Effects of chemical stimulation of the rostral and caudal ventrolateral medulla on cerebral and renal microcirculation in rats

Journal of the

Au~;toi.. System Journal of the Autonomic Nervous System 51 (1995) 77-84

Effects of chemical stimulation of the rostra1 and caudal ventrolateral medulla on cerebral and renal microcirculation in rats Koichi Chida a,*, Mizuo Miyagawa b, Wataru Usui b, Hiroshi Kawamura b, Toshiaki Takasu a, Katsuo Kanmatsuse b aDepartment of Neurology, Nihon University School of Medicine, 30-I Oyaguchi-Kamimachi, Itabashi-ku, Tokyo 173, Japan, b 2nd Department of Internal Medicine, Nihon University School of Medicine, Tokyo, Japan

Received 24 March 1994; revision received and accepted 6 May 1994

Abstract We investigated the effects of neurons in the rostra1 and caudal ventrolateral medulla (RVL and CVL) on cerebral and renal microcirculation in rats. Rats were anesthetized with chloralose, paralyzed with tubocurarine, and artificially ventilated. Cerebral and renal blood flows (CBF and RBF) were measured simultaneously using laser-Doppler flowmetry. Chemical stimulation of the RVL neurons by microinjection of the excitatory amino acid L-glutamate increased arterial pressure CAP), whereas that of the CVL neurons decreased AP. Stimulation of the RVL neurons also elicited a stimulus-locked increase in CBF and a decrease in RBF. The percent change in CBF and RBF was dose-dependent as stimulus intensity was increased. Cerebral and renal vascular resistance (CVR and RVR) levels were calculated from changes in CBF or RBF and changes in mean Ap. The percent reduction in CVR and percent elevation in RVR were also dose-dependent. Chemical stimulation of the CVL neurons elicited a stimulus-locked decrease in CBF and an increase in RBF. The percent reduction in CBF and percent elevation in CVR were dose-dependent. The percent reduction in RVR was also dose-dependent, while the percent elevation in RBF was not significant. Blood withdrawal reduced AP by a similar degree to CVL stimulation, but did not significantly decrease CBF. The results suggest that RVL and CVL neurons integrate cerebral and systemic microcirculation. Keywords:

Rostra1 ventrolateral Renal blood flow

medulla; Caudal ventrolateral

1. Introduction

Two restricted areas in the medulla, one located in the rostra1 and the other in the caudal

* Corresponding author. Tel.: (81-3) 3972-8111. 0165-1838/95/$09.50

medulla; Chemical stimulation;

ventrolateral medulla, tonically regulate resting and reflex control of arterial pressure (API. The rostra1 ventrolateral medulla (RVL) [4-7,1118,201, which is compatible with a cluster of adrenergic neurons of the Cl group, has an excitatory function on cardiovascular regulation. On the other hand, the caudal ventrolateral medulla

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SSDI 0165-1838(94)00115-Z

Cerebral blood flow;

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(CVL) [1,2,7,9,10,19,20], which is compatible with a cluster of noradrenergic neurons of the Al group, has an inhibitory function on the cardiovascular regulation. In conjunction with the nucleus tractus solitarii (NTS), the RVL and CVL act complementarily and integrate the baroreflex function. We partially reported that chemical excitation of the neurons in the ventrolateral medulla affected cerebral blood flow (CBF) and renal blood flow (RBF) in rats [4,13]. In this study, we examined the responses of CBF and RBF elicited by chemical excitation of the RVL and CVL neuron in rats.

2. Materials

and Methods

Studies were performed on 22 male Wistar rats weighing 280-340 g, maintained in a thermally controlled (27”C), light-cycled (07.00 on19.00 off) environment and fed lab chow ad libiturn. First, animals were anesthetized by inhalation of halothane (2% in 100% 0,). Polyethylene catheters (PE-50) were placed in the right femoral artery and vein, and the trachea was cannulated. The left kidney was exposed by a flank incision for laser-Doppler flowmetry. The animals were then placed in a stereotaxic apparatus (Narishige NS-2) with the incisor bar adjusted to - 11 mm below the interaural line. The lower brainstem and the caudal half of the cerebellum were exposed by an occipital craniotomy. After completion of surgery, halothane was discontinued and cr-chloralose (40 mg/kg, i.m., supplemented by 10 mg/kg per h, i.v.> was administered. The tracheal tube was connected to a ventilator (Aika EVM-SOA), then the animals were paralyzed with d-tubocurarine (0.1 mg/kg, i.m.) and artificially ventilated with a mixture of 50% oxygen and nitrogen. The arterial catheter was connected to a strain-gauge transducer (Nihon Koden MUP0.5A) for continuous measurement of arterial pressure (API. Mean AP (MAP) was integrated electrically from AP signals. Throughout the experiment, body temperature was maintained between 37-38°C by a thermostatically controlled infrared lamp, and blood gases were adjusted to

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maintain the arterial pC0, within the normocapnit range. To measure real-time local blood flow changes during chemical stimulation of the RVL or CVL, a laser-Doppler flowmeter (Advance, ALF 21D) capable of monitoring two probes was applied. One small caliber probe (o.d. 0.8 mm> was placed extradurally on the left frontal cortex after a small craniotomy, whereas the other probe was adhered to the left kidney surface with a bonding agent. We used a one second time constant for averaging blood flow. The AP, and the cerebral and renal blood flows (CBF and RBF) were recorded simultaneously on a polygraph chart recorder (Nihon Koden WS-681G). We evaluated CBF and RBF as percentage changes from the prestimulation levels. Cerebral and renal vascular resistance (CVR and RVR) were calculated from recorded values of MAP and blood flow. The RVL and CVL neurons were stimulated chemically by microinjection of L-glutamate, which was made from monosodium glutamic acid (Sigma) dissolved in phosphate-buffered saline (pH 7.35-7.45) at concentrations of 0.0-4.0 nmol in 50 nl. A calibrated glass micropipette (tip diameter 30-50 pm) filled with solutions was carried in a stereo&& micromanipulator, and a custom-made air pressure system was used for the microinjection of solutions. Microinjection was performed over a period of 5-10 s, and the pipettes were remained in place until parameters returned to prestimulation levels to avoid leakage of the solutions. The micropipette was inserted into the left RVL according to the following coordinates: + 2.0- + 2.4 mm anterior, + 1.8- + 2.2 mm lateral, and - 2.8- - 3.4 mm horizontal from the obex, whereas into the left CVL: + 0.5- + 1.0 mm anterior, +2.1- + 2.3 mm lateral, - 1.6-- 2.0 mm horizontal. The pipette was lowered in 0.2-0.4 mm steps while delivering 0.5 nmol in 25 nl glutamate to localize the site. After localizing the site L-glutamate was injected in increasing concentrations at lo-min intervals after responses had returned to prestimulation levels. At the end of each experiment, the stimulation site was marked by an injection of pontamine sky blue dye delivered through the same pipette used

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for delivering solutions. Rats were then killed with a bolus injection of KCl, and the artifactual blood flow secondary to artificial ventilation was determined. All blood flow values were rectified with artifact values. The brain was removed and fixed in a 10% buffered formalin-saline solution. The medulla was cut serially on a vibratome into 70 pm sections, and the small blue spots indicating the sites of microinjection were carefully reconstructed on sections counterstained with neutral red. Multiple comparisons were evaluated by analysis of variance (ANOVA). One-way ANOVA and Ryan’s test were used for testing the significance of changes in dose-response studies. Differences were considered significant at P < 0.05.

3. Results Chemical stimulation of intrinsic neurons in the RVL by microinjection of L-glutamate elicited a pressor response, an increase in cerebral blood flow (CBF) and a decrease in renal blood flow (RBF) (Fig. 1). Conversely, chemical excitation of the CVL neurons elicited a depressor response, a decrease in CBF and an increase in RBF (Fig. 1). These responses were enhanced when the concentration of microinjected L-glutamate was increased (Fig. 2 and 3). Histologically injected sites were located in the rostra1 and caudal ventral medulla, respectively (Fig. 4). The RVL and CVL stimulation changed MAP dose-dependently as stimulus intensity was increased. The percent change in the blood flow, calculated from the change/(baseline - artifact level) x lOO%, showed a dose-dependent curve in CBF and RBF of the RVL stimulation study (Fig. 2). The percent change in CBF on the CVL stimulation was also dose-dependent (Fig. 3). Vascular resistance was calculated from the change in MAP/(the change in blood flow artifact level) x 100%. Cerebral and renal vascular resistance (CVR and RVR) of the RVL stimulation study showed dose-dependent curves (Fig. 2). In the CVL stimulation study, RVR was also dose-dependent, whereas CVR and RBF appeared to be so but not significantly (Fig. 3).

RVL

Ap

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CVL

uA@m

E

0

200

MAP 0E”-

CBF 0

1

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t 0.5 nmol in 50 nl

t 1.0 nmol in 50 nl P -Glutamate

Fig. 1. Typical response of arterial pressure (API, mean arterial pressure (MAP), cerebral blood flow (CBF) and renal blood flow (RBF) elicited by microinjection of L-glutamate into the rostra1 and caudal ventrolateral medulla (RVL and CVL).

Decrease in CBF induced by blood withdrawal was smaller than that by CVL chemical stimulation when the magnitude of AP reduction was similar (Fig. 5). In the CVL chemical stimulation study, the magnitude of reduction in MAP significantly correlated with the percent decrease in CBF (Fig. 6). In contrast, the magnitude of reduction in MAP did not correlate with the percent changes in CBF by blood withdrawal (Fig. 6). Blood withdrawal decreased RBF, while CVL stimulation increased RBF (Fig. 5).

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4. Discussion

In the present study, we demonstrated with laser-Doppler flowmetry and microinjections of L-glutamate that excitation of the RVL neurons increased CBF (decrease in CVR) and decreased RBF (increase in RVR) simultaneously. In contrast, excitation of the CVL neurons decreased CBF (increased CVR) and increased RBF (decreased RVR) simultaneously. 2207

The RVL neurons play a critical role in maintaining baroreflex control of AP, thus have been considered a tonic vasomotor center [3,7,14-181 and a final common pathway of sympathetic flow from the brain [12,14-161. Excitation of the RVL neurons increased AP, inhibition of the neurons decreased AP and destruction of the RVL reduced AP to the spinal level [12,15,161. CVL neurons tonically inhibit the RVL neurons, and information regarding AP fluctuation affects ac-

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Fig. 2. Group data showing responses in blood flow and vascular resistance elicited by microinjections of ascending concentration of L-glutamate into the RVL. (A) Responses in CBF and cerebral vascular resistance (CVR). Both changes were dosedependent by one-way ANOVA. (B) Responses in RBF and renal vascular resistance (RVR). Both changes were dose-dependent by one-way ANOVA.

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tivities of the CVL neurons through the NTS neurons [7,14,20]. The RVL, CVL and NTS are main components of the baroreflex which acts in the homeostasis of AP. Thus, when AF’becomes abruptly elevated, the NTS neurons excite the CVL neurons, which inhibit the RVL neurons, then AF’ decrease to physiological levels [7]. In contrast, when AP is reduced, the NTS neurons

Nervous System 51 (1995) 77-84

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inhibit the CVL neurons, which excite the RVL neurons, then AP is increased [7,14]. It has been recently developed to investigate the effect of excitation of local neurons but fibers of passage by microinjections of L-glutamate on CBF [31. Only one study using radiolabeled microspheres reported that excitation of the CVL neurons decreased ipsilateral CBF [lo]. This was

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Fig. 3. Group data showing responses in blood flow and vascular resistance elicited by microinjections of ascending concentration of L-glutamate into the CVL. (A) Responses in CBF and CVR. The decrease in CBF was dose-dependent, but the increase in CVR was not significant by one-way ANOVA. (B) Responses in RBF and RVR. The decrease in RVR was dose-dependent, but the increase in RBF was not significant by one-way ANOVA.

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not contrary to the present study. Moreover, we demonstrated an increase in RBF (a decrease in RVR) and a decrease in AF’ simultaneously fol-

Fig. 4. A representative picture demonstrating pontamine sky blue dye in the left medulla.

a microinjection

Nervous System 51 (1995) 77-84

lowing CVL stimulation. These results indicate that excitation of the CVL neurons inhibited sympathetic tone and dilated vessels in the systemic

site of L-glutamate

into the RVL (A) and CVL (B). Black spots are

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circulation. Decrease in CBF (increase in CVR) in the same condition indicated contrary responses of the vessels (i.e., vasoconstriction) in the cerebral circulation. Our results indicate that RVL and the CVL neurons have inverse effects on the systemic and cerebral microcirculation. Blood withdrawal reduced AP in a degree similar to CVL stimulation, but decreased CBF to a lesser degree than CVL stimulation. This suggests that AP reduction by blood withdrawal inhibited the CVL neurons and excited the RVL neurons, thus maintaining CBF. We suspect that autoregulation of CBF not only

CVL Chemical Stimulation

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0L

RBF lmin 1 4.0 nmol in 50 nl

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B -Glutamate Fig. 5. Tracings showing the typical response of AP, MAP, CBF and RBF elicited by the CVL chemical stimulation and blood withdrawal.

depends upon local vascular responses [8] but also on neurons in the ventrolateral medulla participating in the baroreflex and tonically regulating circulation. Excitation of the RVL neurons increased CBF measured using the [‘4Cliodoantipyrine technique [18] and laser-Doppler flowmetry 1171, but not that measured by radiolabeled microspheres [ 111. In studies with microspheres, CBF was decreased

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following chemical stimulation of the RVL [17]. This disagreement may be due to differences in the methodology used. Possibly, the microsphere method detects responses in vessels of a particular diameter and in that study, a controlled hemorrhage prior to the RVL stimulation affected the results. Noteworthy, excitation of the CVL neurons decreased CBF measured not only by laser-Doppler flowmetry but also by microspheres [lo]. Considering the connected reversed functions of the RVL and CVL neurons in the baroreflex, it seems rational that excitation of the RVL neurons increased CBF. In conclusion, neurons in the ventral medulla, which tonically regulate resting and reflex control of AI’, may play an important role in the systemic and cerebral microcirculation.

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through the cerebral cortex. J. Neurol. Neurosurg. Psychiatr., 29 (1966) 398-403. [9] Imaizumi, T., Granata, A.R., Benarroch, E.E., Sved, A.F. and Reis, D.J., Contributions of arginine vasopressin and the sympathetic nervous system to fluminating hypertension after destruction of neurons of caudal ventrolateral medulla in the rat. J. Hypertens., 3 (1985) 491-501. [lo] Maeda, M., Krieger, A.J. and Sapru, H.N., Chemical stimulation of the ventrolateral medullary depressor area decreases ipisilateral cerebral blood flow in anesthetized rats. Brain Res., 543 (1991) 61-68. [ll] Maeda, M., Krieger, A.J., Nakai, M. and Sapru, H.N., Chemical stimulation of the rostra1 ventrolateral medullary pressor area decreases cerebral blood flow in anesthetized rats. Brain Res., 563 (1991) 261-269. [12] Miyagawa, M., Chida, K., Kawamura, H. and Takasu, T., Effects of chemical stimulation and lesion of the rostra1 ventrolateral medulla in spontaneously hypertensive rats. Neurosci. Lett., 132 (1991) 1-4. [13] Miyagawa, M., Chida, K., Kawamura, H. and Yasugi, T., Difference in vascular responses elicited from two distinct sites of the rostra1 ventrolateral medulla in rats. Ther. Res., 13 (1992) 285-292. [14] Reis, D.J. Ruggiero, D.A. and Morrison, SF.: The Cl area of the rostra1 ventrolateral medulla oblongata. A critical brainstem region for control of resting and reflex integration of arterial pressure, Am. J. Hypertens., 2 (1989) 3638-374s. [lS] Ross, C.A., Ruggiero, D.A., Joh, T.H., Park, D.H. and Reis, D.J., Adrenaline synthesizing neurons in the ventrolateral medulla: a possible role in tonic vasomotor control, Brain Res., 273 (1983) 356-361. [16] Ross, CA., Ruggiero, D.A., Park, D.H., Joh, T.H., Sved, A.F., Fernandez-Pardal, J., Saavedra, J.M. and Reis, D.J., Tonic vasomotor control by the rostra1 ventrolateral medulla: effect of electrical or chemical stimulation of the area containing Cl adrenaline neurons on arterial pressure, heart rate, and plasma catecholamines and vasopressin, J. Neurosci., 4 (1984) 479-494. [17] Saeki, Y., Sato, A., Sato, Y. and Trzebski, A., Stimulation of the rostra1 ventrolateral medullary neurons increases cortical cerebral blood flow via activation of the intracerebral neural pathway. Neurosci. Lett., 107 (1989) 26-32. [18] Underwood, M.D., Iadecola, C. and Reis D.J., Neurons in the Cl area of rostra1 ventrolateral medulla mediate global cerebrovascular responses to hypoxia but not hypercapnia. J. Cereb. Blood. Flow. Metab., 7 (SuppI. 11 (1987) S226. [19] West, M., Blessing, W.W. and Chalmers, J., Arterial baroreceptor reflex function in the conscious rabbit after brainstem lesions coinciding with the Al group of catecholamine neurons. Circ. Res., 49 (1981) 959-970. [ZO] Willette, R.N. Barcas, P.P., Krieger, A.J. and Sapru, H.N., Vasopressor and depressor areas in the rat medulla. Neuropharmacology, 22 (1983) 1071-1079.