Effect of culture conditions, donor age, and injection site on in vitro development of DNA microinjected porcine zygotes.

A series of experiments evaluated development of porcine zygotes microinjected with DNA in three culture media and two incubation temperatures, from postpubertal and prepubertal donors, and between zygotes injected with DNA into the pronucleus and the cytoplasm. Zygotes recovered from 36 postpubertal gilts in Exp. 1 were injected and cultured in modified NCSU-23, modified NCSU-37, and CZB media at 37 degrees C or 39 degrees C for 7 d. In Exp. 2, zygotes were collected from postpubertal or prepubertal gilts, microinjected with DNA, and cultured in modified NCSU-23. In Exp. 3 superovulated prepubertal gilts had DNA injected into the cytoplasm or pronucleus of zygotes. Mean percentages developing to the expanded or hatched blastocyst stage in modified NCSU-23 (42.9) and modified NCSU-37 (40.1) did not differ, but development was greater than that for zygotes cultured in CZB (8.8; P < .05). Development was greater at 39 degrees C (P < .05) than at 37 degrees C (36.5 vs 24.6%). Microinjection of DNA decreased development (P < .05) from that of noninjected controls (18.1 vs 43.1%). Zygotes from postpubertal gilts had a higher percentage (68.0) of expanded and hatched blastocysts than zygotes from prepubertal donors (29.0; P < .05). No development difference was found between DNA injection into the pronucleus (23.1%) or cytoplasm (17.4%), but development was less than for control embryos (64.9%; P < .05). DNA microinjected porcine zygotes can be successfully cultured to the expanded blastocyst stage in modified NCSU-23 and modified NCSU-37 media at 39 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanics of large and small cerebral arteries in chronic hypertension.

The goal of this study was to investigate factors that contribute to reductions in internal diameter of large and small cerebral arteries during chronic hypertension. We measured diameter of second- and third-order branches of the posterior cerebral artery in vitro during maximal dilation with EDTA in 6-mo-old stroke-prone spontaneously hypertensive rats (SHRSP, n = 7) and Wistar-Kyoto rats (WKY, n = 7). Cross-sectional area of the vessel wall, measured histologically, was not significantly different at 70 mmHg in SHRSP and WKY in large or small branches of posterior cerebral artery. In large branches of posterior cerebral artery, external and internal diameters were significantly less at 70 mmHg in SHRSP than in WKY, whereas external and internal diameters converged at 0 mmHg in the two groups of rats. In small branches, on the other hand, external and internal diameters were significantly less at all levels of intravascular pressure in SHRSP than in WKY. The stress-strain relation in posterior cerebral artery of SHRSP was shifted to the left in large branches and to the right in small branches, which indicates that distensibility was reduced in large cerebral arteries of SHRSP and increased in small cerebral arteries. These findings suggest that different mechanisms are responsible for impairment of maximal dilator capacity in large and small cerebral arteries of SHRSP: reduced distensibility in large arteries and remodeling with reduced external diameter in small arteries. Furthermore the findings provide additional support for the concept that hypertrophy may not be a primary factor in impaired maximal dilation.

Mechanics and composition of cerebral arterioles in renal and spontaneously hypertensive rats.

The purpose of this study was to examine effects of hypertension on mechanics of cerebral arterioles in nongenetic and genetic models of chronic hypertension. Pressure (servo null) and diameter were measured in pial arterioles of anesthetized renal hypertensive rats (one-kidney, one clip), uninephrectomized normotensive rats, spontaneously hypertensive rats, and normotensive Wistar-Kyoto rats. During maximal dilatation with EDTA, external diameter of pial arterioles at 70 mm Hg pial arteriolar pressure was not significantly different in renal hypertensive and normotensive rats (86 +/- 5 [mean +/- SEM] versus 84 +/- 4 microns) but was less in spontaneously hypertensive rats than in Wistar-Kyoto rats (81 +/- 3 versus 92 +/- 3 microns; p < 0.05). Cross-sectional area of the arteriolar wall (histological) was greater in renal hypertensive than in normotensive rats (1,360 +/- 131 versus 952 +/- 89 microns 2; p < 0.05) and in spontaneously hypertensive rats than in Wistar-Kyoto rats (1,294 +/- 97 versus 817 +/- 86 microns 2; p < 0.05). The stress-strain relation obtained from pressure-diameter data during maximal dilatation with EDTA indicated that distensibility of pial arterioles, when fully relaxed, was greater in renal hypertensive and spontaneously hypertensive rats than in normotensive and Wistar-Kyoto rats. We used point-counting stereology to quantitate composition of pial arterioles in renal hypertensive rats. Cross-sectional area of smooth muscle and elastin was significantly greater in renal hypertensive than in normotensive rats (smooth muscle, 947 +/- 108 versus 620 +/- 62 microns 2; elastin, 101 +/- 11 versus 55 +/- 6 microns 2; p < 0.05), whereas cross-sectional area of collagen and basement membrane was not significantly different in the two groups (collagen, 6 +/- 1 versus 5 +/- 1 microns 2; basement membrane, 120 +/- 12 versus 104 +/- 8 microns 2). Thus, we conclude that 1) cerebral arterioles undergo hypertrophy in both renal hypertensive and spontaneously hypertensive rats; 2) cerebral arterioles in renal hypertensive rats do not undergo "remodeling" with a reduction in external diameter, whereas external diameter is smaller in spontaneously hypertensive than in Wistar-Kyoto rats; 3) distensibility of cerebral arterioles, when fully relaxed, is increased in renal hypertensive rats and is greater in spontaneously hypertensive than in Wistar-Kyoto rats; and 4) the distensible components of the arteriolar wall are increased disproportionately in cerebral arterioles of renal hypertensive rats, which may contribute to increases in arteriolar distensibility.

Effects of aging on cerebral vascular responses to serotonin in rats.

The purpose of this study was to examine effects of aging on responses of large cerebral arteries to serotonin. We measured cerebral microvascular pressure (with a micropipette and servo-null method), diameter of pial arterioles, and cerebral blood flow (microspheres) in adult (12- to 14-mo-old, n = 15) and aged (24- to 27-mo-old, n = 14) Wistar rats. Responses to intra-atrial infusion of serotonin (5 and 50 micrograms.kg-1.min-1) were examined. Infusion of the low dose of serotonin decreased mean arterial pressure and pial arteriolar pressure in adult and aged rats to similar levels. Cerebral blood flow was not reduced in adult or aged rats during infusion of the low dose of serotonin. The high dose of serotonin did not affect mean arterial pressure but reduced pial arteriolar pressure [from 46 +/- 4 to 23 +/- 2 (SE) in adult rats and from 52 +/- 3 to 18 +/- 4 mmHg in aged rats]. The high dose of serotonin increased large-artery resistance from 0.9 +/- 0.1 to 1.6 +/- 0.2 in adult rats and from 0.9 +/- 0.1 to 2.7 +/- 0.6 mmHg.ml-1.min.100 g in aged rats. Cerebral blood flow was reduced significantly in aged rats (from 59 +/- 3 to 41 +/- 6 ml.min-1.100 g-1), but not in adult rats, during infusion of the high dose of serotonin. We conclude that aging augments constrictor responses of large cerebral arteries to intravascular serotonin, which results in a reduction of cerebral blood flow in aged but not adult rats.

Effects of antihypertensive treatment on composition of cerebral arterioles.

Treatment of chronic hypertension with cilazapril, but not hydralazine, attenuates changes in distensibility of cerebral arterioles that occur in stroke-prone spontaneously hypertensive rats (SHRSPs). In this study, effects of antihypertensive treatment on composition of cerebral arterioles was determined in SHRSPs. Cilazapril (45 mg/kg/day), an angiotensin converting enzyme (ACE) inhibitor, or hydralazine (18 mg/kg/day) was begun when rats were 3 months of age. Both cilazapril and hydralazine reduced systolic arterial pressure in SHRSPs (from 199 +/- 6 to 122 +/- 7 mm Hg for cilazapril versus 143 +/- 5 mm Hg for hydralazine [mean +/- SEM]; p less than 0.05). Cerebral arterioles were fixed in vivo, and the cross-sectional area of the vessel wall was measured histologically. In SHRSPs, both cilazapril and hydralazine reduced cross-sectional area of the vessel wall to values obtained in Wistar-Kyoto (WKY) rats. Thus, both cilazapril and hydralazine prevented hypertrophy of cerebral arterioles in SHRSPs. Composition of the arteriolar wall was determined with point counting stereology. Cerebral arterioles in SHRSPs contained significantly more smooth muscle and elastin than in WKY rats (1,294 +/- 157 versus 853 +/- 88 microns2, respectively, for smooth muscle and 148 +/- 13 versus 108 +/- 7 microns2, respectively, for elastin (120 +/- 8 microns2) in cerebral arterioles in SHRSPs was similar to that in WKY rats. Treatment with hydralazine was effective in preventing increases in elastin (128 +/- 14 microns2) and in attenuating increases in smooth muscle (1,008 +/- 18 microns2). The ratio of nondistensible (collagen, basement membrane) to distensible (smooth muscle, elastin, endothelium) components was greater in SHRSPs than in WKY rats.(ABSTRACT TRUNCATED AT 250 WORDS)

The interaction of the Bainbridge and Bezold-Jarisch reflexes in the conscious dog.

Experiments were undertaken to determine the efferent path of the Bainbridge reflex and to investigate the interaction of the Bainbridge reflex with the Bezold-Jarisch reflex in conscious, chronically instrumented dogs. The Bainbridge reflex was elicited by distending the left atrium by inflating a chronically implanted balloon catheter. The Bezold-Jarisch reflex was elicited using chemical stimulation of left ventricular receptors with infusions of veratridine (0.1-0.8 micrograms/kg/min) into the left circumflex coronary artery. Heart-rate responses to left atrial balloon inflation were compared before and after either beta-1 antagonism with metoprolol or cholinergic antagonism with atropine, and before and during left ventricular receptor stimulation with intracoronary veratridine. Left atrial balloon inflation alone caused a significant increase in heart rate (70.1 +/- 5 bpm), left atrial pressure (14 +/- 3 mmHg) and mean arterial blood pressure (10 +/- mmHg). Heart-rate responses to left atrial distension were inhibited, but not abolished by either cholinergic or beta-1 antagonism. Left atrial distension after both cholinergic and beta-1 antagonism abolished the heart-rate response to balloon inflation. These results indicate that the efferent component of the Bainbridge reflex has both a vagal and a sympathetic component in conscious dogs. Left atrial distension during simultaneous left ventricular receptor stimulation resulted in a significantly decreased tachycardia than did left atrial distension alone (26 +/- 3 bpm compared to 68 +/- 8 bpm in the control experiments). In addition, the slope of the heart rate vs left atrial pressure relationship was significantly inhibited by left ventricular receptor stimulation (1.8 +/- 0.2 bpm/mmHg compared to 5.7 +/- 0.3 bpm/mm Hg in the control experiments). There were no significant differences in either the left atrial pressure or arterial blood pressure changes between the two groups. These data suggest an interaction between these two reflexes that may be occurring in the central nervous system.

Effects of antihypertensive therapy on mechanics of cerebral arterioles in rats.

The purpose of this study was to examine effects of antihypertensive treatment on structure and mechanics of cerebral arterioles and the incidence of stroke in stroke-prone spontaneously hypertensive rats (SHRSP). Treatment of hypertension was begun at 3 months of age with cilazapril (45 mg/kg/day), an angiotensin converting enzyme (ACE) inhibitor, or with hydralazine (18 mg/kg/day). Cilazapril and hydralazine reduced systolic arterial pressure (from 195 +/- 8 to 125 +/- 5 and 148 +/- 3 mm Hg, respectively [mean +/- SEM]; p less than 0.05). To examine structure and mechanics of cerebral arterioles, we measured pressure (servonull), external diameter, and cross-sectional area of the vessel wall (histologically) in pial arterioles of normotensive Wistar-Kyoto (WKY) rats and SHRSP that were untreated or that were treated for 3 months with cilazapril or with hydralazine. Arterioles were maximally dilated with EDTA. In WKY rats, cilazapril and hydralazine did not alter pial arteriolar pressure, external diameter, or cross-sectional area of the vessel wall. In SHRSP, both cilazapril and hydralazine reduced cross-sectional area of the vessel wall to levels not significantly different from WKY rats (from 1,911 +/- 155 to 1,244 +/- 101 and 1,388 +/- 59 microns 2, respectively, compared with 1,405 +/- 95 microns 2 for untreated WKY rats). Cilazapril was more effective than hydralazine in reducing pial arteriolar pressure (from 110 +/- 6 to 62 +/- 2 mm Hg with cilazapril versus 79 +/- 5 mm Hg for hydralazine compared with 60 +/- 4 mm Hg for untreated WKY rats). Cilazapril, but not hydralazine, attenuated reductions in external diameter of pial arterioles (from 91 +/- 4 to 100 +/- 4 microns for cilazapril versus 91 +/- 3 microns for hydralazine compared with 107 +/- 3 microns for untreated WKY rats).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of aging on mechanics and composition of cerebral arterioles in rats.

The purpose of this study was to examine effects of aging on the mechanics and composition of cerebral arterioles. We measured pressure (servo-null) and diameter in pial arterioles in anesthetized adult (9-12 months old) and aged (24-27 months old) Fischer 344 rats. After deactivation of smooth muscle with EDTA, diameter of pial arterioles at 70 mm Hg pial arteriolar pressure was less in aged than in adult rats (67 +/- 4 vs. 81 +/- 4 microns [mean +/- SEM], p less than 0.05). The stress-strain relation and the slope of tangential elastic modulus versus stress (6.8 +/- 0.6 vs. 5.3 +/- 0.3, p less than 0.05) indicated that distensibility of pial arterioles was reduced in aged rats. Cross-sectional area of the vessel wall, measured histologically, was less in aged than adult rats (1,239 +/- 91 vs. 1,832 +/- 180 microns2, p less than 0.05). Point counting stereology was used to quantitate smooth muscle, elastin, collagen, and basement membrane in the arteriolar wall. Cross-sectional areas of smooth muscle and elastin were significantly less in aged than adult rats (744 +/- 57 vs. 1,291 +/- 119 microns2 for smooth muscle, 52 +/- 6 vs. 113 +/- 15 microns2 for elastin; p less than 0.05), whereas cross-sectional areas of collagen and basement membrane were not significantly different in aged and adult rats (4 +/- 1 vs. 3 +/- 1 microns2 for collagen, 236 +/- 17 vs. 258 +/- 31 microns2 for basement membrane). The ratio of nondistensible (collagen and basement membrane) to distensible (smooth muscle and elastin) components was greater in aged than adult rats (0.30 +/- 0.01 vs. 0.18 +/- 0.01, p less than 0.05). Thus, we conclude that, during aging, cerebral arterioles undergo atrophy, distensibility of cerebral arterioles is reduced, and the relative proportion of distensible elements, elastin and smooth muscle, is reduced in the arteriolar wall.

A central mechanism of acute baroreflex resetting in the conscious dog.

The role of the central nervous system in the mechanism(s) involved in acute carotid baroreflex resetting was studied in six conscious, chronically instrumented, aortic-denervated dogs. Dogs were prepared for reversible vascular isolation of the carotid sinuses. Acute baroreflex resetting was induced by holding the left carotid sinus pressure (LCcsp) at a given value for 20 minutes using a pulsatile pressure control system while at the same time keeping the right carotid sinus pressure (RCSP) at a subthreshold level (approximately 40 mm Hg). At the end of the 20 minutes, the LCcsp) was reduced to approximately 20 mm Hg, and a baroreflex (RCSP-mean arterial pressure [MAP]) curve was generated on the right carotid sinus using static-step increases in carotid sinus pressure. At the control LCcsp of 100 mm Hg, the RCSP-MAP baroreflex had a threshold pressure (Pth) of 86.6 +/- 3.1 mm Hg and a set point pressure (Psp) of 104.7 +/- 2.5 mm Hg. Increasing LCcsp) to 140 mm Hg for 20 minutes caused these parameters for the right carotid baroreflex to increase. Pth and Psp increased by 18.4 +/- 4.0 and 14.2 +/- 3.0 mm Hg, respectively (p less than 0.05). The baroreflex curve, therefore, was shifted upward and to the right. Decreasing LCcsp to 60 mm Hg caused Pth and Psp to decrease by 24.7 +/- 5.0 and 18.1 +/- 2 mm Hg, respectively (p less than 0.05). The baroreflex curve was therefore shift downward and to the left. The percent of resetting of Pth and Psp was 46 +/- 9% and 36 +/- 8%, respectively, when LCcsp was 140 mm Hg.(ABSTRACT TRUNCATED AT 250 WORDS)

PGE2 and arachidonate inhibit the baroreflex in conscious dogs via cardiac receptors.

Prostaglandin (PG) I2 and PGE2 are known to stimulate left ventricular receptors with nonmyelinated vagal afferents. The present experiments were performed to determine the effects of intracoronary infusion of PGE2 (10-50 ng.kg-1.min-1) and arachidonic acid (50-100 micrograms.kg-1.min-1) on the baroreflex control of heart rate in conscious dogs. Dogs were anesthetized with pentobarbital sodium and were instrumented using sterile surgical techniques. After recovery, baroreflex pressure-heart rate curves were constructed by varying arterial pressure with partial occlusions of the descending aorta or inferior vena cava. Intracoronary infusion of PGE2 significantly inhibited the maximum heart rate achieved during unloading of baroreceptors, attenuated the heart rate range, and decreased the maximum slope of the baroreflex curve; PGE2 had no significant effect on the minimum heart rate during hypertension. Intravenous infusion of PGE2 did not cause significant baroreflex inhibition, and pericoronary nerve block in three dogs prevented the effects of intracoronary PGE2. Intracoronary infusion of arachidonic acid had effects on the baroreflex control of heart rate similar to those of PGE2. The effects of arachidonic acid infusion were prevented by cyclooxygenase blockade. Thus intracoronary PGE2 and arachidonic acid inhibit the baroreflex control of heart rate most likely via stimulation of left ventricular receptors with vagal C-fiber afferents. The effects of arachidonic acid were secondary to synthesis of prostaglandins.

Intracoronary infusion of prostaglandin I2 attenuates arterial baroreflex control of heart rate in conscious dogs.

Prostaglandin I2 (PGI2) is known to stimulate ventricular C fiber receptors resulting in a Bezold-Jarisch-like reflex. Also, cardiac receptor stimulation is known to interact with the expression of arterial baroreflexes. Therefore, experiments were performed to determine the effects of left circumflex coronary artery infusion of PGI2 on the baroreflex control of heart rate in conscious instrumented dogs. Dogs were instrumented chronically with an aortic catheter for the measurement of mean aortic pressure, hydraulic occluder cuffs on the descending aorta and inferior vena cava, a left ventricular catheter for the measurement of left ventricular pressure and heart rate, and a nonocclusive catheter in the left circumflex coronary artery. At the time of experimentation, arterial pressure was altered randomly in steps by partially inflating the occluders. Mean arterial pressure-heart curves (baroreflex curves) were constructed by fitting the data to a logistic curve by nonlinear regression. PGI2 infused into the left circumflex coronary artery at doses of 10, 20, and 50 ng/kg/min caused significant (p less than 0.05) inhibition of the maximum heart rate, heart rate range, and maximum slope of the curve compared to the control baroreflex curve obtained during intracoronary infusion of PGI2 vehicle. PGI2 had no significant effect on the minimum heart rate during hypertension. Since PGI2 is known to stimulate left ventricular receptors, these effects were most likely produced via stimulation of cardiac receptors. In additional experiments using beta 1-blockade with metoprolol or cholinergic blockade with atropine methyl bromide, it was shown that PGI2 attenuates baroreflex-mediated tachycardia by preventing parasympathetic withdrawal completely and by attenuating sympathetic stimulation by approximately 50%.(ABSTRACT TRUNCATED AT 250 WORDS)

PGI2 attenuates baroreflex control of renal nerve activity by a vagal mechanism.

The present study was undertaken to determine whether left circumflex coronary artery (ic) administration of prostacyclin (PGI2) caused an inhibition of the baroreflex control of renal sympathetic nerve activity (RSNA). RSNA was recorded in 12 dogs. Baroreflex sensitivity of RSNA was assessed by infusion of either sodium nitroprusside or phenylephrine and by determining the slope of the mean arterial pressure-RSNA relationship. During nitroprusside infusion, intracoronary PGI2 depressed the baroreflex sensitivity by nearly 90% compared with intracoronary tris(hydroxymethyl)aminomethane (Tris) (P less than 0.002). In addition, the peak increase in RSNA during nitroprusside infusion was significantly inhibited during intracoronary PGI2 (57.9 +/- 6.4 vs. 21.2 +/- 3.0 spikes/s, P less than 0.05). There was no significant difference in the inhibition of RSNA during phenylephrine infusion when intracoronary PGI2 was compared with Tris. Both bilateral vagotomy and pericoronary lidocaine blocked the inhibitory effects of PGI2 on the baroreflex increase in RSNA. It is concluded from these data that exogenously administered PGI2 stimulates or sensitizes afferent endings within the supply of the left circumflex coronary artery to inhibit the baroreflex control of RSNA during evoked hypotension. These afferents traverse vagal pathways via the pericoronary nerves. The role of endogenous prostaglandins in modulation of baroreflex function via a cardiac reflex remains to be elucidated.

Prostaglandins mediate the increased sensitivity of left ventricular reflexes after captopril treatment in conscious dogs.

Captopril administration has been shown to result in the release of prostaglandins (PGs) in experimental animals and patients. Also, PGs, particularly prostacyclin (PGI2), have been shown to stimulate left ventricular receptor reflexes. Thus, the hypothesis that captopril administration results in sensitization of left ventricular reflexes via increased circulating levels of PGs was tested in conscious instrumented dogs. Left ventricular reflexes were stimulated by injecting veratridine into the left circumflex coronary artery through a nonocclusive catheter. Under control conditions, injection of veratridine resulted in a decrease in mean arterial pressure of -25 +/- 4.7% from a base line of 97 +/- 4.7 mm Hg and a decrease in heart rate of -28 +/- 3.7% from a base line of 91 +/- 6.6 beats/min. After administration of captopril, veratridine injection resulted in a decrease in mean arterial pressure of -43 +/- 4.9% and a decrease in heart rate of -51 +/- 8.5%, both significantly greater effects than before captopril (P less than .05); N = 7). Subsequent administration of the cyclooxygenase inhibitor, indomethacin (5 mg/kg), in the presence of captopril reversed the potentiation of the response to veratridine. Thus, after indomethacin, injection of veratridine decreased mean arterial pressure -29 +/- 4.4% and decreased heart rate -28 +/- 4.1%; changes not significantly different from the control response. Similar findings were observed in a separate set of experiments in which heart rate was held constant by cardiac pacing (N = 6).(ABSTRACT TRUNCATED AT 250 WORDS)