = 15) 8.0 (0.8)* 108.4 (1 I .O)**
Notes. Propranolol was given for 6 days subcutaneously (5 mgjkg day) or for 28 days via the drinking water (about 10 mg/kg day). N = Number of rats used. Values represent means (fSEM). Level of significance between propranololtreated and control rats: *P < 0.05; **P < 0.01.
VAN
LANSCHOT,
TEN
BERG,
enhancing as well as hemostasis-inhibiting effects of propranolol have been described. From our findings it can be concluded, that in the normotensive rat these enhancing effects are of greater importance than the inhibiting ones. Further studies have to be carried out before this finding can be extrapolated to the clinical situation. Significantly more blood loss occurred in the long-term tail test control group than in the short-term one. A difference in age of the two groups at the time of the performance of the tests has to be taken in account. REFERENCES 1. Dejana, E., Calhoni, A., Quintana, A., and de Gaetano, G. Bleeding time in laboratory animals. II-A comparison of different assayconditions in rats. Thromb. Res. 15: 191, 1979.
2. Dejana, E., Quintana, A., Calliono, A., and de Gaetano, G. Bleeding time in laboratory animals. IIIDo tail bleeding times in rats only measure a platelet defect? Thromb. Rex 15: 199, 1979. 3. Frishman, W. H., Weksler, B., Christodoulou, J. P., Smithen, C., and Killip, T. Reversal of abnormal platelet aggregability and change in exercise tolerance in patients with angina pectoris following oral propranolol. Circulation 50: 887, 1974. 4. Gader, A. M. A., Clarkson, A. R., and Cash, J. D. The plasminogen activator and coagulation factor VIII responses to adrenaline, noradrenaline, isoprenahne and salbutamol in man. Thromb. Res. 2: 9, 1973. 5. Hansson, L., Zweifler, A. J., Julius, S., and Hunyor, S. N. Hemodynamic effects of acute and prolonged
AND
BRUINING:
PROPRANOLOL
b&-adrenergic Acta Med.
&and.
91
blockade in essential hypertension. 196: 27, 1974.
Kral, J. G., Ablad, B., Johnsson, G., and KorsamBengtsen, K. Effects of adrenaline and alprenolol (Apti#) on blood coagulation and fibrinolysis in man. Eur. J. Clin. Pharmacol. 3: 144, 197 1. Krauss, X. H., Schalekamp, M. A. D. H., Koisters, G., Zaal, G. A., and Birkenhager, W. H. Effects of chronic beta-adrenergic blockade on systemic and renal haemodynamic response to hyperosmotic saline in hypertensive patients. Clin. Sci. 43: 385, 1972. 8. Lundgren, Y. Blood pressure and vascular design in renal hypertension in rats after prolonged propranolol treatment. Acta Physiol. Stand. 91: 409, 1974. 9. Meuleman, D. G., Hobbelen, P. M. J., Dedem, G. van, and Moelker, H. C. T. A novel anti-thrombotic heparinoid (Org 10172) devoid of bleeding inducing capacity; a survey of its pharmacological properties in experimental animal models. Thromb. Res. 27: 353, 1982.
10. Michelakis, A. M., and McAllister, R. G. The effect of chronic adrenergic receptor blockade on plasma renin activity in man. J. Clin. Endocrinol. Metab. 34: 386, 1972.
11. Smits, J. F. M. The Antihypertensive Effect of Propranolol. Thesis Maastricht, 1980. 12. Vavra, I., Tom, H., and Greselin, E. Chronic propranolol treatment in young spontaneously hypertensive and normotensive rats. Canad. J. Physiol. Pharmacol.
51: 727, 1973.
13. Weiss, L., Lundgren, Y., and Folkow, B. Effects of prolonged treatment with adrenergic beta-receptor antagonists on blood pressure, cardiovascular design and reactivity in spontaneously hypertensive rats (SHR). Acta Physiol. &and. 91: 447, 1974. 14. Weksler, B. B., Gillick, M., and Pink, J. Effect of propranolol on platelet function. Blood 49: 185, 1977.