Role of the Median Preoptic Nucleus in Arterial Pressure Regulation and Sodium and Water Homeostasis during High Dietary Salt Intake

Abstract

Changes in the osmolality and level of angiotensin II (ANG II) are important peripheral signals modulating appropriate central sympathetic output and maintaining a normal arterial pressure during high salt intake. The median preoptic nucleus (MnPO) receives reciprocal inputs from the subfornical organ (SFO) and organum vasculosum of the lamina terminalis (OVLT), the circumventricular organs that have been shown to be necessary in multiple central effects of changes in the osmolality and circulating ANG II directed toward the maintenance of sodium and water homeostasis. We, therefore, hypothesized that the MnPO is a crucial part of the central neuronal mechanisms mediating the blood pressure control by altered osmolality and/or ANG II signaling during chronic high dietary salt intake. Male Sprague−Dawley rats were randomly assigned to either sham (operation), or electrolytic lesion of the MnPO. After a 7-day recovery, rats were instrumented with radiotelemetric transducers and aortic flow probes for the measurement of the mean arterial pressure + heart rate (HR) and cardiac output (CO), respectively. Femoral venous catheters were also implanted to collect blood for the measurements of plasma osmolality and sodium concentration, as well as plasma renin activity. Rats were given another 10 days to recover and then were subjected to a 28-day-long study protocol that included a 7-day control period (1.0% NaCl diet), followed by 14 days of high salt (4.0% NaCl), and a 7-day recovery period (1.0% NaCl). The data showed, that despite a slight increase in the MAP observed in both MnPO- (n = 12) and sham-lesioned (n = 8) rats during the high-salt period, there were no significant differences between the MAP, HR, and CO in the two groups throughout the study protocol. These findings do not support the hypothesis that the MnPO is necessary to maintain normal blood pressure during high dietary salt intake. However, MnPO-lesioned rats showed less sodium balance than sham-lesioned rats during the first 4 days of high salt intake. Although, these results may be explained partly by the plasma hyperosmolarity and hypernatremia observed in MnPO-lesioned rats; they also shed light on the role of the MnPO in central neuronal control of renal sodium handling during chronic high dietary salt intake.

INTRODUCTION

An appropriate adjustment of sympathetic nerve activity is believed to be one of the major mechanisms providing a normal physiological response to high dietary salt intake [1–3]. Failure of this homeostatic mechanism has been found to accompany salt-sensitive hypertension in a subset of essential hypertensive patients and several animal models of hypertension [4–8]. In addition, salt-sensitive hypertension can be induced in rats by clamping of sympathetic nerve activity using α-adrenergic blockade [9]. Together, these reports provide strong evidence of the link between the sympathetic nerve activity and salt intake. It remains unclear, however, how dietary salt chronically modulates sympathetic nerve activity.

High salt intake is thought to exert both inhibitory and excitatory effects on the sympathetic nerve activity [2, 10, 11]. An increased plasma sodium level and/or hyperosmolality, secondary to high dietary salt intake, are believed to enhance sympathetic nerve activity via central sodium/osmoreceptor activation [11–13]. On the other hand, sodium and water retention following high salt intake is known to suppress the circulating angiotensin II (ANG II) level resulting in an inhibition of the central sympathetic nerve output. Activation of the arterial baroreflex following salt-induced volume expansion is also known to participate in an inhibitory pathway of sympathetic nerve activity [14]. A balance of these central neural mechanisms is believed to be crucial in maintaining a normal blood pressure during changes in dietary salt. However, how such balance is achieved, leading to appropriate levels of the central sympathetic outflow during high salt intake, remains a topic of interest.

Several brain regions are thought to be integrative sites where peripheral ANG II, osmotic, and baroreflex signals converge. Prominent among these is the median preoptic nucleus (MnPO), a group of hypothalamic neurons located along the rostral border of the third ventricle. First, neuroanatomical studies have revealed reciprocal connections between the MnPO and multiple areas known as primary central sites that receive peripheral ANG II, osmolality, and baroreflex information. These include the subfornical organ (SFO) and organum vasculosum of the lamina terminalis (OVLT), two circumventricular organs shown to be important as primary central targets of circulating ANG II and osmotic substances [15, 16]. In addition, MnPO neurons receive neuronal inputs from the nucleus of the solitary tract and ventrolateral medulla, central sites of projections of peripheral baroreceptor afferent inputs [16–20]. Second, uni- or multisynaptic connections have been shown between the MnPO and peripheral sympathetic nervous system [21], as well as several brain regions proposed to contribute to central sympathetic excitation, including the parvocellular subdivision of the hypothalamic paraventricular nucleus (PVN) and rostral ventrolateral medulla (RVLM) [15, 16, 22, 23]. Third, numerous electrophysiological and Fos- immunocytochemistry studies have shown that peripheral administration of both ANG II and hypertonic saline activate MnPO neurons, a subset of which responds to both of these stimuli [24–28]. Interestingly, the activity of MnPO neurons, including those sensitive to ANG II and hyperosmolality, are also affected by direct stimulation of peripheral baroreflex afferents, as well as baroreflex challenges such as hemorrhage [29–31]. Fourth, a large body of lesion studies has implicated the MnPO in the central neuronal pathway that mediates drinking and vasopressin secretion, central neuronal effects elicited by both peripheral ANG II and hypertonic saline [32–35]. Finally, we have shown previously that the MnPO is necessary for the chronic hypertensive effect of ANG II, suggesting a contribution of the MnPO in the sympathoexcitatory pathway that likely mediates chronic ANG II-induced hypertension [36, 37]. Although the central neural pathway mediating sympathetic excitation following central sodium/osmoreceptor activation has not been completely elucidated, it seems probable, when taken together with previous observations, that the MnPO is a mutual site where parallel sympathoexcitatory pathways activated by ANG II and hyperosmolality converge and are integrated. As a result, we proposed that the MnPO is important in the central neuronal mechanisms that control sympathetic nerve activity and, therefore, blood pressure during changes in dietary salt intake.

In our study, the role of the MnPO in chronic blood pressure control during high salt intake was investigated. We hypothesized that the MnPO is necessary for arterial pressure to be maintained within a normal range during high dietary salt intake. To test this hypothesis, rats were subjected to either MnPO lesion or sham lesion operations. Subsequently, blood pressure and cardiac output to 4% dietary salt (14 days) were continuously monitored in MnPO- and sham-lesioned rats.

METHODS

All procedures followed the Guide for Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the University of Minnesota.

Surgical Procedures

Male Sprague−Dawley rats (Charles River Laboratories, Wilmington, USA) weighing 300–350 g were randomly subjected to either electrolytic lesion of the MnPO or a sham lesion operation. Rats were anesthetized with pentobarbital sodium (50 mg/kg, i.p.) and then positioned in a Kopf stereotaxic apparatus. They received tobramycin (4 mg, i.m.) for antimicrobial surgical prophylaxis. Electrolytic lesions of the MnPO were performed as previously described [36]. Briefly, a dorsal midline incision was made through the scalp, and bregma and lambda were leveled. A 3-mm hole centered on bregma was drilled through the skull. A teflon-insulated monopolar tungsten electrode with a 1.5-mm exposed tip was inserted at the midline into four predetermined coordinates. The coordinates used, caudal to bregma and ventral to the sagittal sinus surface, respectively, were (mm): (–0.25, –7.4), (–0.25, –7.6), (–0.4, –6.1), and (–0.4, –7.2). At each coordinate, a 1 mA current was passed through the electrode for 5 sec. Sham-lesion surgeries were similar to electrolytic lesion, except that all ventral coordinates were 2 mm less with no current passed.

Rats were allowed to recover for 7 days. Then, they were implanted with radiotelemetry blood pressure transducers (model TA11PA-C40, Data Sciences International, USA) and aortic flow probes (model 2.5PSB, Transonic Systems, USA) for continuous 24 h sampling of the mean arterial pressure (MAP) + heart rate (HR) and cardiac output (CO), respectively. The anesthesia was induced by pentobarbital sodium (50 mg/kg, i.p.), and maintained with 1–2% isoflurane via an endotracheal tube. Artificial ventilation was provided using a small animal respirator (Harvard Apparatus Co, USA) at a tidal volume of 2.5 to 3.5 ml, and frequency of 55 to 60 min⁻¹. The pressure transducer was implanted as has been described previously [36]. The venous catheter was implanted into the left femoral vein and exteriorized from the skin between the scapulae. The implantation technique for the aortic flow probe was adopted from the previous report [38]. Briefly, a median sternotomy was made in rats positioned in the dorsal recumbency. A blunt dissection was performed between the ascending aorta and main pulmonary artery, to create a small space to place a flow probe around the ascending aorta. The probe cable was passed through the sternotomy wound and exteriorized at the interscapular region. The thoracic wound was closed, and intrathoracic negative pressure was reestablished. The flow probe cable and venous catheter were passed through a mesh tether and its attached spring. Promptly after surgery, ampicillin (15 mg, i.v.) + tobramycin (4 mg, i.v.) and butorphanol tartrate (0.075 mg, i.m.) were given for antimicrobial prophylaxis and analgesia, respectively.

Rats were then placed individually in metabolic cages with telemetry receivers mounted behind. Probe cables were connected to a flowmeter (model T402, Transonic System, USA) via electronic swivels (model SL6C, Kent Scientific, USA). The housing facility was maintained at a temperature of approximately 23°C with 12/12 light/dark cycle (light on at 7.00 am). Ampicillin (15 mg, i.v.) and tobramycin (4 mg, i.v.) were given once daily for another 3 days after surgery. Another dose of butorphanol tartrate (0.075 mg, i.v.) was given at day one after surgery. Rats were allowed to recover at least 10 days before the experimental protocol was started. During this period, 1.0% NaCl diet (all diets were purchased from Research Diets, USA) and distilled water were provided ad libitum.

Experimental Protocol

The 28-day diet protocol was as follows: a 7-day-long control period of 1.0% NaCl diet, a 14-day-long period of high-salt diet (4.0% NaCl), and a 7-day-long recovery period identical to the control period. Throughout the protocol, rats were provided with ad libitum diet and distilled water.

The MAP, HR, and CO data were sampled and recorded at 500 sec⁻¹ digitizing every 4 min for 10 sec using data acquisition and analysis software (Dataquest ART version 2.2, Data Sciences International, USA). The 24-h average total peripheral resistance (TPR) was calculated as a ratio of 24-h average of the MAP and CO, assuming that the mean right atrial pressure was equal to zero.

Daily food intake, water intake, and urine output data were collected at the same time every day (2.00 p.m.). Sodium intake was calculated as a product of total food intake and sodium content in the diet (1.0% NaCl diet, 0.175 mmol/g and 4.0% NaCl diet, 0.7 mmol/g). Sodium excretion was calculated as a product of the urine output and urine sodium concentration. The urine sodium concentration was measured with an ion-specific electrode (Nova Biomedical, USA).

Measurement of the Plasma Renin Activity (PRA)

Plasma samples for the PRA measurement were collected at 10.00 p.m. on day 4 of the control period and day 7 of high-salt period. Whole blood (500 μl) was drawn from the femoral venous catheter and promptly mixed with 1 mg EDTA (20 μl) in a chilled 1-ml syringe. Five hundred microliters of 0.9% NaCl was infused through the intravenous catheter, to replace the volume of blood removed. Whole blood was centrifuged at 1000g for 20 min at 4°C. Plasma was isolated and stored at –70°C for later radioimmunoassay, as previously described [39].

Measurements of the Plasma Osmolarity and Sodium Concentration

Plasma samples were collected for the measurements of plasma osmolality and sodium concentration at 10.00 p.m. on day 6 of the control and recovery periods, and day 12 of the high-salt period. Five hundred microliters of whole blood was drawn and placed into blood tubes containing lithium heparin. The tube was gently mixed and centrifuged at 1000g for 20 min at 4°C. Plasma was promptly isolated and stored at –70 °C.

The plasma sodium concentration was later measured using a sodium analyzer (NOVA Biomedical, USA). The plasma osmolality was measured with a vapor pressure osmometer (model 5500, Wescor, Inc., USA).

Verification of MnPO Lesions

At the completion of the experimental protocol, the rats were anesthetized with pentobarbital sodium (50 mg/kg) and perfused intracardially with heparinized saline (20 U/ml heparin in 0.9% NaCl solution), followed by 4% paraformaldehyde in phosphate-buffered saline (PBS). Whole brains were removed and submersed in 4% paraformaldehyde (overnight at 4°C). Then, the brains were transferred to 30% sucrose in PBS at 4°C for 3 days. Sagittal sections of the brains (40 μm thick) were made using a freezing microtome (Lipshaw Mfg., USA). The sections were mounted on slides, allowed to dry for 24 h, and then stained with cresyl violet. Complete lesions of the MnPO were verified by light microscopy. MnPO-lesioned rats were included in the analyses if they had at least 90% of the MnPO ablated, with no or slight damage to the adjacent brain regions.

Statistical Analyses

All statistical procedures were performed with statistical software (NCSS, USA). Two-way ANOVA with repeated measures was conducted to compare each parameter between MnPO- and sham-lesioned groups. The Tukey−Kramer multiple comparison test was used to determine on which specific day of the protocol the groups were different from each other after two-way ANOVA determined significant interaction between two main factors (lesion vs treatment day). Values from the day 7 control measurement were averaged to obtain baseline values. Then, one-way ANOVA followed by Dunnett’s multiple comparison with control was performed to determine differences within groups between baseline control values and those for 14 days of high salt intake. The plasma sodium concentration and osmolality, as well as PRA, were analyzed with two-way ANOVA followed by the Tukey−Kramer multiple comparison test. Differences within groups were determined by Dunnett’s multiple comparison with control. A P value of 0.05 was set as the level of statistical significance for all statistical analyses. All values were presented as mean ± s.e.

RESULTS

After lesion verification, 12 MnPO-lesioned rats and 8 sham-lesioned rats were included in the analyses. All of the 12 MnPO-lesioned rats had at least 90% damage to the MnPO with no damage to either the SFO or OVLT. Figure 1 shows representative examples of sham and MnPO lesions.

Fig. 1.

Photomicrographs of 40-μm-thick mid-sagittal sections of the median preoptic nucleus (MnPO). A) Section from a sham-lesioned rat demonstrating dorsal (*) and ventral (#) MnPO. B) Section from a MnPO-lesioned rat demonstrating ablated dorsal and ventral MnPO. 3V is the third venricle.

Plasma Renin Activity

The mean PRA during the control period (1.0% NaCl diet) in MnPO-lesioned animals was 1.39 ± 0.16 ng ANG I • ml⁻¹ • h⁻¹, while in sham group it was 1.32 ± 0.11 ng ANG I • ml⁻¹ • h⁻¹. On day 7 of the high-salt period (4.0% NaCl diet), PRA in the MnPO lesioned rats was 0.29 ± 0.11 ANG I • ml⁻¹ • h⁻¹ and in sham animals it was 0.52 ± 0.11 ANG I • ml⁻¹ • h⁻¹. There were no significant differences in the PRA between MnPO- and sham-lesioned rats at either normal or high salt periods.

Plasma Osmolality and Sodium Concentration

The plasma osmolality in four MnPO-lesioned and four sham-lesioned rats is shown in Fig. 2A. The baseline plasma osmolality during the control period (1.0% NaCl diet) was significantly higher in MnPO-lesioned compared to sham-lesioned rats (292 ± 2 and 285 ± 1 mOsm/kg H₂O, respectively). This index in MnPO-lesioned rats increased significantly from the baseline on day 12 of the high-salt period (4.0% NaCl), while that in sham-lesioned animals remained comparable to the baseline value (297 ± 2 vs 287 ± 1 mOsm/kg H₂O, respectively). The plasma osmolality in MnPO-lesioned rats returned to the baseline during the recovery period (1.0% NaCl diet) when diet had been changed back to 1% salt.

Fig. 2.

Plasma osmolality (A) and plasma sodium concentration (B) on day 6 of the control and recovery periods (1.0% NaCl diet) and on day 12 of the high-salt period (4.0% NaCl diet) in MnPO- and sham-lesioned rats (filled and open symbols, respectiively). *P < 0.05 between groups. #P < 0.05 vs control.

The plasma sodium concentration in MnPO- and sham lesioned rats is shown in Fig. 2B. The baseline plasma sodium concentration during the normal-salt period in the above groups was 147.2 ± 0.5 and 145.5 ± 0.6 mEq/liter, respectively. The plasma sodium concentrations were not significantly different from each other in both groups at all three time points. However, on day 12 of the high-salt period, the above index in MnPO-lesioned rats increased significantly from the baseline (148.8 ± 0.5 mEq/liter), while this value remained nearly unchanged in sham-lesioned rats (146.3 ± 0.9 mEq/liter).

Cardiovascular Characteristics upon High Dietary Salt Intake

Figure 3 shows MAP and HR values throughout the experimental protocol. The average baseline MAP values during the control period (1.0% NaCl diet) in MnPO- and sham-lesioned rats were 99 ± 1 and 101 ± 2 mm Hg, respectively. In these animal groups, the mean MAP values on the last day of the high-salt period (4.0% NaCl diet) were 102 ± 1 and 106 ± 2 mm Hg, respectively. The MAP values in MnPO- and sham-lesioned rats were not significantly different throughout the study protocol (Fig. 3A). However, these indices in both MnPO- and sham-lesioned rats increased significantly from their average baselines during the high-salt period (statistics not shown). This increase in the MAP in both groups also occurred on the 1st day of the recovery period (1.0% NaCl diet).

Fig. 3.

Average 24-h mean arterial pressure (MAP, mm Hg, A) and heart rate (HR, min⁻¹, B) values during the control (1.0% NaCl diet), high-salt (4.0% NaCl diet), and recovery (1.0% NaCl diet) periods in MnPO- and sham-lesioned rats. Other designations are the same as in Fig. 2.

The average baseline HR during the 7-day control period was 397±10 and 397 ± 23 min⁻¹ in MnPO- and sham-lesioned rats, respectively. There were no significant differences between the HR values in MnPO- and sham-lesioned rats throughout the protocol (Fig. 3B). In addition, the HR in both groups remained comparable to their baseline values within the high-salt period.

Figure 4 shows 24-h average of the CO and TPR in MnPO- and sham-lesioned rats throughout the study protocol. The CO value was obtained from nine MnPO-lesioned and five sham-lesioned animals. The average baseline CO was 90 ± 4 and 99±7 ml/min in these groups, respectively. No significant differences in the CO were found between MnPO- and sham-lesioned rats throughout the study protocol (Fig. 4A). The CO in both groups was not significantly influenced by high salt intake.

Fig. 4.

Average 24-h cardiac output (CO, ml/min, A) and total peripheral resistance (TPR, mm Hg/ml/min, B) during the control, high-salt, and recovery periods in MnPO- and sham-lesioned rats. Other designations are the same as in Figs. 2 and 3.

The average baseline TPR was 1.11 ± 0.04 and 1.00 ± 0.10 mm Hg/ml/min in MnPO- and sham-lesioned animals, respectively. There were no significant differences in the TPR between the two groups throughout the study (Fig. 4B). The TPR values in both groups during the high-salt period were not significantly different from the average baselines.

Sodium and Water Balance under Conditions of High Salt Intake

Figure 5 shows daily water balance data. Average baseline water intake during the control period in MnPO- and sham-lesioned rats was 25.0 ± 2.5 and 25.1 ± 2.3 ml/day, respectively. The average baseline urine output was 9.9 ± 1.5 and 8.6 ± 0.8 ml/day in the examined groups, respectively. Water intake and urine output increased significantly in both groups during the high-salt period. Water intake within the last day of the high-salt period was 45.1 ± 4.4 and 46.3 ± 5.1 ml/day, respectively, in MnPO- and sham-lesioned rats. However, there were no significant differences between the groups in water intake, urine output, and water balance throughout the protocol.

Fig. 5.

Average 24-h water intake (ml/day, A), urine output (ml/day, B), and water balance (ml, C) during the control, high-salt, and recovery periods in MnPO- and sham-lesioned rats. Other designations are the same as in Figs. 2 and 3.

Figure 6 shows daily sodium balance data. In MnPO- and sham-lesioned rats, average baseline sodium intake was 3.8 ± 0.2 and 4.4 ± 0.3 Eq/day, and average baseline sodium excretion was 3.1 ± 0.3 and 3.4 ± 0.3 mEq/day, respectively. Sodium intake and excretion increased significantly in both groups when dietary salt was increased. However, while sodium intake in MnPO-lesioned rats was significantly lower than that in sham-lesioned animals during the high-salt period, sodium excretion in these two groups was quite comparable. By the end of the high-salt period, sodium intake was 14.1 ± 0.8 and 16.8 ± 1.1 mEq/day in MnPO- and sham-lesioned rats, respectively. No significant differences in daily sodium balance were detected between the two groups throughout the study protocol.

Fig. 6.

Average 24-h sodium intake (mEq/day, A), sodium excretion (mEq/day, B), and sodium balance (mEq/day, C) during the control, high-salt, and recovery periods in MnPO- and sham-lesioned rats. Other designations are the same as in Figs. 2 and 3.

In addition to daily water and sodium balance, cumulative values were also calculated in order to detect subtle differences in sodium and water homeostasis between the MnPO- and sham-lesioned rats. Cumulative water and sodium balance data are shown in Fig. 7. No significant differences in cumulative water balance were observed between the two groups throughout the study protocol (Fig. 7A). However, although cumulative sodium balance was nearly the same in the two groups during the 7-day control period, it was found to be higher in sham-lesioned rats from day 6 of the high-salt period until the end of study protocol (Fig. 7B). Since the slope of the cumulative sodium balance plots becomes flatter during the recovery period, the significant differences between groups observed during this period probably were misleadingly influenced by cumulative differences that occurred earlier in the study protocol [40]. Therefore, to avoid a carried over cumulative effect and to determine more specifically, within which time period subtle differences of sodium balance occurred, statistical analyses using sums of sodium balance from two consecutive days of the high-salt period were also performed and compared with the average values of the control and recovery periods (Fig. 8). These analyses showed that MnPO-lesioned rats had significantly lower sodium balance during the first 4 days of high salt intake, compared to that in sham-lesioned rats.

Fig. 7.

Cumulative water and sodium balance in MnPO- and sham-lesioned rats. Other designations are the same as in Figs. 2 and 3.

Fig. 8.

Average 2-day sodium balance during the control, high-salt, and recovery periods in MnPO- and sham-lesioned rats. Other designations are the same as in Figs. 2 and 3.

DISCUSSION

In our study, we reasoned that if the integration of neurohumoral signals in the MnPO is necessary for the formation of appropriate central sympathetic output and maintenance of the arterial pressure during chronic high salt intake, then the blood pressure would be abnormally elevated in MnPO-lesioned compared to sham-lesioned rats. Our results demonstrate a subtle increase in blood pressure in both MnPO- and sham-lesioned animals during high salt intake; however, no significant difference in arterial pressure was observed between the two groups throughout the study protocol. Therefore, the results of our study do not support the hypothesis that the MnPO is necessary to maintain a normal blood pressure during high dietary salt intake.

Compared to those obtained from sham-lesioned rats, plasma samples of the MnPO-lesioned rats were found to be hypernatremic and hypertonic. In addition, while sham-lesioned rats were able to maintain their plasma sodium concentration and osmolality within relatively stable limits when dietary salt was elevated, these parameters were raised significantly in the MnPO-lesioned rats during high salt intake. Similar findings were reported earlier with respect to MnPO-lesioned rats that received acute hypertonic saline infusion; it was shown that such animals have impaired ability to secrete vasopressin and oxytocin [33, 35]. However, it should be noted that blood samples in our study were collected at a single time point during the dark period, when the rats were active and eating. Thus, it is not known whether hypertonicity was sustained chronically in MnPO-lesioned rats. Also, any transient elevation of plasma osmolality in sham-lesioned rats may escape detections. Nevertheless, this finding suggests that postprandial plasma hyperosmolality and hypernatremia were sustained longer in MnPO-lesioned rats.

In our present study, a subtle but statistically significant elevation of the arterial pressure was observed in both MnPO- and sham-lesioned rats during the high-salt period (4.0% NaCl diet) and on the 1st day of the recovery period (1.0% NaCl diet). This finding is in agreement with the previous study where an increase in the arterial pressure was observed in rats fed a highly concentrated sodium diet (8.0% NaCl) for a longer duration (5 weeks) [41]. Hypothetically, high dietary salt is believed to chronically induce sodium and water retention and/or increased plasma osmolality and sodium concentration. However, the mechanism(s) mediating increases in the arterial pressure by chronic plasma hyperosmolality and/or hypernatremia is(are) not clear. In fact, most of the present knowledge relating to this topic is based on acute studies using rats that received hypertonic saline either via i.v. or intracarotid arterial (i.c.a.) infusion [11, 13, 42]. Water deprivation techniques have also been used to generate more prolonged hyperosmotic conditions [10, 43, 44]. Although there is some discrepancy between the study approaches, overall, the knowledge based on these aforementioned studies suggests that plasma hyperosmolality increases the blood pressure via central sympathetic excitation [10, 11, 13, 43, 44]. Moreover, due to possible opposing buffering effects of the baroreflex, intravenous hypertonic saline infusion has been shown to produce non-uniform regional sympathetic nerve responses. In fact, while the blood pressure and lumbar sympathetic nerve activity were found to be elevated, renal and splanchnic sympathetic nerve activities are reduced after i.v. hypertonic saline infusion [42]. In our study, no significant changes in the CO and TPR were observed during chronic high salt intake in both studied groups, although there was a trend toward increased CO in sham-lesioned animals compared to MnPO-lesioned rats. Since the increase of arterial pressure observed was subtle, and only specific vascular beds, e.g., muscular vascular beds, could be affected by sympathetic nerve excitation, a change in the TPR might not occur or be detectable. In addition, the increase in arterial pressure in sham-lesioned rats could also be partly due to a subtle increase in the CO.

Based upon the available evidence to date, sympathetic excitation by plasma hyperosmolality and a rise in the ANG II level likely share common central neural pathway(s). In fact, the OVLT, PVN, and RVLM, the cardiovascular brain regions known to contribute to the sympathetic excitatory circuitry of circulating ANG II, have also been shown to participate in mediating sympathetic excitation secondary to i.c.a. hypertonic saline injection or water deprivation [13, 44–46]. The MnPO has also been implicated in ANG II-induced hypertension [36, 37], and it is activated by plasma hyperosmolality [47–49]. In our study, we did not show a contribution of the MnPO to the neuronal mechanism(s) that is(are) necessary to counteract sympathoexcitatory effects of chronic hyperosmolality. Conversely, our findings seem to be more consistent with the role of the MnPO in mediating sympathetic excitation following chronic plasma hyperosmolality. In fact, while the plasma osmolality and sodium concentration were elevated in MnPO-lesioned rats during high dietary salt intake, their arterial pressure values remained comparable to those of sham-lesioned rats. Therefore, these findings appear to suggest a necessary role of central neuronal pathway(s) encompassing the MnPO in mediating sympathetic excitation and increased blood pressure effects of plasma hyperosmolality.

Despite no difference in the arterial pressure, less positive sodium balance was observed in MnPO-lesioned animals compared to sham-lesioned ones during the first 4 days of high salt intake. This finding is in line with our earlier results showing less positive cumulative sodium balance in SFO-lesioned rats fed high salt relative to their sham-lesioned controls [50]. In the current study, sodium intake was lower in MnPO-lesioned rats, while sodium excretion was comparable to that in sham-lesioned animals. Thus, less positive sodium balance in MnPO-lesioned rats appears to be accounted for by their inappropriately high renal sodium excretion. High plasma osmolality and a high sodium concentration observed in MnPO-lesioned rats likely trigger negative feedback causing these rats to have less appetite for the high-sodium diet [51]. The mechanism(s) mediating enhanced renal excretion of sodium in MnPO-lesioned rats is(are) less clear. However, although the PRA decreased significantly in both MnPO- and sham-lesioned rats during high salt intake, comparable PRA values were observed in the two groups during both baseline and high-salt periods. So, the exaggerated sodium excretion seen in MnPO-lesioned rats does not seem to involve a difference in RAS activity between the two groups. In addition, this finding unlikely involves the impaired secretion of vasopressin and oxytocin that occurs in MnPO-lesioned rats [33, 35]. Although the set point of vasopressin secretion may be increased, no change of water balance seen in the above rats suggests an adequate secretion of vasopressin during high salt intake. In addition, if this was the case (considering the known facilitating effect of vasopressin on the renal baroreflex sensitivity [52] and the natriuresis effect of oxytocin [53]), renal sodium retention would rather likely be observed in MnPO-lesioned rats. In fact, administration of a vasopressin V1 receptor blocker was actually shown to increase (instead of decrease) the renal sympathetic nerve activity in water-deprivated rats [54]. Other possibilities include that the renal sympathetic nerve response to high salt intake might be unsuitably low in MnPO-lesioned rats, causing them to excrete larger amounts of sodium relative to their lower sodium intake [55].

From this perspective, high dietary salt intake and plasma hyperosmolality have been shown to stimulate the hepatorenal reflexes via peripheral osmoreceptors located in the hepatic portal veins causing a decrease of renal sympathetic nerve activity [56–58]. Therefore, it is probable that the greater and/or more prolonged increased plasma osmolality and sodium concentration observed in MnPO-lesioned rats during high salt intake reached the stimulation threshold of these peripheral osmoreceptors and/or caused more intense (and/or prolonged) stimulation. On the other hand, while activation of osmoreceptors located peripherally suppresses renal sympathetic nerve activity, stimulation of central osmoreceptors was shown to produce the opposite effect [11]. In fact, i.c.a. infusion of hypertonic saline produces an increase in the renal sympathetic nerve activity [13]. Therefore, the possible lower renal sympathetic nerve activity found in MnPO-lesioned rats, despite higher plasma osmolality, appears to support the aforementioned idea that the MnPO probably contributes to the central neural pathway mediating sympathetic excitation by central osmoreceptor excitation. This view is also supported by an acute study performed by Yasuda et al. [59] showing that lidocaine microinjected into the MnPO attenuates the increased arterial pressure and renal sympathetic nerve activity in response to intracerebroventricular injection of hypertonic saline. Nevertheless, the relative contribution of the central and peripheral osmoreceptor mechanisms to the sympathetic nerve response to the chronic dietary salt intake remains to be investigated, as well as the role of the MnPO in these intricate mechanisms.

In conclusion, we have shown in our study that the arterial pressure in MnPO-lesioned rats during high salt intake is comparable to that in sham-lesioned rats. These findings fail to support the hypothesis that the MnPO is necessary to maintain arterial pressure within a normal range during high dietary salt intake. However, in line with our previous study examining SFO- lesioned rats, MnPO-lesioned rats displayed less renal sodium retention during high salt diet compared to that in sham-lesioned rats. These findings could be accounted in part by the exaggerated increase of plasma osmolality and sodium concentration in response to high salt intake observed in the MnPO-lesioned rats. However, the exact role of the MnPO in the central neural mechanism(s) mediating sympathetic excitation during chronic high dietary salt intake remains to be investigated.