Rostral Ventrolateral Medulla

It is well known that pharmacological inhibition of nitric oxide synthase (NOS) produces acute and chronic hypertension, but the mechanisms mediating the hypertension are not completely understood.¹²³⁴⁵ Although this hypertension was first attributed solely to inhibition of endothelial nitric oxide (NO), more recently a large body of evidence suggests the involvement of the central nervous system. In particular, inhibition of neuronal NO may contribute to the hypertension induced by long-term treatment with N^G-nitro-l-arginine methyl ester (L-NAME).⁶⁷⁸

Contradictory results have been reported about the role of the sympathetic nervous system (SNS) in the maintenance of hypertension induced by NOS blockade. In some studies, blockade of the SNS had no effect on this hypertension,⁹¹⁰ whereas other studies using similar methods showed a significant reduction in hypertension.⁶⁸¹¹

Because the rostral ventrolateral medulla (RVLM) contains sympathetic premotor neurons and is considered to be the final common pathway for several cardiovascular responses and for the control of sympathetic vasomotor tone, it is reasonable to suggest that these premotor neurons may be involved in the maintenance of hypertension due to NOS inhibition.¹² To test this hypothesis, we performed a study in which we compared the effects of pharmacological inhibition of the RVLM with the effects of ganglionic blockade on mean arterial pressure (MAP) and heart rate (HR) in hypertensive and control groups. We also tested the role of glutamatergic synapses within the RVLM in this model.

Methods

Experiments were performed on 51 male Wistar rats (weight 250 to 300 g) provided by the Central Animal House of the Federal University of São Paulo, Brazil. All animal procedures were conducted according to the “Guidelines for the Ethical Care of Experimental Animals” and were approved by the Institutional Animal Care and Use Committee. On arrival, the rats were randomly assigned to 1 of 2 groups: normotensive or hypertensive. Hypertensive animals were treated orally with L-NAME (70 mg/kg) for 7 days. Normotensive animals received treatment with vehicle (distilled water) only. After the end of treatment, rats were anesthetized with ketamine and xylazine (40 and 20 mg/kg IP, respectively) and instrumented with femoral venous and arterial catheters constructed from PE-50 and PE-10 tubing filled with heparinized saline for drug injection and arterial pressure recording, respectively. Catheters were externalized through the nape of the animal’s neck. After surgery, the rats were returned to their home cage for recovery. All experiments were performed ≥24 hours after surgery. On the day of the experiment, arterial blood pressure and HR were recorded in awake, free-moving rats, and the animals were then anesthetized very slowly with urethane (1.2 to 1.4 g/kg IV). Animals showing a decrease in blood pressure with anesthesia were not used in the experiment. The trachea was cannulated for artificial ventilation, and pulsatile arterial blood pressure (ABP) was recorded with a P23XL transducer (Statham Instruments Division, Gould Inc) connected to an RS3400 recorder (Record System Division, Gould Inc). We obtained MAP by filtering the ABP signal, and we obtained HR from a cardiotachometer (ECG/Biotach, Gould Inc) triggered by the pulse wave. Body temperature was maintained at 37°C with the use of a heating table.

Rats were placed prone in a stereotaxic apparatus (David Kopf Instruments) with the bite bar 12 mm below the interaural line. An occipital craniotomy was performed to expose the dorsal surface of the brain stem and cerebellum. We then opened the dura and retracted it by exposing the obex, whose vertex was taken as a landmark for the stereotaxic coordinates.

Microinjection Procedures

The rostral ventrolateral medulla was identified by injection of l-glutamate with stereotaxic coordinates 3.0 mm rostral, 1.8 mm lateral, and 3 mm ventral to the obex. Microinjections into the RVLM were performed with glass micropipettes (tip diameter ≈20 μm) connected to a handheld syringe. We monitored the injected volume by observing the meniscus through a dissecting microscope with a calibrated graticule. Drugs were bilaterally injected in a volume of 100 nL over a period of 10 to 20 seconds. Microinjections of l-glutamate (10 nmol), glycine (50 nmol), and kynurenic acid (2 nmol) were diluted in saline, and the pH of all solutions was adjusted to 7.4. Microinjections of vehicle alone produced no changes in blood pressure or HR in hypertensive or normotensive animals. All drugs used were from Sigma Chemical Co.

At the end of the experiments, 100 nL of 2% Evans blue dye was injected bilaterally into the RVLM, and the brain stem was removed and stored in 10% formalin. The brain stem was sectioned along the coronal plane (50 μm) and stained with neutral red. Microinjection sites were identified by deposition of Evans blue. Figure 1 is representative of the dye distribution.

Experimental Protocol

The RVLM was identified by injection of 5 nmol of l-glutamate, and only 1 more injection was made thereafter (glutamate, glycine, or kynurenic acid). At the end of the experiment, hexamethonium bromide (10 mg/kg) was injected intravenously.

Statistical Analysis

All values are expressed as mean±SEM. The significance of changes in MAP or HR after microinjection was determined within each group by Student’s paired t test. Differences between groups were assessed by 1-way ANOVA followed by the Kruskal-Wallis test. Differences were considered significant at P<0.05.

Results

Rats chronically treated with L-NAME developed a significant increase in MAP (157±5 mm Hg) compared with the vehicle-treated group (105±4 mm Hg).

Effects of Microinjections of Glycine Into the RVLM

Microinjections of the inhibitory amino acid glycine into the RVLM of hypertensive rats (n=7) resulted in a significant decrease in MAP (from 158±4 to 71±4 mm Hg; P<0.05) and HR (from 401±32 to 330±31 bpm; P<0.05), as shown in Figure 2. MAP began to decrease immediately after the microinjection, reached a minimum value within 6.17±1.83 minutes, and then gradually recovered over 45±16.31 minutes.

In control normotensive rats (n=6), microinjections of glycine into the RVLM decreased MAP (from 106±4 to 60±3 mm Hg; P<0.05), and bradycardia was also observed (from 440±19 to 377±55 bpm; P<0.05). The response peaked at 2.2±0.7 minutes, and recovery occurred within 10.6±4.8 minutes. An important finding was that the MAP levels reached by normotensive and hypertensive rats were not significantly different (60±3 mm Hg in normotensive animals and 71±4 mm Hg in hypertensive animals), as shown in Figure 2.

Effects of Microinjection of Glutamate Into the RVLM

A significant pressor response followed glutamate microinjections into the RVLM of hypertensive rats (n=7), as shown in Figure 3. The increase in MAP (from 157±3 to 201±6 mm Hg; P<0.05) was accompanied by bradycardia (from 407±18 to 312±19 bpm; P<0.05). The response began during the microinjection period, peaked at 2 minutes, and remained above the basal level for 33 minutes. When glutamate was injected into normal rats (n=6), it also produced a significant increase in MAP (from 105±5 to 148±9 mm Hg) and a reduction in heart rate from 380±49 to 363±53 bpm (P<0.05). An important result was that no significant difference in pressor response was observed between the hypertensive and normotensive groups.

Effects of Microinjections of the Broad-Spectrum Glutamate Antagonist Kynurenic Acid Into the RVLM

Bilateral microinjection of kynurenic acid into the RVLM of hypertensive animals (n=7) did not elicit any change in MAP or HR, which indicates that glutamatergic synapses within the RVLM are not important for the maintenance of high blood pressure in this model. The same result was observed when kynurenic acid was microinjected into control animals (n=6).

Intravenous Injection of the Ganglion Blocker Hexamethonium

Figure 4 shows the response to intravenous injection of hexamethonium in hypertensive (n=7) and normotensive (n=5) rats. Hexamethonium produced a significant decrease in MAP in both groups (from 140±8 to 69±4 mm Hg [P<0.05] in hypertensive rats and from 100±4 to 60±5 mm Hg [P<0.05] in control rats). There was no significant difference between groups in the level of MAP that was reached. Another finding was that the maximum hypotensive effect of hexamethonium was the same as that of glycine in both normotensive and hypertensive animals.

Discussion

The present study clearly shows that the SNS plays a major role in the maintenance of hypertension induced by long-term L-NAME treatment. It also seems reasonable to suggest that the RVLM is the main source of this sympathetic activation. Importantly, all vasomotor tone generation in the chronic L-NAME model is dependent on the sympathetic system, because either intravenous hexamethonium administration or glycine microinjection into the RVLM reduced MAP to a level comparable to that seen after acute spinal cord transection in both groups (normotensive and hypertensive).

Even though the SNS is primarily involved in this model of hypertension, we observed no evidence of tonic activity of glutamatergic synapses within the RVLM. In a previous study of rats with chronic renal hypertension,¹³ we showed that RVLM activity was also involved in the maintenance of hypertension, although in that study, glutamatergic synapses in the RVLM appeared to have strong tonic activity.

There is increasing evidence to suggest that the SNS may play a primary role in the pathogenesis of essential hypertension and in the long-term regulation of arterial pressure and that effects may be dependent, in part, on the activity of a small group of premotor neurons localized in the RVLM.^13141516 In spontaneously hypertensive rats, for example, there is enhanced sympathetic reactivity in response to RVLM stimulation with glutamate.¹⁴

The role of the SNS in hypertension due to NOS inhibition is controversial. Acute inhibition of the SNS in this model has been shown to produce either a substantial fall or a small change in blood pressure.⁶⁸⁹¹⁰ However, acute or chronic ganglionic blockade induces a fall in blood pressure in rats subjected to long-term treatment with L-NAME, which suggests that an enhanced sympathetic drive must be involved in this model of hypertension.⁶⁸ At some point, the hypertension produced by NOS inhibition changes from an endothelial peripheral vasoconstriction to a model that apparently is mainly dependent on activity of the SNS. Why and when this happens remain to be determined.

We should also consider the involvement of the brain NO system in this model. Iadecola et al¹⁷ found that systemic administration of L-NAME leads to partial inhibition of brain NOS catalytic activity over a period of 1 to 2 hours that persists for several days. Recent immunocytochemical studies¹⁸¹⁹²⁰ reported high concentrations of NOS within specific regions of the brain, some of which are involved in cardiovascular regulation. Microinjection of NO or NO donors in the paraventricular nucleus results in decreases in blood pressure, which indicates the potential of NO to influence cardiovascular control mechanisms through actions in this area.²¹ It has been shown that inhibition of NOS in the nucleus tractus solitarii increases sympathetic tone and blood pressure in rabbits.²² It has also been suggested that NO modulates the baroreceptor reflex control of HR in spontaneously hypertensive and Wistar-Kyoto rats at the level of the nucleus tractus solitarii.²³

On the other hand, several studies have related the hypertension induced by NOS inhibition to the angiotensin system.²⁹²⁴²⁵ Long-term angiotensin II inhibition seems to be able to prevent and reverse hypertension.⁹²⁴ It has also been suggested that hypertension induced by L-NAME treatment is sustained by an interaction of several mechanisms, including activation of the SNS and the renin-angiotensin system.¹⁰²⁶²⁷ Because it is well known that the brain angiotensin system has excitatory effects in several areas, including the RVLM, this system may be involved in this model of hypertension.²⁸²⁹³⁰ Another possibility is that NO could be acting as a neurotransmitter or neuromodulator in the RVLM to produce cardiovascular effects.³¹³²

We know that sympathetic vasomotor tone depends critically on tonic activity of RVLM premotor neurons.¹²³³ There is a large body of evidence showing that the functional integrity of the RVLM is essential for the maintenance of basal vasomotor tone.¹²³³ Electrolytic lesions or chemical inactivation of RVLM neurons by inhibitory amino acids such as glycine or γ-aminobutyric acid (GABA) results in a collapse of blood pressure similar to that usually obtained in animals with acute spinal injury.¹²³³³⁴

In summary, the present study demonstrated that in rats made hypertensive by long-term L-NAME treatment, the RVLM is the major source of vasomotor tone. However, these data provide no definitive answer as to where L-NAME acts in the central nervous system.

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Footnotes