Nancy L. Alsip

A new technique for studying the uterine microvasculature in the rat

OBJECTIVE Our purpose was to develop a method for direct measurement of rat uterine microvessels and to test their viability. STUDY DESIGN In anesthetized female Sprague-Dawley diestrus rats, one uterine hom was isolated from the body cavity with its nerve and blood supply intact. A small fiberoptic probe inserted into the lumen of the uterus served as a light source for transillumination of uterine vessels. Diameters of circumferential arterioles were observed while increasing concentrations of vasoactive agonists were suffused over the uterus. RESULTS No significant diameter changes occurred in circumferential arterioles of the control group (100-minute suffusion of Krebs solution). Dilation with papaverine (100 mumol/L) demonstrated that vessels possessed basal tone. Circumferential arterioles showed concentration-dependent constriction to phenylephrine and angiotensin II and dilation to acetylcholine and serotonin. CONCLUSIONS This uterine microcirculatory preparation provides a stable, reproducible model of a unique microcirculatory bed that responds to vasoactive agents in a manner similar to other microcirculatory beds.

Serotonin-induced dilation of small arterioles is not mediated via endothelium-derived relaxing factor in skeletal muscle

Serotonin (5-HT) dilates precapillary arterioles in skeletal muscle. The purpose of this study was to determine if 5-HT releases endothelium-derived relaxing factor (EDRF) in this tissue. Diameters of third-order arterioles (A3) in the cremaster muscle of pentobarbital-anesthetized rats were measured via videomicroscopy. Concentration-response curves for acetylcholine, nitroprusside and 5-HT were obtained before and after the application of either hydroquinone (5 x 10(-4) M) or NG-nitro-L-arginine (10(-4) M). The involvement of prostaglandins was eliminated by ibuprofen (10(-4) M). In one group, 5-HT (20 micrograms/kg) and NG-nitro-L-arginine were given i.v. (30 mg/kg). The non-EDRF-dependent vasodilator papaverine (10(-5) M) was applied at the end of the protocol to determine the maximal resting diameter. When applied topically, both hydroquinone and NG-nitro-L-arginine significantly inhibited the dilation induced by acetylcholine, but neither agent affected the dilation to nitroprusside or 5-HT. NG-Nitro-L-arginine (i.v.) attenuated acetylcholine-induced dilation but not the dilation to intravenous 5-HT. These data suggest that 5-HT-induced dilation of small arterioles in skeletal muscle is EDRF-independent.

Prostaglandin-related Microvascular Dilation in Pentobarbital- and Etomidate-anesthetized Rats

Etomidate is characterized by minimal systemic cardiovascular effects, but its effect on the microvasculature has not been assessed. We compared the microvasculature of etomidate-anesthetized animals to that of animals anesthetized with pentobarbital, since its effects on the microvasculature are known. Male Sprague-Dawley rats were anesthetized with etomidate or pentobarbital. The cremaster muscle was prepared for microscopic viewing, leaving the neural and vascular supply intact. Small arterioles were near their maximal diameters in etomidate-anesthetized rats, whereas the pentobarbital group had a large dilator capacity (maximal diameter-basal diameter/basal diameter). The effect on resting arteriolar diameters of endothelium-derived relaxing factor (EDRF) and prostaglandin synthesis inhibitors was tested. Dilator capacity was not affected by the EDRF inhibitor nitro-L-arginine, but it was significantly increased by mefenamic acid and ibuprofen in etomidate-anesthetized animals. To test whether dilator and constrictor mechanisms were normal, serotonin concentration-response curves were obtained in pentobarbital and etomidate-anesthetized animals with and without mefenamate or ibuprofen present. The dilation of small arterioles to serotonin in the etomidate groups with mefenamate or ibuprofen was not significantly different from that of the pentobarbital groups. Serotonin produced a comparable constriction of large arterioles in both anesthetic groups. The topical application of etomidate to the cremaster muscle did not affect arteriolar diameters. Thus, etomidate appears to trigger the release of dilator prostaglandins in striated muscle through a central or indirect mechanism.

Systemic vascular reactivity in an aortic coarctation model of preeclampsia in the rat

Preeclampsia (preECL), a hypertensive disorder of pregnancy, which occurs only in humans, is dangerous for mother and fetus. It may be caused by placental hypoxia triggering the release of a circulating factor that damages the maternal endothelium leading to vasoconstriction and hypertension. Our primary objective was to determine if systemic vascular reactivity is altered in a rat aortic coarctation (ACOR) model of preECL. We hypothesized that reduced blood flow to the rat utero-placental unit would lead to increased responsiveness to vasoconstrictors. On day 8 of pregnancy, rats were anesthetized and subjected to sham (SHAM) or aortic coarctation (ACOR) surgery by placing a 0.4 mm diameter silver clip distal to the renal arteries. Systemic pressor responses to bolus doses of phenylephrine (PE), angiotensin II (AII), and 5-hydroxytryptamine (5-HT) were determined in these animals on gestation days 14-15 (mid-pregnancy) or 19-20 (late pregnancy). Virgin rats were subjected to sham or ACOR surgery and arterial pressure measured 10-11 days after surgery. Arterial pressure was elevated in late pregnant ACOR animals (mean of 120+/-4 mmHg) compared with SHAM (mean of 101+/-6), but mid-pregnant and virgin groups were not different. Proteinuria was present more frequently in late pregnant ACOR animals (86%) than in SHAM (41%), but there were no differences in average fetal weight, fetal number, or number of reabsorbed fetuses. Pressor responses were not different in ACOR and SHAM groups in mid- or late pregnancy. These data indicate that the aortic coarctation to 0.4 mm in the rat mimics the clinical presentation of mild preECL in humans.

Human chorionic gonadotropin directly and indirectly alters uterine arteriolar diameters in cycling rats

OBJECTIVE Our purpose was to investigate whether uterine microvascular responses to human chorionic gonadotropin application depend on route of administration and estrous cycle day. STUDY DESIGN One uterine horn was exteriorized in pentobarbital-anesthetized cycling and ovariectomized rats and superfused with Krebs solution Uterine arterioles (64 +/- 2.1 microns) were viewed by videomicroscopy. Diameters were measured during a 20-minute baseline period and for 60 minutes during human chorionic gonadotropin suffusion (20 IU/60 ml) or 60 minutes after intraperitoneal injection of 50 IU of human chorionic gonadotropin. Papaverine (100 mumol/L) suffusion maximally dilated the uterine arterioles (80 +/- 2.6 microns). RESULTS Suffusion of human chorionic gonadotropin-dilated arterioles on diestrus-1 (122% +/- 2% baseline) and diestrus-2 (118% +/- 4% baseline) but constricted arterioles on proestrus (78% +/- 7% baseline). Intraperitoneal injection of human chorionic gonadotropin resulted in arteriolar constriction on diestrus-2 (76% +/- 5% baseline) and proestrus (82% +/- 3% baseline). Ovariectomy eliminated the effects of injected but not suffused human chorionic gonadotropin. All results are significant at p < 0.05. CONCLUSIONS Results indicate estrous cycle day-dependent direct and indirect effects of human chorionic gonadotropin on the resistance of uterine arterioles.

The microphotohemolytic response of erythrocytes is altered by streptozotocin-induced diabetes and copper deficiency in rats

Microphotohemolysis is a new technique that has been used to determine the presence of alterations in the erythrocyte membrane. The method involves light activation through a microscope of a fluorescent dye-erythrocyte-buffer solution in a hemocytometer. The interaction of the light and dye result in the generation of toxic oxygen products which attack the membrane allowing water to enter the cell. As hemolysis occurs the optical density of the microscopic field decreases and this is recorded for later quantitation with an image analysis system. Maximal effect, time to half maximal effect and the slope of the hemolysis curve are determined. The goal of this study was to determine if microphotohemolysis could be used to detect differences in erythrocytes from animals with altered physiological states such as hypercholesterolemia, diabetes, and copper deficiency. These are conditions that alter the lipid or protein structure of the erythrocyte membrane and/or the antioxidative capacity of the erythrocyte. There were no effects of hypercholesterolemia on the microphotohemolytic response of the erythrocyte. Streptozotocin-induced diabetes resulted in a decreased maximum effect, a significant shift of the hemolysis curve to the right (increased T 1/2) and a significant decrease in the slope of the hemolysis curve. Copper deficiency resulted in a significant decrease in the slope of the hemolysis curve. These results in diabetes and copper deficiency are consistent with an altered protein structure in the erythrocyte membrane that occurs in these conditions. The data demonstrate that this technique may be used to detect differences between normal and altered erythrocytes. As such, it could be useful in monitoring the course of a disease or its treatment.

In vivo effect of naftidrofuryl on 5-hydroxytryptamine-mediated constriction in rat peripheral microcirculation

Naftidrofuryl is commonly used in treatment of peripheral vascular disease. Its vasodilator action has been partly explained by its inhibitory effect of 5-HT2 receptors on peripheral arteries in vitro. The purpose of this study was to test in vivo whether naftidrofuryl selectively inhibits 5-hydroxytryptamine (5-HT)-mediated constriction of large arterioles in the peripheral microcirculation. This constriction appears to be 5-HT2 receptor-mediated. Three separate protocols were used to test the effects of naftidrofuryl: chronic injection (15 mg/kg, i.p., twice daily for 5-6 days; n = 7), acute intravenous (i.v.) infusion (15 mg/kg over 30 min; n = 7), or topical application (5 x 10(-8) M, n = 6; 5 x 10(-7) M, n = 5; 5 x 10(-6) M, n = 5; 10(-5) M, n = 7). Male Sprague-Dawley rats (145-185 g body weight) were anesthetized with sodium pentobarbital (50 mg/kg) and the cremaster muscle was prepared for intravital video microscopy. Diameter response of arterioles (70-120 microns) to increasing concentrations of locally applied 5-HT (10(-8)-10(-4) M) was assessed. In rats receiving no drug treatment, 5-HT caused vasoconstriction of arterioles beginning at 10(-6) M and reaching approximately 40% constriction at 10(-4) M. These vasoactive responses were not altered by chronic daily doses or an acute infusion of naftidrofuryl. 5-HT responses obtained with and without naftidrofuryl applied directly into the cremaster-bath also had little effect on the arteriole response at each of the four concentrations tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple Serotonin Receptors on Large Arterioles in Striated Muscle

In rat striated muscle, serotonin (5-hydroxytryptamine; 5-HT) constricts large arterioles (first-order; A1) via 5-HT2 receptor activation but dilates smaller arterioles via a 5-HT1-like receptor. In this preparation, A1 arterioles possess little basal tone. The purpose of this study was to determine if 5-HT would elicit A1 dilation if arteriolar tone was first induced. In anesthetized rats, A1 diameters of the cremaster muscle were measured via in vivo videomicroscopy. Topical application of angiotensin II caused a 26 +/- 3% constriction of the vessels. 5-HT dilated the preconstricted A1s by 36 +/- 7% while constricting normal tone A1s by 33 +/- 5%. This dilation was enhanced by blocking 5-HT2 receptors with LY53857, but abolished with methysergide, a 5-HT1 and 5-HT2 receptor antagonist indicating that the dilation was mediated by a 5-HT1-like receptor. Thus, A1 arterioles possess both 5-HT2 and 5-HT1-like receptors. The net result of 5-HT application in striated muscle, dilation or constriction, will depend on the initial tone of the vessels.

Halothane attenuates small arteriole vasodilation to serotonin (5-HT) in skeletal muscle

Inhaled anesthetics may have an effect on the micro-circulation through selective alteration of receptor-mediated control. Halothane was chosen because its actions in the microcirculation have been determined previously. Serotonin has received recent attention as a potential mediator of vascular changes in a number of disease states. We obtained concentration-response curves for serotonin-induced constriction of large arterioles and dilation of small arterioles (< 60 μm diameter) in the cremaster muscle of halothane-anesthetized and decerebrate rats. Cremaster muscles were prepared for microscopic viewing, leaving the neural and vascular supply intact. Serotonin concentration-response curves were obtained before and after receptor antagonist application. Large arteriole constriction was not affected by halothane. Dilation of small arterioles was decreased in halothane-anesthetized animals but enhanced in the presence of methysergide, a nonspecific antagonist. These data indicate that halothane interferes with occupancy of 5-hydroxytryptamine receptors.

Alteration of arteriolar responses to serotonin by two intravenous anesthetics.

The effects of serotonin (5-HT) on the microvasculature of cremaster muscles were compared in decerebrate, ketamine- and pentobarbital-anesthetized rats. 5-HT-induced constriction of large distributing arterioles was enhanced in the ketamine- and pentobarbital-anesthetized rats. Precapillary arterioles of pentobarbital-anesthetized animals were more sensitive to 5-HT-induced dilation than either decerebrate or ketamine-anesthetized animals. The response of vessels to two 5-HT receptor antagonists (methysergide and LY53857) were unchanged by the anesthetics. Our findings suggest that both ketamine and pentobarbital enhance the microvascular response to 5-HT, but that these changes are not due to an alteration of receptor sensitivity.