- Email: [email protected]
Effects of estrogen on the microcirculation and thrombus formation in pial vessels of the rat
International Congress Series 1235 (2002) 413 – 418
Effects of estrogen on the microcirculation and thrombus formation in pial vessels of the rat Yasuto Sasaki a,*, Hiroaki Ono a, Junji Seki b, John C. Giddings c, Junichiro Yamamoto a a
Laboratory of Physiology, Faculty of Nutrition, Kobe Gakuin University, 518 Arise Ikawadani-Cho, Nishi-Ku, Kobe, 651-2180, Japan b Department of Biomedical Engineering, National Cardiovascular Center Research Institute, Suita, 565-8565, Japan c Department of Haematology, University of Wales College of Medicine, CF4 4XN, Cardiff, UK
Abstract Female Wistar/ST rats were ovariectomized at 14 weeks of age. Ethynylestradiol, 17h-estradiol (E2h), medroxyprogesterone acetate (MPA) and 4-hydroxy-tamoxifen were administered subcutaneously every other day for 3 weeks from 1 week after ovariectomy. Closed cranial windows were created and the thrombotic potential was measured using the He – Ne laser-induced thrombosis method. The mean red cell velocity was measured with a fiber-optic laser-Doppler anemometer microscope. The plasma concentrations of nitrite/nitrate (nitric oxide (NO) metabolites) were determined using the Griess reagent. The diameters mean red cell velocity and blood flow in pial arterioles were reduced after ovariectomy, however, the thrombotic tendency was increased after ovariectomy. These parameters were reversed after E2h treatment. In contrast, MPA had the opposite effects. The plasma concentrations of nitrite/nitrate were significantly decreased following ovariectomy and were increased after treatment with E2h. MPA did not affect the concentration of NO metabolites. These results strongly indicated that estrogen in the female rat mediated beneficial effects on the cerebral microcirculation and moderated cerebral thrombotic mechanisms by enhancing the plasma levels of NO. The findings suggest that increased blood flow and inhibition of thrombosis might contribute to the prevention of cerebrovascular disease in a normal female. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Estrogen; Progesterone; NO; Thrombosis; Microcirculation
*
Corresponding author. Tel.: +81-78-974-1551; fax: +81-78-974-5689. E-mail address: [email protected] (Y. Sasaki).
0531-5131/02 D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 2 ) 0 0 2 0 3 - 0
414
Y. Sasaki et al. / International Congress Series 1235 (2002) 413–418
1. Introduction Estrogen is believed to contribute to the reduced incidence of cardiovascular disease and the lower risk of cerebrovascular disease seen in females compared with males [1]. Estrogen is widely acknowledged to be beneficial to arterial wall function. Estrogen increases the production of prostacyclin and nitric oxide (NO) by enhancing endothelial NO synthase (eNOS) activity. Prostacyclin and NO are known to be potent vasodilators and strong inhibitors of platelet aggregation [2]. Infusion of L-arginine and nitroprusside reduced ischemic brain damage after middle cerebral artery ligation, probably by increasing the cerebral blood flow, and 17h-estradiol assisted recovery of the postischemic cerebral blood flow after an experimental stroke in the rat [3]. Moreover, NO donors inhibited thrombus formation induced by He – Ne laser irradiation in pial vessels, and treatments that increased NO synthesis, such as ingestion of L-arginine and exercise, reduced the incidence of stroke and cerebral thrombosis in stroke-prone spontaneously hypertensive rats (SHRSP) [4]. In addition, estrogen has been reported to moderate the development of atherosclerosis by changing lipoprotein profiles in plasma [5]. The precise role of estrogen in the cerebrovascular circulation and the mechanisms by which estrogen mediates the beneficial effects remain to be fully clarified. It seems likely, therefore, that the NO produced in response to estrogen alters the intravascular physical properties and moderates the thrombotic mechanisms in the cerebral blood vessels. Several steroid hormones, such as ethynylestradiol, medroxyprogesterone acetate (Depo-provera) and others, alone or in combination, are used widely in hormone replacement therapy (HRT) in post-menopausal women. It is generally believed that progesterone antagonizes the beneficial effects of estrogen. The purpose of the present study was to clarify the mechanisms of action of these hormones on the microcirculation and thrombosis in cerebral vessels.
2. Materials and methods 2.1. Ovariectomy and hormones Ovaries were surgically removed from female Wistar/ST rats at 14 weeks of age. 17aEthynylestradiol (EE; 1.0 mg/kg), 17h-estradiol 3-benzoate (E2h; 1.0 mg/kg), medroxyprogesterone acetate (MPA; 1.0 mg/kg) and 4-hydroxy-tamoxifen (TAM; 5.0 mg/kg) were dissolved in ethanol, diluted in sesame oil and administered subcutaneously every other day in the back of the animal for 3 weeks from 1 week after ovariectomy. The blood pressure was measured by the tail-cuff method. Body weights, blood pressures and blood flow parameters were measured once a week. 2.2. Measurement of thrombotic potential assessed by the He –Ne laser-induced thrombus formation method Animals were anesthetized with sodium pentobarbital and were artificially ventilated. Blood samples were obtained from one of the femoral arteries for the measurement of blood gases and pH. The other femoral artery was used for the measurement of the mean
Y. Sasaki et al. / International Congress Series 1235 (2002) 413–418
415
arterial blood pressure. Rates of respiration and the stroke volume of the respirator were adjusted to maintain constant levels of arterial blood gases and pH. The animals were immobilized in a stereotaxic frame, and a craniotomy was performed using a hand drill to form a cranial window with 5 mm in diameter in the center of right parietal bone [6]. Artificial cerebrospinal fluid was continuously infused within the cranial window, and the intracranial pressure was adjusted to 3 –5 mm Hg to avoid brain herniation. The animal was placed on the stage of a microscope and the cerebral vessels were monitored with a CCD camera. Images were recorded continuously on videotape. A He – Ne laser beam was focused on the center of the selected arterioles, and thrombi were formed by repeated irradiation. The number of laser pulses needed to form an occlusive thrombus was used as an index of thrombotic potential [4,7]. 2.3. Measurements of mean red cell velocity and calculation of blood flow The mean red cell velocity in the cerebral arterioles was measured with a fiber-optic laserDoppler anemometer microscope (FLDAM) [8]. Wall shear rates were calculated according to the equation, 8mean red cell velocity/inner diameter of the blood vessel. The mean blood flow of each arteriole was calculated from the mean red cell velocity and the diameter. 2.4. Measurements of arteriolar vessel diameter The diameter of the branch of the middle cerebral artery was measured from video images, captured on a personal computer. The arterioles were delineated from the branch of the middle cerebral artery, which was outlined as a large Y shape in the same position at the right side of the cranial window. The diameters of the arterioles were measured initially in pixels and then converted to micrometers. 2.5. Measurement of regional cerebral blood flow (rCBF) The regional cerebral blood flow was measured using a laser-Doppler flow meter (ALF21; Advance Tokyo) with a needle type probe (0.55 mm diameter). The probe was positioned over the surface of the parietal cortex and cortical blood flow in the parietal lobe was measured continuously. The signals of ALF-21 were analyzed by a personal computer. 2.6. Measurement of plasma concentrations of nitrite/nitrate Blood samples were collected from the femoral artery and centrifuged at 1500g for 15 min. The plasma was stored at 70 jC until assayed. The concentrations of nitrite/ nitrate and the metabolites of NO were determined using the Griess reagent (Assay kit-C; Dojindo Laboratory, Kumamoto, Japan). 2.7. Measurement of serum concentrations of estradiol (E2) and progesterone The plasma samples were prepared and stored at 70 jC as described above. The serum concentrations of E2 and progesterone were determined by radioimmunoassay.
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Y. Sasaki et al. / International Congress Series 1235 (2002) 413–418
3. Results 3.1. Effects of hormones on various parameters The body weights and systolic blood pressures of the control rats (sesame oil) were 292F4 g and 128F3 mm Hg, respectively. Treatment with MPA did not change the body weights and blood pressures but EE and E2h decreased these parameters (Table 1). On the other hand, the body weights were increased up to 2 weeks after treatment with TAM but were depressed after this time. 3.2. Changes in uterine wet weight after treatment with hormones The mean uterine wet weight in the control animals given sesame oil was 222F10 mg. MPA and TAM did not affect this (Table 1). Treatment with EE and E2h increased the uterine wet weight significantly. 3.3. Changes in serum concentrations of estradiol, progesterone and NO metabolites Serum concentrations of estradiol (E2) were increased significantly after E2h treatment. Treatment with other hormones, however, did not affect the serum concentrations of E2. The serum concentrations of progesterone were increased after treatment with MPA. Furthermore, metabolites of NO were increased significantly after treatment with EE, E2h and TAM.
Table 1 Changes in parameters after administration of various hormones Measurements
Sesame oil
EE (1.0 mg/kg)
Body weight (g) Systolic pressure (mm Hg) Uterine wet weight (mg) Serum estradiol (pg/ml) Serum progesterone (ng/ml) Nitrite/nitrate (Amol/l) Number of laser pulses
292F4 (6) 128F3 (6)
247F3 (6)* 108F3 (6)*
222F10 (5)
E2h (1.0 mg/kg)
Progesterone (1.0 mg/kg)
TAM (5.0 mg/kg)
242F5 (6)* 103F3 (6)*
300F10 (6) 132F2 (6)
252F3 (6)* 114F2 (6)
2198F33 (5)**
875F24 (6)**
210F31 (5)
316F43 (5)
10.0F0.9 (4)
8.2F2.0 (4)
946.6F29.5 (4)**
8.8F0.3 (4)
11F2.5 (4)
17.7F2.5 (4)
18.0F3.0 (4)
13.2F5.0 (4)
27.3F2.7 (4)*
13.2F4.4 (4)
14.2F0.5 (6)
27.4F2.6 (6)*
34.8F2.6 (6)**
14.9F1.0 (6)
23.3F3.1 (6)*
7.7F0.4 (6)
10.1F0.7 (6)**
10.8F0.5 (6)**
MeanFS.E.M. (N). * p < 0.05 vs. sesame oil. ** p < 0.01 vs. sesame oil.
5.4F0.2 (4)**
9.7F0.3 (5)**
Y. Sasaki et al. / International Congress Series 1235 (2002) 413–418
417
3.4. Effects of hormones on thrombotic potential in pial arterioles The thrombotic potential was assessed by the He – Ne laser method. The number of laser pulses needed to occlude pial arterioles was used as an index of thrombotic potential. The number of laser pulses needed to occlude the vessel was increased in rats treated with EE, E2h, TAM, but was decreased by the MPA treatment. These findings indicated that treatment with EE, E2h and TAM decreased the thrombotic potential. MPA treatment enhanced the thrombotic mechanisms significantly (Table 1). 3.5. Effects of hormones on cerebral microcirculation The mean red cell velocities measured by FLDAM increased significantly after treatment with EE, E2h and TAM but decreased after MPA. Similarly, arteriole diameters were significantly increased after treatment with EE and E2h, but were significantly decreased after MPA. Wall shear rates and blood flow were calculated from red cell velocity. The arteriole diameters were measured from video images. The blood flow was increased significantly after treatment with EE (6.3F0.9 (N = 6) nl/s; p< 0.05) and E2h (12.7F1.0 (N = 6) nl/s; p < 0.01), compared with sesame oil (4.2F0.3 (N = 6) nl/s), however, MPA significantly decreased the blood flow (2.9F0.3 (N =6) nl/s; p <0.05). Laser Doppler measurement (ALF-21), after treatment with hormones, indicated that the cortical blood flow increased significantly after EE, E2h and TAM, but reduced significantly after MPA ( p< 0.05).
4. Discussion In the current study, the effects of hormones on the cerebral microcirculation and thrombotic potential were studied. The thrombotic potential decreased after treatment with EE, E2h and TAM, but was enhanced by MPA. The plasma concentration of the NO metabolites was significantly increased in rats treated with EE, E2h and TAM, but was unchanged in animals given MPA. Rosselli et al. [9] found that the administration of estrogen alone to post-menopausal women increased the concentration of NO metabolites, whereas the combined progestin/17h-estradiol preparation was not found to increase nitrite/nitrate levels. In the present study, the diameters of pial vessels, red cell velocities and blood flow were increased significantly in rats treated with EE, E2h and TAM. These effects of the hormones might be due to increased plasma NO levels. We found that the treatment, which increased plasma NO levels in the present study, was similar to those seen previously in spontaneously hypertensive rats (SHRSP/Izm) [4]. In contrast, the opposite effect of MPA might be due to decreased levels of plasma NO. The thrombotic potential, assessed by the He –Ne laser method, was decreased by treatment with EE, E2h and TAM, but was increased by MPA. We have previously demonstrated that NO donors increased plasma NO levels and decreased the thrombotic potential in normal rats and SHRSP [4,7]. The increased plasma levels of NO, therefore, seem to contribute at least in part to the beneficial effects of hormones. Moreover, progesterone has been reported to antagonize estrogen binding to the estrogen receptor in nuclei [10]. In HRT, combined
418
Y. Sasaki et al. / International Congress Series 1235 (2002) 413–418
estrogen and progesterone preparations are used widely to reduce the risk of carcinoma of the endometrium and breast. It appears that the beneficial effects of estrogen might be moderated by progesterone. Further studies are required to assess the use of these hormones in the prevention of cerebral vascular diseases.
References [1] E. Barrett-Connor, T.L. Bush, Estrogen and coronary heart disease in women [see comments], JAMA, J. Am. Med. Assoc. 265 (1991) 1861 – 1867. [2] S. Moncada, The 1991 Ulf von Euler Lecture. The L-arginine: nitric oxide pathway, Acta Physiol. Scand. 145 (1992) 201 – 227. [3] N.J. Alkayed, I. Harukuni, A.S. Kimes, E.D. London, R.J. Traystman, P.D. Hurn, Gender-linked brain injury in experimental stroke, Stroke 29 (1998) 159 – 165, discussion 166. [4] T. Noguchi, Y. Sasaki, J. Seki, J.C. Giddings, J. Yamamoto, Effects of voluntary exercise and L-arginine on thrombogenesis and microcirculation in stroke-prone spontaneously hypertensive rats, Clin. Exp. Pharmacol. Physiol. 26 (1999) 330 – 335. [5] X. Zhu, B. Bonet, H. Gillenwater, R.H. Knopp, Opposing effects of estrogen and progestins on LDL oxidation and vascular wall cytotoxicity: implications for atherogenesis, Proc. Soc. Exp. Biol. Med. 222 (1999) 214 – 221. [6] S. Morii, A.C. Ngai, H.R. Winn, Reactivity of rat pial arterioles and venules to adenosine and carbon dioxide: with detailed description of the closed cranial window technique in rats, J. Cereb. Blood Flow Metab. 6 (1986) 34 – 41. [7] Y. Sasaki, J. Seki, J.C. Giddings, J. Yamamoto, Effects of NO-donors on thrombus formation and microcirculation in cerebral vessels of the rat, Thromb. Haemostasis 76 (1996) 111 – 117. [8] J. Seki, Y. Sasaki, T. Oyama, J. Yamamoto, Fiber-optic laser-Doppler anemometer microscope applied to the cerebral microcirculation in rats, Biorheology 33 (1996) 463 – 470. [9] M. Rosselli, B. Imthurn, P.J. Keller, E.K. Jackson, R.K. Dubey, Circulating nitric oxide (nitrite/nitrate) levels in postmenopausal women substituted with 17 beta-estradiol and norethisterone acetate. A two-year followup study, Hypertension 25 (4 Pt 2) (1995) 848 – 853. [10] P. Colburn, V. Buonassisi, Estrogen-binding sites in endothelial cell cultures, Science 201 (1978) 817 – 819.
Effects of estrogen on the microcirculation and thrombus formation in pial vessels of the rat Yasuto Sasaki a,*, Hiroaki Ono a, Junji Seki b, John C. Giddings c, Junichiro Yamamoto a a
Laboratory of Physiology, Faculty of Nutrition, Kobe Gakuin University, 518 Arise Ikawadani-Cho, Nishi-Ku, Kobe, 651-2180, Japan b Department of Biomedical Engineering, National Cardiovascular Center Research Institute, Suita, 565-8565, Japan c Department of Haematology, University of Wales College of Medicine, CF4 4XN, Cardiff, UK
Abstract Female Wistar/ST rats were ovariectomized at 14 weeks of age. Ethynylestradiol, 17h-estradiol (E2h), medroxyprogesterone acetate (MPA) and 4-hydroxy-tamoxifen were administered subcutaneously every other day for 3 weeks from 1 week after ovariectomy. Closed cranial windows were created and the thrombotic potential was measured using the He – Ne laser-induced thrombosis method. The mean red cell velocity was measured with a fiber-optic laser-Doppler anemometer microscope. The plasma concentrations of nitrite/nitrate (nitric oxide (NO) metabolites) were determined using the Griess reagent. The diameters mean red cell velocity and blood flow in pial arterioles were reduced after ovariectomy, however, the thrombotic tendency was increased after ovariectomy. These parameters were reversed after E2h treatment. In contrast, MPA had the opposite effects. The plasma concentrations of nitrite/nitrate were significantly decreased following ovariectomy and were increased after treatment with E2h. MPA did not affect the concentration of NO metabolites. These results strongly indicated that estrogen in the female rat mediated beneficial effects on the cerebral microcirculation and moderated cerebral thrombotic mechanisms by enhancing the plasma levels of NO. The findings suggest that increased blood flow and inhibition of thrombosis might contribute to the prevention of cerebrovascular disease in a normal female. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Estrogen; Progesterone; NO; Thrombosis; Microcirculation
*
Corresponding author. Tel.: +81-78-974-1551; fax: +81-78-974-5689. E-mail address: [email protected] (Y. Sasaki).
0531-5131/02 D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 2 ) 0 0 2 0 3 - 0
414
Y. Sasaki et al. / International Congress Series 1235 (2002) 413–418
1. Introduction Estrogen is believed to contribute to the reduced incidence of cardiovascular disease and the lower risk of cerebrovascular disease seen in females compared with males [1]. Estrogen is widely acknowledged to be beneficial to arterial wall function. Estrogen increases the production of prostacyclin and nitric oxide (NO) by enhancing endothelial NO synthase (eNOS) activity. Prostacyclin and NO are known to be potent vasodilators and strong inhibitors of platelet aggregation [2]. Infusion of L-arginine and nitroprusside reduced ischemic brain damage after middle cerebral artery ligation, probably by increasing the cerebral blood flow, and 17h-estradiol assisted recovery of the postischemic cerebral blood flow after an experimental stroke in the rat [3]. Moreover, NO donors inhibited thrombus formation induced by He – Ne laser irradiation in pial vessels, and treatments that increased NO synthesis, such as ingestion of L-arginine and exercise, reduced the incidence of stroke and cerebral thrombosis in stroke-prone spontaneously hypertensive rats (SHRSP) [4]. In addition, estrogen has been reported to moderate the development of atherosclerosis by changing lipoprotein profiles in plasma [5]. The precise role of estrogen in the cerebrovascular circulation and the mechanisms by which estrogen mediates the beneficial effects remain to be fully clarified. It seems likely, therefore, that the NO produced in response to estrogen alters the intravascular physical properties and moderates the thrombotic mechanisms in the cerebral blood vessels. Several steroid hormones, such as ethynylestradiol, medroxyprogesterone acetate (Depo-provera) and others, alone or in combination, are used widely in hormone replacement therapy (HRT) in post-menopausal women. It is generally believed that progesterone antagonizes the beneficial effects of estrogen. The purpose of the present study was to clarify the mechanisms of action of these hormones on the microcirculation and thrombosis in cerebral vessels.
2. Materials and methods 2.1. Ovariectomy and hormones Ovaries were surgically removed from female Wistar/ST rats at 14 weeks of age. 17aEthynylestradiol (EE; 1.0 mg/kg), 17h-estradiol 3-benzoate (E2h; 1.0 mg/kg), medroxyprogesterone acetate (MPA; 1.0 mg/kg) and 4-hydroxy-tamoxifen (TAM; 5.0 mg/kg) were dissolved in ethanol, diluted in sesame oil and administered subcutaneously every other day in the back of the animal for 3 weeks from 1 week after ovariectomy. The blood pressure was measured by the tail-cuff method. Body weights, blood pressures and blood flow parameters were measured once a week. 2.2. Measurement of thrombotic potential assessed by the He –Ne laser-induced thrombus formation method Animals were anesthetized with sodium pentobarbital and were artificially ventilated. Blood samples were obtained from one of the femoral arteries for the measurement of blood gases and pH. The other femoral artery was used for the measurement of the mean
Y. Sasaki et al. / International Congress Series 1235 (2002) 413–418
415
arterial blood pressure. Rates of respiration and the stroke volume of the respirator were adjusted to maintain constant levels of arterial blood gases and pH. The animals were immobilized in a stereotaxic frame, and a craniotomy was performed using a hand drill to form a cranial window with 5 mm in diameter in the center of right parietal bone [6]. Artificial cerebrospinal fluid was continuously infused within the cranial window, and the intracranial pressure was adjusted to 3 –5 mm Hg to avoid brain herniation. The animal was placed on the stage of a microscope and the cerebral vessels were monitored with a CCD camera. Images were recorded continuously on videotape. A He – Ne laser beam was focused on the center of the selected arterioles, and thrombi were formed by repeated irradiation. The number of laser pulses needed to form an occlusive thrombus was used as an index of thrombotic potential [4,7]. 2.3. Measurements of mean red cell velocity and calculation of blood flow The mean red cell velocity in the cerebral arterioles was measured with a fiber-optic laserDoppler anemometer microscope (FLDAM) [8]. Wall shear rates were calculated according to the equation, 8mean red cell velocity/inner diameter of the blood vessel. The mean blood flow of each arteriole was calculated from the mean red cell velocity and the diameter. 2.4. Measurements of arteriolar vessel diameter The diameter of the branch of the middle cerebral artery was measured from video images, captured on a personal computer. The arterioles were delineated from the branch of the middle cerebral artery, which was outlined as a large Y shape in the same position at the right side of the cranial window. The diameters of the arterioles were measured initially in pixels and then converted to micrometers. 2.5. Measurement of regional cerebral blood flow (rCBF) The regional cerebral blood flow was measured using a laser-Doppler flow meter (ALF21; Advance Tokyo) with a needle type probe (0.55 mm diameter). The probe was positioned over the surface of the parietal cortex and cortical blood flow in the parietal lobe was measured continuously. The signals of ALF-21 were analyzed by a personal computer. 2.6. Measurement of plasma concentrations of nitrite/nitrate Blood samples were collected from the femoral artery and centrifuged at 1500g for 15 min. The plasma was stored at 70 jC until assayed. The concentrations of nitrite/ nitrate and the metabolites of NO were determined using the Griess reagent (Assay kit-C; Dojindo Laboratory, Kumamoto, Japan). 2.7. Measurement of serum concentrations of estradiol (E2) and progesterone The plasma samples were prepared and stored at 70 jC as described above. The serum concentrations of E2 and progesterone were determined by radioimmunoassay.
416
Y. Sasaki et al. / International Congress Series 1235 (2002) 413–418
3. Results 3.1. Effects of hormones on various parameters The body weights and systolic blood pressures of the control rats (sesame oil) were 292F4 g and 128F3 mm Hg, respectively. Treatment with MPA did not change the body weights and blood pressures but EE and E2h decreased these parameters (Table 1). On the other hand, the body weights were increased up to 2 weeks after treatment with TAM but were depressed after this time. 3.2. Changes in uterine wet weight after treatment with hormones The mean uterine wet weight in the control animals given sesame oil was 222F10 mg. MPA and TAM did not affect this (Table 1). Treatment with EE and E2h increased the uterine wet weight significantly. 3.3. Changes in serum concentrations of estradiol, progesterone and NO metabolites Serum concentrations of estradiol (E2) were increased significantly after E2h treatment. Treatment with other hormones, however, did not affect the serum concentrations of E2. The serum concentrations of progesterone were increased after treatment with MPA. Furthermore, metabolites of NO were increased significantly after treatment with EE, E2h and TAM.
Table 1 Changes in parameters after administration of various hormones Measurements
Sesame oil
EE (1.0 mg/kg)
Body weight (g) Systolic pressure (mm Hg) Uterine wet weight (mg) Serum estradiol (pg/ml) Serum progesterone (ng/ml) Nitrite/nitrate (Amol/l) Number of laser pulses
292F4 (6) 128F3 (6)
247F3 (6)* 108F3 (6)*
222F10 (5)
E2h (1.0 mg/kg)
Progesterone (1.0 mg/kg)
TAM (5.0 mg/kg)
242F5 (6)* 103F3 (6)*
300F10 (6) 132F2 (6)
252F3 (6)* 114F2 (6)
2198F33 (5)**
875F24 (6)**
210F31 (5)
316F43 (5)
10.0F0.9 (4)
8.2F2.0 (4)
946.6F29.5 (4)**
8.8F0.3 (4)
11F2.5 (4)
17.7F2.5 (4)
18.0F3.0 (4)
13.2F5.0 (4)
27.3F2.7 (4)*
13.2F4.4 (4)
14.2F0.5 (6)
27.4F2.6 (6)*
34.8F2.6 (6)**
14.9F1.0 (6)
23.3F3.1 (6)*
7.7F0.4 (6)
10.1F0.7 (6)**
10.8F0.5 (6)**
MeanFS.E.M. (N). * p < 0.05 vs. sesame oil. ** p < 0.01 vs. sesame oil.
5.4F0.2 (4)**
9.7F0.3 (5)**
Y. Sasaki et al. / International Congress Series 1235 (2002) 413–418
417
3.4. Effects of hormones on thrombotic potential in pial arterioles The thrombotic potential was assessed by the He – Ne laser method. The number of laser pulses needed to occlude pial arterioles was used as an index of thrombotic potential. The number of laser pulses needed to occlude the vessel was increased in rats treated with EE, E2h, TAM, but was decreased by the MPA treatment. These findings indicated that treatment with EE, E2h and TAM decreased the thrombotic potential. MPA treatment enhanced the thrombotic mechanisms significantly (Table 1). 3.5. Effects of hormones on cerebral microcirculation The mean red cell velocities measured by FLDAM increased significantly after treatment with EE, E2h and TAM but decreased after MPA. Similarly, arteriole diameters were significantly increased after treatment with EE and E2h, but were significantly decreased after MPA. Wall shear rates and blood flow were calculated from red cell velocity. The arteriole diameters were measured from video images. The blood flow was increased significantly after treatment with EE (6.3F0.9 (N = 6) nl/s; p< 0.05) and E2h (12.7F1.0 (N = 6) nl/s; p < 0.01), compared with sesame oil (4.2F0.3 (N = 6) nl/s), however, MPA significantly decreased the blood flow (2.9F0.3 (N =6) nl/s; p <0.05). Laser Doppler measurement (ALF-21), after treatment with hormones, indicated that the cortical blood flow increased significantly after EE, E2h and TAM, but reduced significantly after MPA ( p< 0.05).
4. Discussion In the current study, the effects of hormones on the cerebral microcirculation and thrombotic potential were studied. The thrombotic potential decreased after treatment with EE, E2h and TAM, but was enhanced by MPA. The plasma concentration of the NO metabolites was significantly increased in rats treated with EE, E2h and TAM, but was unchanged in animals given MPA. Rosselli et al. [9] found that the administration of estrogen alone to post-menopausal women increased the concentration of NO metabolites, whereas the combined progestin/17h-estradiol preparation was not found to increase nitrite/nitrate levels. In the present study, the diameters of pial vessels, red cell velocities and blood flow were increased significantly in rats treated with EE, E2h and TAM. These effects of the hormones might be due to increased plasma NO levels. We found that the treatment, which increased plasma NO levels in the present study, was similar to those seen previously in spontaneously hypertensive rats (SHRSP/Izm) [4]. In contrast, the opposite effect of MPA might be due to decreased levels of plasma NO. The thrombotic potential, assessed by the He –Ne laser method, was decreased by treatment with EE, E2h and TAM, but was increased by MPA. We have previously demonstrated that NO donors increased plasma NO levels and decreased the thrombotic potential in normal rats and SHRSP [4,7]. The increased plasma levels of NO, therefore, seem to contribute at least in part to the beneficial effects of hormones. Moreover, progesterone has been reported to antagonize estrogen binding to the estrogen receptor in nuclei [10]. In HRT, combined
418
Y. Sasaki et al. / International Congress Series 1235 (2002) 413–418
estrogen and progesterone preparations are used widely to reduce the risk of carcinoma of the endometrium and breast. It appears that the beneficial effects of estrogen might be moderated by progesterone. Further studies are required to assess the use of these hormones in the prevention of cerebral vascular diseases.
References [1] E. Barrett-Connor, T.L. Bush, Estrogen and coronary heart disease in women [see comments], JAMA, J. Am. Med. Assoc. 265 (1991) 1861 – 1867. [2] S. Moncada, The 1991 Ulf von Euler Lecture. The L-arginine: nitric oxide pathway, Acta Physiol. Scand. 145 (1992) 201 – 227. [3] N.J. Alkayed, I. Harukuni, A.S. Kimes, E.D. London, R.J. Traystman, P.D. Hurn, Gender-linked brain injury in experimental stroke, Stroke 29 (1998) 159 – 165, discussion 166. [4] T. Noguchi, Y. Sasaki, J. Seki, J.C. Giddings, J. Yamamoto, Effects of voluntary exercise and L-arginine on thrombogenesis and microcirculation in stroke-prone spontaneously hypertensive rats, Clin. Exp. Pharmacol. Physiol. 26 (1999) 330 – 335. [5] X. Zhu, B. Bonet, H. Gillenwater, R.H. Knopp, Opposing effects of estrogen and progestins on LDL oxidation and vascular wall cytotoxicity: implications for atherogenesis, Proc. Soc. Exp. Biol. Med. 222 (1999) 214 – 221. [6] S. Morii, A.C. Ngai, H.R. Winn, Reactivity of rat pial arterioles and venules to adenosine and carbon dioxide: with detailed description of the closed cranial window technique in rats, J. Cereb. Blood Flow Metab. 6 (1986) 34 – 41. [7] Y. Sasaki, J. Seki, J.C. Giddings, J. Yamamoto, Effects of NO-donors on thrombus formation and microcirculation in cerebral vessels of the rat, Thromb. Haemostasis 76 (1996) 111 – 117. [8] J. Seki, Y. Sasaki, T. Oyama, J. Yamamoto, Fiber-optic laser-Doppler anemometer microscope applied to the cerebral microcirculation in rats, Biorheology 33 (1996) 463 – 470. [9] M. Rosselli, B. Imthurn, P.J. Keller, E.K. Jackson, R.K. Dubey, Circulating nitric oxide (nitrite/nitrate) levels in postmenopausal women substituted with 17 beta-estradiol and norethisterone acetate. A two-year followup study, Hypertension 25 (4 Pt 2) (1995) 848 – 853. [10] P. Colburn, V. Buonassisi, Estrogen-binding sites in endothelial cell cultures, Science 201 (1978) 817 – 819.