Characterization of coronary vasoconstriction produced by rostra1 ventrolateral medulla stimulation in rats LESLIE F. JONES AND MICHAEL J. BRODY-/Department of Pharmacology and Cardiovascular Center, University of Iowa, Iowa City, Iowa 52242 Jones, Leslie F., and Michael J. Brody. Characterization of coronary vasoconstriction produced by rostra1 ventrolateral medulla stimulation in rats. Am. J. PhysioZ. 262 (Heart Circ. Physiol. 31): H437-H442, 1992.-Previous studies have demonstrated that coronary vasoconstriction can be produced by activation of specific central nervous system sites in the cat. The present study was undertaken 1) to develop a rat model for studying central influences on coronary circulation and 2) to utilize this model for characterization of the changes in coronary blood flow (CBF) produced by stimulation of rostra1 ventrolateral medulla (RVLM). Electrical stimulation of right RVLM in chloralose-anesthetized rats with bilateral vagotomy produced a transient decrease in CBF followed by an increase in CBF concomitant with a decrease in hindquarter blood flow, a pressor response, and tachycardia. After atenolol the tachycardia and increase in CBF were abolished, whereas the decrease in CBF was enhanced and prolonged. Phentolamine (1 mg/kg iv) or removal of the stellate ganglia inhibited the decrease in CBF but did not totally abolish the increase in coronary vascular resistance. Inhibition of nitric oxide synthesis with N-nitro-L-arginine (10 PM/kg iv) enhanced the decrease in CBF produced by stimulation in RVLM. These results indicate that, in a rat model, the centrally induced decrease in CBF is 1) mediated by cardiac sympathetic innervation but only partially through a-adrenoceptors and 2) enhanced by removal of the inhibitory effect of the endothelium. central neural endothelium
control;
coronary
blood
flow;
stellate
ganglia;
CIRCULATION is influenced by neural mechanisms. For example, enhancing sympathetic activity by chemoreceptor reflex activation, severe exercise, or behavioral stress increases coronary vascular resistance (CVR) (2, 7, 23). Although the predominant effect of sympathetic excitation in the canine model is to produce an increase in coronary blood flow (CBF) as a result of metabolic autoregulation, the neurogenically mediated coronary vasoconstriction has been shown to reduce metabolically induced coronary vasodilation (21). The involvement of the central nervous system in the regulation of CBF was suggestedby the early finding that electrical stimulation in periaqueductal gray and hypothalamus produced a transient decrease in CBF (31). Intracerebroventricular administration of picrotoxin, a y-aminobutyric acid antagonist, has also been shown to increase CVR (29). Previous studies from this laboratory have shown that coronary vasoconstriction can be produced by site-specific activation of the cat central nervous system (1, 3, 12). The central pathways that mediate this response have been defined. In the feline model the decrease in CBF is observed only after administration of a ,&adrenoceptor antagonist, is peripherally mediated by cul-adrenoceptors, and is blocked by removal of the stellate CORONARY
“f Deceased
3 December
1990.
0363-6135/92 $2.00 Copyright
0
ganglion ipsilateral to the central site of stimulation (1, 3, 12). In the present study, a rat model for studying central influences on coronary circulation was developed by modifying a technique described previously by Wangler et a1.(35). Several important advantages of using the rat as a model for studying central regulation of CBF include the following: 1) much of the work in this and other laboratories concerning the role of the central nervous system in the control of cardiovascular function has been done in the rat, 2) the brain of the rat has been extensively mapped stereotaxically, 3) more information is known about the background of the rat (strain, age, diet, and exposure to disease) compared with mongrel dogs or cats, 4) the life span of the rat is relatively short, allowing accurate age-related studies to be performed, 5) the coronary circulation can be studied in models of hypertension such as the spontaneously hypertensive rat, and 6) the rat is less expensive than larger laboratory animals. Centrally mediated coronary vasoconstriction in the rat has been suggested by the finding that electrical stimulation of lateral hypothalamus results in elevation of the S-T segment on an electrocardiogram (12). To more directly assessneurally induced changes in coronary circulation, we have continuously measured CBF during stimulation of rostra1 ventrolateral medulla (RVLM) in the anesthetized rat. RVLM was chosen as the initial site for stimulation because it is known to play an important role in cardiovascular regulation (9). Electrical and chemical stimulation of RVLM in the anesthetized rat has been shown to produce sympathetic excitation, including increases in arterial pressure (AP) and heart rate (HR) and femoral and renal vasoconstriction (6). Additionally, previous studies in our laboratory have shown that electrical and chemical stimulation of specific medullary sites decreases CBF in the cat (26). In this study, we describe the technique used to continuously measure CBF velocity during central stimulation of RVLM in the anesthetized rat. The characteristics and mechanisms for the changes in CBF are defined, and the role of the endothelium in modulating the centrally induced coronary vasoconstriction is described. METHODS General preparation. Male Sprague-Dawley rats (300-350 g) were anesthetized with an intramuscular injection of ketamine (100 mg/kg) and acepromazine (3 mg/kg). After cannulation of the left femoral vein, a-chloralose (60 mg/kg iv) was administered. Body temperature was maintained with a constant temperature heating pad, and rats were intubated, mechanically ventilated, and then paralyzed with gallamine (7 mg/kg iv) to prevent respiratory or movement artifacts that can be induced by central stimulation. Arterial blood gases and pH were main-
1992 the American
Physiological
Society
H437
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on January 18, 2019.
H438
CENTRALLY
MEDIATED
CORONARY
tained within physiological limits by adjusting respiratory volume or rate, ventilating with 95% O&% Con, or administering sodium bicarbonate (100 meq/l iv) as needed. The left femoral artery was cannulated for recording AP. HR was derived with a cardiotachometer triggered from the AP pressure pulse. After bilateral cervical vagotomy, a left thoracotomy was performed. In one group of animals, stellate ganglionectomy was performed. The middle cervical-stellate ganglion complexes (middle and inferior cervical ganglia) were isolated bilaterally. The ganglion complexes were approached retropleurally at the level of the first and second intercostal space, and 6-O surgical suture was positioned around all branches immediately distal and proximal to the ganglion complex before placing the rat in the stereotaxic apparatus. After finding a site in RVLM where a repeatable decrease in CBF could be elicited, the 6-O surgical suture was threaded through polyethylene tubing (0.28 mm inner diameter; 0.61 mm outer diameter) (PE-lo), and the branches of the ganglion complex were then cut by pulling the 6-O surgical suture through the PE-10 tubing. The complete removal of the ganglion complex was verified microscopically at the end of the experiment. Blood flow velocity measurements. A pulsed Doppler flow probe was placed around the abdominal aorta and held in place with 6-O surgical suture for measurement of hindquarter blood flow velocity (HqBF). CBF was measured with a specially designed flow probe consisting of a 20-mHz pulsed Doppler crystal in a dome-shaped Silastic suction cup. The probe was positioned over the left anterior descending (LAD) coronary artery and held in place by vacuum. With this technique the coronary blood vessel and nerves located below the surface of the myocardium are not damaged. This flow probe was used in the rat by Wangler et al. (35). Our modifications of their technique include performing a left rather than midsternal thoracotomy so that the probe could be positioned after placing the rat in a stereotaxic apparatus. Additionally, a better signal was obtained while the rat was in the stereotaxic apparatus by positioning the probe over the LAD coronary artery more proximal to the aorta rather than midway between the base and apex of the heart. Probes were connected to a pulsed Doppler flowmeter as described previously (13). AP, HR, and blood flow velocities were continuously monitored on a Beckman recorder. Electrical stimulation. Rats were positioned in a stereotaxic apparatus. A portion of the skull and dura were removed, exposing the obex, and a tungsten microelectrode (Micro Probe, X-pm-diameter tip) was stereotaxically positioned in right RVLM. Stimulations were made in an area 1.7-2.3 mm lateral and 1.7-2.3 mm rostra1 to obex, and the electrode was lowered through this area in 0.5-mm increments. Stimulation parameters consisted of 100 PA cathodal pulses of 0.5 ms duration at 50 Hz, and current was delivered for 10 s. At the end of the experiment a focal lesion was made at the most responsive site by stimulating at 1 mA cathodal pulses of 0.5 ms duration at 50 Hz for 10 s. Histological identification of stimulation sites. Rats were killed by intravenous administration of saturated KC1 solution and perfused intracardially with 0.9% saline followed by 10% formaldehyde. Brains were stored in 10% formaldehyde and then sectioned and cut in 40-pm-thick slices that were mounted and dried on glass slides and stained with cresyl violet. Electrode tip placement was verified by the electrolytic lesion, and stimulation sites were mapped on a representative coronal section taken from the atlas of Paxinos and Watson (27). Protocol. In the first experiment, AP, HR, CBF and HqBF were measured during electrical stimulation of RVLM before and after blockade of ,&-adrenoceptors with atenolol (1 mg/kg iv). Atenolol was used to inhibit the metabolic vasodilation produced by the positive chronotropic and inotropic effects of
CONSTRICTION
IN
RATS
RVLM stimulation. All remaining experiments were performed in the presence of blockade of ,&-adrenoceptors with atenolol. To assess the possibility that the decrease in CBF was due to autoregulatory or pressure-induced changes in coronary diameter, an aortic snare was used to elicit an increase in AP similar to that produced by electrical stimulation. For these experiments the cannula measuring AP was placed in the left carotid artery. The change in CBF produced by the aortic snare was then compared with the change produced by central stimulation. Another set of experiments was used to determine whether coronary vasoconstriction was centrally mediated and whether noradrenergic mechanisms were involved. After finding a site in RVLM where electrical stimulation elicited a repeatable decrease in CBF, phentolamine (1 mg/kg iv) was administered to one group of rats while stellate ganglionectomy was performed in another group of rats before restimulation in the same site. Blockade of a-adrenoceptors was confirmed by the absence of a pressor response to phenylephrine (15 rug iv). Finally, to assess the role of nitric oxide release in modifying or masking a component of the coronary vasoconstriction, central stimulation was performed before and after administration of p-nitro-L-arginine (L-NNA; 10 PM/kg iv), an inhibitor of nitric oxide synthesis (22). Data analysis. Values reported in this study, unless otherwise noted, were taken at the point corresponding to the maximum change in blood flow velocity and are expressed as percent change in vascular resistance, which was calculated using the following formula % AVR = [ (% AAP + lOO)/( % ABFV + lOO)1] X 100, where VR is vascular resistance and BFV is blood flow velocity. Changes in coronary and HqBF velocities measured with the pulsed Doppler flow probe are directly and linearly proportional to changes in blood flow measured by the microsphere and electromagnetic flow probe techniques (13, 35). All data are reported as means & SE. Data were analyzed by repeated-measures analysis of variance with covariance (ANCOVA) followed by the Student’s modified t test using the modified error mean square (EMS) terms from the ANCOVA with the Bonferroni correction for multiple comparisons between means (33). SE was calculated from the formula SE = w) where n is the number of rats. RESULTS
Stimulation before and after ,&adrenoceptor blockade. As illustrated in Fig. 1, left, electrical stimulation in one site of RVLM before administration of atenolol produced an increase in HR, AP, and CBF and a decrease in HqBF. Stimulation 1 mm ventral to this site within RVLM also produced tachycardia, a pressor response, and a decrease in HqBF, but a transient decrease in CBF was observed before the increase in CBF. Blockade of ,&adrenoceptors with atenolol (1 mg/kg iv) before restimulation in the same site that transiently reduced CBF inhibited the tachycardia, enhanced the initial decrease in CBF, and abolished the later increase in CBF. The relationship between changes in AP a.nd regional vascular resistance was analyzed usling the formula in Data analysis. Figure 2 summarizes the changes in vascular resistance produced by electrical stimulation in RVLM before and after administration of atenolol. In Fig. 2, AP, CBF, and CVR are shown at two different time points that correspond to the maximum decrease and the maximum increase in CBF produced by stimulation in RVLM before atenolol administration. The
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on January 18, 2019.
CENTRALLY
HR (beatdmin)
Before Atenolol
r
600
MEDIATED
Before Atenolol 1 mm ventral
CORONARY
AfterAteno
12oL
2ow AP mm)
100
L
t
Ob 8
CBF (kHz)
4
7 a HqBF W-M
031 WC.
7
-
Stim (10 set)
Fig. 1. Representative tracings from single rat showing hemodynamic responses elicited by electrical stimulation in RVLM before and after atenolol. Time scale under panel 2 refers to panels 2, 3, and 4. Site eliciting responses shown in panels 3 and 4 is 1 mm ventral to site eliciting responses shown in panel 2. HR, heart rate; AP, arterial pressure; CBF, coronary blood flow; HqBF, hindquarter blood flow.
decrease in CBF averaged 17 t 2%, had a latency to onset of l-3 s and lasted 10-25 s. Additionally, the decrease in CBF was found to be frequency dependent in a range of lo-75 Hz at a current intensity of 100 PA and a pulse duration of 0.5 ms. Histology. A schematic representation of the sites from which a decrease in CBF could be elicited after administration of atenolol is shown in Fig. 3. Microscopic analysis revealed that sites that produced a decrease in CBF and a pressor response ~30 mmHg were located in the rostra1 and caudal portions of RVLM; however, some sites were found surrounding this area within RVLM where only a pressor response was observed with no change in CBF. Effect of aortic snare. The finding of a pressor response with no change in CBF indicates that the decrease in CBF was not due to autoregulatory changes. This was further clarified by using an aortic snare to elicit an increase in AP similar to that produced by electrical 30 ’
10
% A
%
AP
A
HR
12026
30 % A
% A
HqVR 4o
CVR 0
0
’
-20 352 t 14
60
A
CBF
0 11227
29027
c
H439
* A*
20
5
RATS
DISCUSS1oN The medulla oblongata is necessary for the maintenance of AP and also integrates baroreceptor and chemoreceptor reflexes (17,20,28). Increases in sympathetic activity in response to exercise, fear, pain, and brain stem ischemia are also integrated by RVLM (6, 15, 19). Several of these stimuli have been shown to alter CBF. For example, activation of the carotid chemoreceptor reflex produced an increase in CVR mediated by sympathetic innervation in dogs (7). Severe exercise, anger,
.
%
IN
stimulation. With the aortic snare, an increase in CBF and a small increase in CVR (5 t 1%) were observed (Fig. 4). In comparison, electrical stimulation in RVLM produced a similar increase in AP, a decrease in CBF, and an increase in CVR (85 t 10%). Effect of stellate ganglionectomy. Removal of the stellate ganglion ipsilateral to the site of stimulation attenuated but did not abolish the decrease in CBF produced by stimulation in RVLM after administration of atenolol (Fig. 5). Bilateral stellate ganglionectomy completely inhibited the decrease in CBF. The increase in AP to RVLM stimulation after ipsilateral and bilateral stellate ganglionectomy was not significantly different from control. Effect of phentolamine. Intravenous phentolamine (1 mg/kg) did not significantly alter the pressor response produced by RVLM activation after atenolol; however, the decrease in CBF, the increase in CVR, and the increase in hindquarter vascular resistance (HqVR) were attenuated. A summary of the effect of phentolamine on the changes in vascular resistance produced by stimulation is shown in Fig. 6. Effect of L-NNA. Administration of L-NNA (10 PM/ kg iv) produced an increase in AP (14 t 4 mmHg) and a decrease in CBF (14 t 5%). Electrical stimulation in RVLM 20 min after L-NNA produced a significantly enhanced decrease in CBF and increase in CVR; however, the decrease in CBF was not prolonged. A summary of the effect of L-NNA on the hemodynamic response to stimulation in RVLM is illustrated in Fig. 7. Administration of L-NNA did not significantly alter the pressor response produced by stimulation in RVLM.
1 mm ventral
-
240 t
CONSTRICTION
q
Before
=
After
Atenolol Atenolol
* pco.05
Fig. 2. Summary of hemodynamic responses elicited by electrical stimulation in rostra1 ventrolateral medulla (RVLM) before and after atenolol. Percent changes in AP, CBF, and CVR are shown at 2 time points that correspond to maximum decrease and maximum increase in biphasic CBF response before ateno101. *Significant difference from stimulation before atenolol. Baseline AP and HR are shown below bars. CVR, coronary vascular resistance; HqVR, hindquarter vascular resistance; n, no. of rats. Values represent means, and error bars represent SE among means.
n=7
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on January 18, 2019.
H440
CENTRALLY
MEDIATED
o
pressor
response
>
30
mmHg
o
pressor
response
>
30
mmHg
and
decrease
in
CORONARY
CBF
Fig. 3. Schematic representation of coronal sections through 3 levels of RVLM showing sites from which a decrease in CBF was elicited (n = 14). Amb, ambiguous nucleus; RVL, rostroventrolateral reticular nucleus; Rob, raphe obscurus nucleus; Py, pyramidal tract.
‘;;
%A CVR
25
5.
%A
CBF
’
q n
Control Aortic
Constriction
*p
control;
coronary
blood
flow;
stellate
ganglia;
CIRCULATION is influenced by neural mechanisms. For example, enhancing sympathetic activity by chemoreceptor reflex activation, severe exercise, or behavioral stress increases coronary vascular resistance (CVR) (2, 7, 23). Although the predominant effect of sympathetic excitation in the canine model is to produce an increase in coronary blood flow (CBF) as a result of metabolic autoregulation, the neurogenically mediated coronary vasoconstriction has been shown to reduce metabolically induced coronary vasodilation (21). The involvement of the central nervous system in the regulation of CBF was suggestedby the early finding that electrical stimulation in periaqueductal gray and hypothalamus produced a transient decrease in CBF (31). Intracerebroventricular administration of picrotoxin, a y-aminobutyric acid antagonist, has also been shown to increase CVR (29). Previous studies from this laboratory have shown that coronary vasoconstriction can be produced by site-specific activation of the cat central nervous system (1, 3, 12). The central pathways that mediate this response have been defined. In the feline model the decrease in CBF is observed only after administration of a ,&adrenoceptor antagonist, is peripherally mediated by cul-adrenoceptors, and is blocked by removal of the stellate CORONARY
“f Deceased
3 December
1990.
0363-6135/92 $2.00 Copyright
0
ganglion ipsilateral to the central site of stimulation (1, 3, 12). In the present study, a rat model for studying central influences on coronary circulation was developed by modifying a technique described previously by Wangler et a1.(35). Several important advantages of using the rat as a model for studying central regulation of CBF include the following: 1) much of the work in this and other laboratories concerning the role of the central nervous system in the control of cardiovascular function has been done in the rat, 2) the brain of the rat has been extensively mapped stereotaxically, 3) more information is known about the background of the rat (strain, age, diet, and exposure to disease) compared with mongrel dogs or cats, 4) the life span of the rat is relatively short, allowing accurate age-related studies to be performed, 5) the coronary circulation can be studied in models of hypertension such as the spontaneously hypertensive rat, and 6) the rat is less expensive than larger laboratory animals. Centrally mediated coronary vasoconstriction in the rat has been suggested by the finding that electrical stimulation of lateral hypothalamus results in elevation of the S-T segment on an electrocardiogram (12). To more directly assessneurally induced changes in coronary circulation, we have continuously measured CBF during stimulation of rostra1 ventrolateral medulla (RVLM) in the anesthetized rat. RVLM was chosen as the initial site for stimulation because it is known to play an important role in cardiovascular regulation (9). Electrical and chemical stimulation of RVLM in the anesthetized rat has been shown to produce sympathetic excitation, including increases in arterial pressure (AP) and heart rate (HR) and femoral and renal vasoconstriction (6). Additionally, previous studies in our laboratory have shown that electrical and chemical stimulation of specific medullary sites decreases CBF in the cat (26). In this study, we describe the technique used to continuously measure CBF velocity during central stimulation of RVLM in the anesthetized rat. The characteristics and mechanisms for the changes in CBF are defined, and the role of the endothelium in modulating the centrally induced coronary vasoconstriction is described. METHODS General preparation. Male Sprague-Dawley rats (300-350 g) were anesthetized with an intramuscular injection of ketamine (100 mg/kg) and acepromazine (3 mg/kg). After cannulation of the left femoral vein, a-chloralose (60 mg/kg iv) was administered. Body temperature was maintained with a constant temperature heating pad, and rats were intubated, mechanically ventilated, and then paralyzed with gallamine (7 mg/kg iv) to prevent respiratory or movement artifacts that can be induced by central stimulation. Arterial blood gases and pH were main-
1992 the American
Physiological
Society
H437
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on January 18, 2019.
H438
CENTRALLY
MEDIATED
CORONARY
tained within physiological limits by adjusting respiratory volume or rate, ventilating with 95% O&% Con, or administering sodium bicarbonate (100 meq/l iv) as needed. The left femoral artery was cannulated for recording AP. HR was derived with a cardiotachometer triggered from the AP pressure pulse. After bilateral cervical vagotomy, a left thoracotomy was performed. In one group of animals, stellate ganglionectomy was performed. The middle cervical-stellate ganglion complexes (middle and inferior cervical ganglia) were isolated bilaterally. The ganglion complexes were approached retropleurally at the level of the first and second intercostal space, and 6-O surgical suture was positioned around all branches immediately distal and proximal to the ganglion complex before placing the rat in the stereotaxic apparatus. After finding a site in RVLM where a repeatable decrease in CBF could be elicited, the 6-O surgical suture was threaded through polyethylene tubing (0.28 mm inner diameter; 0.61 mm outer diameter) (PE-lo), and the branches of the ganglion complex were then cut by pulling the 6-O surgical suture through the PE-10 tubing. The complete removal of the ganglion complex was verified microscopically at the end of the experiment. Blood flow velocity measurements. A pulsed Doppler flow probe was placed around the abdominal aorta and held in place with 6-O surgical suture for measurement of hindquarter blood flow velocity (HqBF). CBF was measured with a specially designed flow probe consisting of a 20-mHz pulsed Doppler crystal in a dome-shaped Silastic suction cup. The probe was positioned over the left anterior descending (LAD) coronary artery and held in place by vacuum. With this technique the coronary blood vessel and nerves located below the surface of the myocardium are not damaged. This flow probe was used in the rat by Wangler et al. (35). Our modifications of their technique include performing a left rather than midsternal thoracotomy so that the probe could be positioned after placing the rat in a stereotaxic apparatus. Additionally, a better signal was obtained while the rat was in the stereotaxic apparatus by positioning the probe over the LAD coronary artery more proximal to the aorta rather than midway between the base and apex of the heart. Probes were connected to a pulsed Doppler flowmeter as described previously (13). AP, HR, and blood flow velocities were continuously monitored on a Beckman recorder. Electrical stimulation. Rats were positioned in a stereotaxic apparatus. A portion of the skull and dura were removed, exposing the obex, and a tungsten microelectrode (Micro Probe, X-pm-diameter tip) was stereotaxically positioned in right RVLM. Stimulations were made in an area 1.7-2.3 mm lateral and 1.7-2.3 mm rostra1 to obex, and the electrode was lowered through this area in 0.5-mm increments. Stimulation parameters consisted of 100 PA cathodal pulses of 0.5 ms duration at 50 Hz, and current was delivered for 10 s. At the end of the experiment a focal lesion was made at the most responsive site by stimulating at 1 mA cathodal pulses of 0.5 ms duration at 50 Hz for 10 s. Histological identification of stimulation sites. Rats were killed by intravenous administration of saturated KC1 solution and perfused intracardially with 0.9% saline followed by 10% formaldehyde. Brains were stored in 10% formaldehyde and then sectioned and cut in 40-pm-thick slices that were mounted and dried on glass slides and stained with cresyl violet. Electrode tip placement was verified by the electrolytic lesion, and stimulation sites were mapped on a representative coronal section taken from the atlas of Paxinos and Watson (27). Protocol. In the first experiment, AP, HR, CBF and HqBF were measured during electrical stimulation of RVLM before and after blockade of ,&-adrenoceptors with atenolol (1 mg/kg iv). Atenolol was used to inhibit the metabolic vasodilation produced by the positive chronotropic and inotropic effects of
CONSTRICTION
IN
RATS
RVLM stimulation. All remaining experiments were performed in the presence of blockade of ,&-adrenoceptors with atenolol. To assess the possibility that the decrease in CBF was due to autoregulatory or pressure-induced changes in coronary diameter, an aortic snare was used to elicit an increase in AP similar to that produced by electrical stimulation. For these experiments the cannula measuring AP was placed in the left carotid artery. The change in CBF produced by the aortic snare was then compared with the change produced by central stimulation. Another set of experiments was used to determine whether coronary vasoconstriction was centrally mediated and whether noradrenergic mechanisms were involved. After finding a site in RVLM where electrical stimulation elicited a repeatable decrease in CBF, phentolamine (1 mg/kg iv) was administered to one group of rats while stellate ganglionectomy was performed in another group of rats before restimulation in the same site. Blockade of a-adrenoceptors was confirmed by the absence of a pressor response to phenylephrine (15 rug iv). Finally, to assess the role of nitric oxide release in modifying or masking a component of the coronary vasoconstriction, central stimulation was performed before and after administration of p-nitro-L-arginine (L-NNA; 10 PM/kg iv), an inhibitor of nitric oxide synthesis (22). Data analysis. Values reported in this study, unless otherwise noted, were taken at the point corresponding to the maximum change in blood flow velocity and are expressed as percent change in vascular resistance, which was calculated using the following formula % AVR = [ (% AAP + lOO)/( % ABFV + lOO)1] X 100, where VR is vascular resistance and BFV is blood flow velocity. Changes in coronary and HqBF velocities measured with the pulsed Doppler flow probe are directly and linearly proportional to changes in blood flow measured by the microsphere and electromagnetic flow probe techniques (13, 35). All data are reported as means & SE. Data were analyzed by repeated-measures analysis of variance with covariance (ANCOVA) followed by the Student’s modified t test using the modified error mean square (EMS) terms from the ANCOVA with the Bonferroni correction for multiple comparisons between means (33). SE was calculated from the formula SE = w) where n is the number of rats. RESULTS
Stimulation before and after ,&adrenoceptor blockade. As illustrated in Fig. 1, left, electrical stimulation in one site of RVLM before administration of atenolol produced an increase in HR, AP, and CBF and a decrease in HqBF. Stimulation 1 mm ventral to this site within RVLM also produced tachycardia, a pressor response, and a decrease in HqBF, but a transient decrease in CBF was observed before the increase in CBF. Blockade of ,&adrenoceptors with atenolol (1 mg/kg iv) before restimulation in the same site that transiently reduced CBF inhibited the tachycardia, enhanced the initial decrease in CBF, and abolished the later increase in CBF. The relationship between changes in AP a.nd regional vascular resistance was analyzed usling the formula in Data analysis. Figure 2 summarizes the changes in vascular resistance produced by electrical stimulation in RVLM before and after administration of atenolol. In Fig. 2, AP, CBF, and CVR are shown at two different time points that correspond to the maximum decrease and the maximum increase in CBF produced by stimulation in RVLM before atenolol administration. The
Downloaded from www.physiology.org/journal/ajpheart by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on January 18, 2019.
CENTRALLY
HR (beatdmin)
Before Atenolol
r
600
MEDIATED
Before Atenolol 1 mm ventral
CORONARY
AfterAteno
12oL
2ow AP mm)
100
L
t
Ob 8
CBF (kHz)
4
7 a HqBF W-M
031 WC.
7
-
Stim (10 set)
Fig. 1. Representative tracings from single rat showing hemodynamic responses elicited by electrical stimulation in RVLM before and after atenolol. Time scale under panel 2 refers to panels 2, 3, and 4. Site eliciting responses shown in panels 3 and 4 is 1 mm ventral to site eliciting responses shown in panel 2. HR, heart rate; AP, arterial pressure; CBF, coronary blood flow; HqBF, hindquarter blood flow.
decrease in CBF averaged 17 t 2%, had a latency to onset of l-3 s and lasted 10-25 s. Additionally, the decrease in CBF was found to be frequency dependent in a range of lo-75 Hz at a current intensity of 100 PA and a pulse duration of 0.5 ms. Histology. A schematic representation of the sites from which a decrease in CBF could be elicited after administration of atenolol is shown in Fig. 3. Microscopic analysis revealed that sites that produced a decrease in CBF and a pressor response ~30 mmHg were located in the rostra1 and caudal portions of RVLM; however, some sites were found surrounding this area within RVLM where only a pressor response was observed with no change in CBF. Effect of aortic snare. The finding of a pressor response with no change in CBF indicates that the decrease in CBF was not due to autoregulatory changes. This was further clarified by using an aortic snare to elicit an increase in AP similar to that produced by electrical 30 ’
10
% A
%
AP
A
HR
12026
30 % A
% A
HqVR 4o
CVR 0
0
’
-20 352 t 14
60
A
CBF
0 11227
29027
c
H439
* A*
20
5
RATS
DISCUSS1oN The medulla oblongata is necessary for the maintenance of AP and also integrates baroreceptor and chemoreceptor reflexes (17,20,28). Increases in sympathetic activity in response to exercise, fear, pain, and brain stem ischemia are also integrated by RVLM (6, 15, 19). Several of these stimuli have been shown to alter CBF. For example, activation of the carotid chemoreceptor reflex produced an increase in CVR mediated by sympathetic innervation in dogs (7). Severe exercise, anger,
.
%
IN
stimulation. With the aortic snare, an increase in CBF and a small increase in CVR (5 t 1%) were observed (Fig. 4). In comparison, electrical stimulation in RVLM produced a similar increase in AP, a decrease in CBF, and an increase in CVR (85 t 10%). Effect of stellate ganglionectomy. Removal of the stellate ganglion ipsilateral to the site of stimulation attenuated but did not abolish the decrease in CBF produced by stimulation in RVLM after administration of atenolol (Fig. 5). Bilateral stellate ganglionectomy completely inhibited the decrease in CBF. The increase in AP to RVLM stimulation after ipsilateral and bilateral stellate ganglionectomy was not significantly different from control. Effect of phentolamine. Intravenous phentolamine (1 mg/kg) did not significantly alter the pressor response produced by RVLM activation after atenolol; however, the decrease in CBF, the increase in CVR, and the increase in hindquarter vascular resistance (HqVR) were attenuated. A summary of the effect of phentolamine on the changes in vascular resistance produced by stimulation is shown in Fig. 6. Effect of L-NNA. Administration of L-NNA (10 PM/ kg iv) produced an increase in AP (14 t 4 mmHg) and a decrease in CBF (14 t 5%). Electrical stimulation in RVLM 20 min after L-NNA produced a significantly enhanced decrease in CBF and increase in CVR; however, the decrease in CBF was not prolonged. A summary of the effect of L-NNA on the hemodynamic response to stimulation in RVLM is illustrated in Fig. 7. Administration of L-NNA did not significantly alter the pressor response produced by stimulation in RVLM.
1 mm ventral
-
240 t
CONSTRICTION
q
Before
=
After
Atenolol Atenolol
* pco.05
Fig. 2. Summary of hemodynamic responses elicited by electrical stimulation in rostra1 ventrolateral medulla (RVLM) before and after atenolol. Percent changes in AP, CBF, and CVR are shown at 2 time points that correspond to maximum decrease and maximum increase in biphasic CBF response before ateno101. *Significant difference from stimulation before atenolol. Baseline AP and HR are shown below bars. CVR, coronary vascular resistance; HqVR, hindquarter vascular resistance; n, no. of rats. Values represent means, and error bars represent SE among means.
n=7
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H440
CENTRALLY
MEDIATED
o
pressor
response
>
30
mmHg
o
pressor
response
>
30
mmHg
and
decrease
in
CORONARY
CBF
Fig. 3. Schematic representation of coronal sections through 3 levels of RVLM showing sites from which a decrease in CBF was elicited (n = 14). Amb, ambiguous nucleus; RVL, rostroventrolateral reticular nucleus; Rob, raphe obscurus nucleus; Py, pyramidal tract.
‘;;
%A CVR
25
5.
%A
CBF
’
q n
Control Aortic
Constriction
*p
