Brain Research, 557 (1991) 202-209 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 000689939116912N

202

BRES 16912

Role of medullary lateral reticular formation in baroreflex coronary vasoconstriction David D. Gutterman, John M. Arthur, Peter D. Pardubsky, Gerald F. Gebhart, Melvin L. Marcus* and Michael J. Brody** Veterans Administration Medical Center and the Department of Internal Medicine and Pharmacology, College of Medicine, and the Cardiovascular Center, University of Iowa, Iowa City, IA 52242 (U.S.A.)

(Accepted 9 April 1991) Key words: Brain; Sympathetic; Reflex; Cat; Coronary; Blood pressure

We have recently identified a polysynaptic pathway traversing discrete regions of the hypothalamus, midbrain, and medulla, along which site-specific electrical and chemical activation produces coronary vasoconstriction as part of a sympathoexcitatory response. We tested for the potential functional significance of this pathway by examining the hypothesis that a medullary component is involved in carotid baroreflex induced coronary vasoconstriction. Coronary flow velocity was measured with a Doppler probe in anesthetized cats. Following vagotomy and propranolol, bilateral carotid occlusion produced an increase in mean arterial pressure (56 _+ 14%, means _+ S.E.M) and in coronary vascular resistance (51 + 13%) which was greater than that (29 + 6%) expected from the concurrent rise in arterial pressure during aortic constriction. Bilateral microinjections of lidocaine into the medullary lateral reticular formation attenuated the reflex increase in pressure (11 _+ 2%) and virtually abolished the rise (8 + 2%) in coronary resistance. After one hour of recovery, carotid occlusion again increased aortic pressure (56 -+ 13%) and coronary vascular resistance (47 + 15%). Microinjections of lidocaine outside this medullary region did not impair the coronary vasoconstrictor response to carotid occlusion. We conclude that the medullary lateral reticular formation contains neural elements which participate in baroreflex-induced changes in arterial pressure and coronary vascular resistance. Components of the previously described central coronary vasoconstrictor pathway may play a role in pathophysiological conditions associated with increased coronary vasomotor tone.

INTRODUCTION Neural factors contribute to the regulation of coronary blood flow in a variety of diverse h e m o d y n a m i c conditions 8'24. Little is known about central nervous system pathways which initiate sympathetic coronary vasoconstrictor responses. Previous studies have shown that activation of selected sites in the central nervous system produces coronary vasoconstriction that is a c o m p o n e n t of a general sympathoexcitatory response. Electrical stimulation of discrete regions within hypothalamus 3'2°, m e d u l l a 13, and pons 27 produces transient coronary vasoconstriction. We have recently p e r f o r m e d n e u r o a n a t o m ical and physiological studies which suggest that these coronary vasoconstrictor sites are part of a neuroanatomical pathway that extends from anterior hypothalamus, and through medullary lateral reticular formation is en route to rostromediai lateral medulla 36. Electrical activation at several sites along this polysynaptic pathway

can p r o d u c e coronary constriction, but a physiological role for these regions in regulation of coronary blood flow has not previously been addressed. The purpose of the present study was to d e t e r m i n e whether the central coronary vasoconstrictor pathway participates in physiological regulation of coronary flow; namely, b a r o r e f l e x - m e d i a t e d c o r o n a r y vasoconstriction. We examined a caudal c o m p o n e n t of the pathway, the medullary lateral reticular formation, because projections from nucleus tractus solitarius t e r m i n a t e in this area of the brain stem 23'28'3°. The magnitude of coronary constriction elicited during electrical stimulation of this region is greater than that p r o d u c e d from m o r e rostral areas I'3. W e tested the hypothesis that interruption of neural transmission in medullary lateral reticular formation would attenuate the coronary vasoconstrictor response p r o d u c e d by bilateral occlusion of the c o m m o n carotid arteries.

t Melvin L. Marcus died on 10/29/89. ** Michael J Brody died on 12/03/90. Correspondence: D.D. Gutterman, Dept. Internal Medicine, University of Iowa Hospital, Iowa City, IA 52242, U.S.A.

203 MATERIALS AND METHODS

stem, for subsequent histological analysis.

General preparation

Histological analysis

Adult mongrel cats of either sex were sedated with an intramuscular injection of ketamine (10 mg/kg) and acepromazine (1 mg/kg), and anesthetized with a-chloralose (60 mg/kg i.v.). Supplemental doses of 30 mg were given when a corneal reflex or pressor response to surgical manipulation was present. Catheters were placed into the right femoral vein and brachial artery for administration of drugs, and measurement of arterial pressure, respectively. Body temperature was maintained at 37 °C with a thermostatically controlled heating pad. Animals were intubated and mechanically ventilated. Respiratory rate and tidal volume were varied, and sodium bicarbonate administered (1 mg/kg i.v.), to maintain the following arterial blood parameters: pH = 7.35-7.42; p O 2 = 70--110; and pCO 2 = 35-42. Through a ventral neck incision the cervical vagi were isolated and tagged for later transection. Each carotid artery was isolated and loosely snared using silk suture and PE-200 tubing. Bilateral carotid occlusions were performed by tightening both snares to the point that carotid blood flow ceased. Hemodynamic responses using this method are similar to those observed when inflatable balloon occluder devices are used for baroreflex deactivation. Carotid occlusion was performed following bilateral vagotomy and infusion of propranoloi hydrochloride (1 mg/kg i.v.; Sigma). A left fifth thoracotomy was performed, the pericardium incised, and the heart suspended in a pericardial cradle formed by suturing the pericardium to intercostal muscles. This procedure exposed the left anterior descending coronary artery for placement of an epicardial Doppler probe held onto the surface of the heart with suction. A snare was placed around the descending aorta for mechanical adjustments in arterial pressure.

Measurements of flow velocity Blood flow velocity in the left anterior descending coronary artery was continuously measured using a 20 mHz piezoelectric crystal mounted within a cupped silicon housing and attached to the epicardial surface with suction (4 Torr) 3'13. Changes in flow velocity using this probe correlate well with absolute changes in blood flow (r = 0.97-0.99) in a variety of species and over a wide range of flows 17"25'37. A principal advantage of this method is that surgical dissection of the tissue surrounding the epicardial coronary artery is not necessary, thus minimizing the risk of denervation. The right femoral artery was isolated in the proximal hindlimb and a circumferential Doppler flow probe was placed around the artery. Signals from this and the coronary piezoelectric crystal were relayed through a 4-channel Doppler signal analyzer (University of Iowa Bioengineering) and recorded on a dynograph chart recorder.

Inhibition of central neurotransmission Animals were positioned in a Kopf stereotaxic apparatus and a 1 cm diameter midline craniotomy was made over the occipital ridge, exposing the cerebellum. The craniotomy was extended laterally 0.5 cm in each direction. The dura was incised and reflected. An array of 6 stainless steel guide cannulae (0.5 mm outside diameter) were arranged 0.7 mm apart, 3 on each side of the brain stem. Using stereotaxic coordinates 2, the array was lowered (30° cranial angulation) into the brain so that both center cannulae were located 3.5 mm above the goal coordinates (region where electrical stimulation produces coronary vasoconstriction) 13. Next a stainless steel microinjector cannula (0.25 mm outside diameter) was inserted into the center guide on each side of the brain stem, protruding 2.5 mm beyond the tip of the guide cannulae. Using a Hamilton microsyringe, 500-1000 nl of 4% lidocaine (Elkin-Sinn Inc.) was injected bilaterally into the brain, above the coronary vasoconstrictor region of the medullary lateral reticular formation. Additional microinjections were made into adjacent electrodes or at other vertical levels as described in the Protocol. The centers of effective sites of injection of lidocaine were marked with Evans Blue dye (500 hi) injected bilaterally into the brain

Following dye injection, animals were sacrificed with an intravenous bolus of chloralose (100 mg) followed by potassium chloride (5 ml saturated solution). Brains were perfused through an aortic catheter with 4% buffered formaldehyde (300-500 ml at a rate of 50 ml/min) and stored in formaldehyde for 24 h. The medulla was sectioned (40 microns thick) on a freezing stage microtome. Sections were mounted onto glass slides and stained with Cresyl violet for microscopic identification of dye injection sites:.

Data analysis Analog recordings of aortic pressure, heart rate, and coronary and femoral flow velocities were digitized on-line and stored on an IBM-PC computer. Baseline values for each parameter were averaged during the 5 s immediately preceding the stimulus, and data were collected at one second intervals during carotid occlusion for 20 s, and for 20 s following release of the occlusion. Changes in hemodynamic parameters were measured between 15 and 18 s of carotid artery occlusion, at the time of peak increase in coronary vascular resistance. Two or three carotid occlusions were performed before and after each intervention, and averages of the responses were used for statistical analysis. A one-way ANOVA (random block) was used to compare percent changes in heart rate, arterial pressure, and regional vascular resistances to carotid occlusion. Comparisons were made among data obtained before and after lidocaine injection into the medullary reticular formation, and one hour later, following recovery. Each of these measurements was made after vagotomy and infusion of propranolol. Significant differences (P < 0.05) among the 3 groups were determined using Tukey's test. Other comparisons were made using a two-tailed t-test. All data are expressed as mean _+ S.E.M.

Protocol Animals were instrumented as described above, and injection cannulae were inserted so that they were located 1 mm dorsal to the desired stereotaxic coordinates. Bilateral vagotomy was performed to eliminate both efferent parasympathetic activity and the buffering effects of the aortic baroreceptors. Propranolol was administered to attenuate the myocardial metabolic response to reflex chronotropic and inotropic activation. The average hemodynamic response to two or three 20 s bilateral carotid occlusions was recorded. Next, lidocaine was injected through each of the 6 guide carmulae using an injector cannula. Bilateral carotid occlusion was then repeated. If the degree of coronary constriction to bilateral carotid occlusion was reduced by less than 50% of the control value, the electrode array was lowered 1 mm, the lidocaine injections repeated, and another carotid occlusion performed. This process was repeated until the coronary constriction was reduced by more than 50%, or until the injection site was 2 mm below goal (i.e. up to 4 injections). In one animal it was necessary to move the electrode array 1 mm caudally and repeat the injection sequence. Carotid occlusions were then performed every 30 min following an effective dose of lidocaine. By one hour, the pressor response to bilateral carotid occlusion had returned to within 70% of the pre-lidocaine value in 8 animals. These were used for statistical analysis. Of the remaining 5 cats, in 3 cases the pressor response to carotid occlusion did not return to within 70% of the pre-lidocaine value, and in 2 cases carotid occlusion was not performed following recovery from lidocaine. It was unclear why recovery of the pressor response did not occur in 3 cats. In 3 of the 8 animals included for statistical analysis, a second and more discrete injection of lidocaine was made bilaterally into the center cannula following recovery of the cardiovascular response to carotid occlusion. If coronary constriction to subsequent bilateral carotid occlusion was not reduced by 50% of the recovery value, a second injection was made into an adjacent cannula. In each of these 3 animals one or 2 repeat injections again blocked the

204

HR (bpm)

AP (mmHg)

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Aortic Pressure

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Coronary Vascular Resistance Index

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gemoral Vascular Resistance Index

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Fig. 1. Coronary vascular response to bilateral carotid occlusion (BCO). Mean values of heart rate (HR), aortic pressure (AP), left anterior descending coronary blood flow velocity (CBFV), femoral blood flow velocity (FBFV), and calculated percent change in coronary vascular resistance index (CR) are shown. Phasic tracings are seen in the first panel, and the response to a 20 s BCO after propranolol (1 mghtg i.v.) and vagotomy is shown in the second panel from the left. Carotid occlusion produced a marked increase in CR, peaking 15-18 s into the stimulation. Bilateral administration of iidocaine (1000 nl) into the lateral reticular formation (third panel) did not alter baseline HR, AP, or CBFV, but prevented the pressor and coronary constrictor response to BCO. One hour later, the response to BCO recovered (fourth panel).

coronary constriction to bilateral carotid occlusion. T h u s a more specific region of the brain stem was identified where microinjection of lidocaine inhibited baroreflex coronary vasoconstriction. Evan's blue was injected to mark the effective injection sites for subsequent histological analysis. In 11 animals (including 7 not in the above experiments) an aortic snare was constricted to elevate arterial pressure to a level comparable to that achieved during bilateral carotid occlusion. Changes in coronary vascular resistance were compared between these groups over a range of changes in arterial pressure.

Criteria for an acceptable experiment Data were analyzed only from animals where the following conditions were met: Arterial pH, pCO 2, and pO 2 were consistently

Fig. 2. Summary of coronary and hemodynamic responses to bilateral carotid occlusion. Percent changes in arterial pressure, coronary vascular resistance, femoral vascular resistance, and heart rate are shown for control measurements (following propranolol and vagotomy) in open bars, following injection of lidocalne into the reticular formation (hatched bars), and following recovery of responses (one hour after lidocaine) in solid bars. The increase in coronary vascular resistance to bilateral carotid occlusion was markedly attenuated following lidocaine. Full recovery of pressor and coronary constrictor responses was achieved one hour later.

within physiological range (see Materials and Methods); mean arterial pressure was greater than 65 mmHg throughout the study; a significant increase in coronary vascular resistance (>20%) was observed during each of 2 or 3 sequential occlusions of the carotid artery following propranolol and bilateral vagotomy; following lidocalne, the pressor response to carotid occlusion recovered to at least 70% of the pre-licodaine value; and lidocaine injection sites were located in the region of lateral reticular formation where electrical stimulation has been shown to produce coronary vasoconstriction. Of the 29 animals entered into this study, 15 demonstrated less than 20% increase in coronary vascular resistance to bilateral carotid occlusion (with little or no change in arterial pressure). In one of these 15 animals, mean arterial pressure remained below 65 mmHg. In the remaining 14 animals, carotid occlusion produced a significant increase in coronary vascular resistance, and lidocaine was injected into medullary lateral reticular formation. In one of these experiments, multiple lidocaine injections did not attenuate the coronary vasoconstriction to bilateral carotid occlusion. Histo-

TABLE I

Hemodynamic effects of carotid occlusion before and after lidocaine injected into medullary lateral reticularformation (LRF) All data were obtained after vagotomy and propranolol and are presented as mean + S.E.M. BCO = bilateral carotid artery occlusion; HR = heart rate; AP = mean aortic pressure; CVRI = coronary vascular resistance index; FVRI = femoral vascular resistance index.

Baseline

Baseline BCO BCO after lidocaine into LRF Recovery BCO (45-75 rain later) BCO after repeat injection of lidocaine *P < 0.05 vs Baseline BCO.

8 8 8 3

Response to bilateral carotid occlusion (% change from baseline)

HR (bpm)

AP (mmHg)

HR

AP

CVR1

FVRI

138 + 5 134 -+ 5 136 _+5 135 + 1.5

87+4 81 + 4 83 -+ 3 71+5

1.4+0.4 0 -+ 0.5 1.2 + 0.4 0.6_+0.3

56__+ 14 11 + 2* 56 + 13 22_+7

51 + 13 7.6 + 2* 47 -+ 15 13+8

76+27 14 + 4* 61 _+20 14+5

205

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Role of medullary lateral reticular formation in baroreflex coronary vasoconstriction.

We have recently identified a polysynaptic pathway traversing discrete regions of the hypothalamus, midbrain, and medulla, along which site-specific e...
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