Original Article

Contribution of Orexin to the Neurogenic Hypertension in BPH/2J MiceNovelty and Significance

Kristy L. Jackson, Bruno W. Dampney, John-Luis Moretti, Emily R. Stevenson, Pamela J. Davern, Pascal Carrive, Geoffrey A. Head
https://doi.org/10.1161/HYPERTENSIONAHA.115.07053
Hypertension. 2016;67:959-969
Originally published March 14, 2016

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Abstract

BPH/2J mice are a genetic model of hypertension associated with an overactive sympathetic nervous system. Orexin is a neuropeptide which influences sympathetic activity and blood pressure. Orexin precursor mRNA expression is greater in hypothalamic tissue of BPH/2J compared with normotensive BPN/3J mice. To determine whether enhanced orexinergic signaling contributes to the hypertension, BPH/2J and BPN/3J mice were preimplanted with radiotelemetry probes to compare blood pressure 1 hour before and 5 hours after administration of almorexant, an orexin receptor antagonist. Mid frequency mean arterial pressure power and the depressor response to ganglion blockade were also used as indicators of sympathetic nervous system activity. Administration of almorexant at 100 (IP) and 300 mg/kg (oral) in BPH/2J mice during the dark-active period (2 hours after lights off) markedly reduced blood pressure (−16.1±1.6 and −11.0±1.1 mm Hg, respectively; P<0.001 compared with vehicle). However, when almorexant (100 mg/kg, IP) was administered during the light-inactive period (5 hours before lights off) no reduction from baseline was observed (P=0.64). The same dose of almorexant in BPN/3J mice had no effect on blood pressure during the dark (P=0.79) or light periods (P=0.24). Almorexant attenuated the depressor response to ganglion blockade (P=0.018) and reduced the mid frequency mean arterial pressure power in BPH/2J mice (P<0.001), but not BPN/3J mice (P=0.70). Immunohistochemical labeling revealed that BPH/2J mice have 29% more orexin neurons than BPN/3J mice which are preferentially located in the lateral hypothalamus. The results suggest that enhanced orexinergic signaling contributes to sympathetic overactivity and hypertension during the dark period in BPH/2J mice.

Introduction

BPH/2J mice are a genetic model of hypertension which were selectively bred for elevated blood pressure (BP) in the 1970s alongside a normotensive strain (BPN/3J).1 Recent studies suggest that the hypertension in BPH/2J mice is caused by enhanced activation of the sympathetic nervous system (SNS) because ganglion blockade causes a greater depressor response in BPH/2J mice compared with BPN/3J controls.2,3 Importantly, cardiovascular regulatory forebrain regions within the hypothalamus and amygdala display markedly greater neuronal activity in BPH/2J compared with BPN/3J mice during the dark-active period of the 24-hour light cycle.2 Furthermore, lesions of the medial amygdala reduced the hypertension and SNS overactivity in BPH/2J mice.4 Thus the central nervous system seems to play a crucial role in driving the sympathetically mediated hypertension in this model.

A gene array approach has been used to identify differential expression of genes in the hypothalamus between BPH/2J and BPN/3J mice.5,6 An important finding was that expression of the orexin precursor gene (hcrt) in BPH/2J mice was more than double that of normotensive mice in early and established hypertension. Thus, hcrt could potentially contribute to the development and maintenance of BPH/2J hypertension.5 Furthermore, BPH/2J mice have ≈4-fold greater expression of the hcrt gene in the hypothalamus during the dark-active period compared with light period when mice are predominantly inactive or asleep.6 Characteristics of the BPH/2J mouse strain, such as high BP, tachycardia, greater locomotor activity, overactivity of the SNS,2 and exaggerated cardiovascular reactivity to stressful stimuli,7 could all be reflective of a greater activity of the orexinergic neurons. Indeed, orexin is capable of increasing BP, heart rate (HR), and SNS activity.8 Orexinergic neurons originate in the hypothalamus and project to a wide range of brain regions, but in terms of sympathetic control of BP, the most well-studied projections are to premotor neurons in the rostral ventrolateral medulla,9 as well as direct projections to the sympathetic preganglionic neurons in the spinal cord, thus classifying some orexinergic neurons as premotor neurons.10 Although orexin does not seem to contribute to basal BP maintenance in normal animals, it does contribute to the hypertension in certain models of hypertension, including spontaneously hypertensive rats (SHR) and a model of stress-induced hypertension.11–13 Most importantly, Li et al11 found that blockade of the orexinergic system using the orally active dual orexin receptor antagonist, almorexant, an antagonist of the 2 subtypes of orexin receptor (orexin 1 and orexin 2), was capable of lowering BP in conscious SHR, but not in normotensive Wistar-Kyoto controls, through mechanisms consistent with an effect of the SNS. Also, immunohistochemical labeling of orexin has revealed that SHR have 25% more orexin neurons than Wistar-Kyoto rats, which is consistent with the different effect of almorexant in these 2 strains.14,15

Although it is clear that central orexin is capable of influencing BP and contributes to other genetic models of hypertension, it is not known whether the greater activity of the central orexinergic system in BPH/2J mice contributes to the hypertension in this model. Therefore, the major aim of the present study was to determine whether overactivity of the central orexinergic system contributes to the sympathetically mediated hypertension in BPH/2J mice. This was tested pharmacologically with almorexant and anatomically by immunohistochemical labeling of orexin using the same approach as in the SHR.

Methods

Animals

Experiments were performed on normotensive BPN/3J (n=18) and hypertensive BPH/2J (n=18) 12 to 17-week–old male mice. The experiments were approved by the Alfred Medical Research Education Precinct Animal Ethics Committee and conducted in accordance with the Australian Code of Practice for Scientific Use of Animals, in line with international standards. To readily assess mice during both the dark and light phases of the 24-hour light cycle, the light cycle was set to 1 am to 1 pm and mice were allowed at least 10 days to acclimatize.

Telemetry Transmitters

BP telemetry transmitters (model TA11PA-C10; Data Sciences International, St Paul, MN) were implanted as detailed in the online-only Data Supplement.

Protocol and Experimental Procedures

After a 10-day recovery period from telemetry surgery, baseline cardiovascular parameters of mean arterial pressure (MAP), HR, and locomotor activity were recorded continuously for 48 hours in freely moving mice in their home cage.

Almorexant Administration During the Dark Period

After a 1-hour control period, BPN/3J and BPH/2J mice (n=7 per strain) were administered the dual orexin receptor antagonist, almorexant (Actelion Pharmaceuticals), during the dark period of the 24-hour light cycle (2 hours after lights off). Almorexant was administered via an intraperitoneal injection (0, 30, 100 mg/kg) and orally via gavage (0, 100, 300 mg/kg). The effect of almorexant was analyzed in the 5 hours after administration to allow comparison with the effect during the light period. In addition, the 6- to 10-hour period post administration was also analyzed during this dark period. The different doses/routes were administered on separate days with at least a day recovery before the next treatment. Doses were based on those reported previously.16

Almorexant Administration During the Light Period

After a 1-hour control period, BPN/3J (n=5) and BPH/2J (n=6) mice were administered almorexant (0 and 100 mg/kg, IP) during the light period of the 24-hour light cycle (5 hours before lights off).

Cardiovascular Variability and the Cardiac Baroreceptor Sensitivity

Spectral analysis of cardiovascular variability and the baroreceptor HR reflex gain were measured as described previously4 in BPN/3J (n=5–6) and BPH/2J (n=7) mice treated with vehicle and almorexant (100 mg/kg, IP) during the dark period.

Cardiovascular Response to Angiotensin-Converting Enzyme Inhibition and Ganglion Blockade

BPN/3J (n=3–5) and BPH/2J mice (n=3–5) were administered a ganglion blocker, pentolinium (5 mg/kg, IP; Sigma-Aldrich), 30 minutes after administration of the angiotensin-converting enzyme inhibitor, enalaprilat (1.5 mg/kg, IP; Merck & Co), as described previously.3 The cardiovascular responses to these drugs were measured during the dark period in mice 6 hours after an injection of almorexant (100 mg/kg, IP) and in untreated mice.

BP-Activity Relation

To assess the relation between BP and locomotor activity levels in BPN/3J and BPH/2J mice, log-locomotor activity was plotted against average MAP using 2-second intervals and a 6-second delay (to account for the temporal relation between variables17) for 10 hours after administration of vehicle or almorexant (100 mg/kg, IP) injected during the light period. This 10-hour period encompasses 5 hours of the light period followed by 5 hours of the dark period.

Histology

Naïve BPN/3J (n=8) and BPH/2J (n=8) mice were used for immunohistochemical labeling of orexin neurons. Details are available in Methods in the online-only Data Supplement.

Statistical Analysis

Both cardiovascular and histological data were expressed as mean±SEM. Cardiovascular data were analyzed by multifactor, nested split-plot ANOVA, which allowed for within animal and between animal contrasts.18 A combined residual was used that pooled between and within animal variance as described previously.19 Histological data were analyzed with a simple t test or 2-way ANOVA followed by Bonferroni multiple comparison post hoc tests. A probability of P<0.05 was considered significant.

Results

Twenty-Four–Hour Cardiovascular Measurements

MAP in BPH/2J mice was higher than BPN/3J mice during a 24-hour period (P<0.001; n=10/strain). During the dark period, MAP in BPH/2J mice was 20% greater, HR was 38% greater, and locomotor activity was 4.5-fold greater than BPN/3J mice (Pstrain<0.01 all; Figure 1). During the light period, MAP in BPH/2J mice was 8% greater than in BPN/3J mice (Pstrain=0.04), whereas HR and locomotor activity were comparable between strains (Pstrain=0.2; Figure 1).

Figure 1.
Figure 1.

Hourly averaged data showing the circadian variation of mean arterial pressure (MAP, mm Hg), heart rate (HR, beats/min), and activity (units) during the dark (outer panels) and light (middle) phases in BPN/3J (n=10, white circles) and BPH/2J mice (n=10, black circles). Shaded columns represent time of drug administration during the light (5 hours before lights off) and dark (2 hours after lights off) periods. Bar graphs on right represent average MAP, HR, and locomotor activity during the light and dark periods in BPN/3J (N, white) and BPH/2J (H, black) mice. Values are mean±SEM for comparisons between strains across the light and dark periods; *P<0.05; ***P<0.001.

Effect of Intraperitoneal Administered Almorexant During the Dark Period

During the dark period, MAP, HR, and locomotor activity during the 1-hour control period before treatments were greater in BPH/2J mice than BPN/3J mice (Pstrain<0.001).

During the 5 hours after injection, almorexant (30 and 100 mg/kg, IP) reduced MAP in BPH/2J mice by 8% (P=0.001) and 12% from baseline, respectively (P<0.001), which was markedly greater than the effect of vehicle in BPH/2J mice (P<0.001; Figure 2). By contrast, almorexant did not reduce MAP from baseline in BPN/3J mice at either 30 or 100 mg/kg (IP; P>0.43). Thus, MAP in BPH/2J mice was reduced to levels comparable with BPN/3J mice (P=0.80). During the period 6 to 10 hours after almorexant injection (100 mg/kg, IP), there was a reduction in MAP compared with the 1-hour control period in BPH/2J mice (P=0.025), but this was comparable with the effect of vehicle (P=0.18; Table S2). In contrast, during the 6- to 10-hour period post almorexant (100 mg/kg, IP) administration, MAP was comparable with the 1-hour control period in BPN/3J mice (P=0.44), but this effect was different compared with vehicle (P=0.006). During the 5 hours after administration, almorexant (30 and 100 mg/kg, IP) reduced HR in BPH/2J mice by 11% (P=0.004) and 21% (P<0.001) from baseline, respectively, which was greater than after vehicle (P<0.011), whereas almorexant (30 mg/kg, IP) had no effect and almorexant (100 mg/kg) actually increased HR by 11% from baseline in BPN/3J mice (P=0.03). Almorexant (30 and 100 mg/kg, IP) reduced locomotor activity in BPH/2J mice by 69% and 83% from baseline, respectively (P<0.001), which was greater than the effect in BPH/2J mice treated with vehicle (P<0.033; Figure 2). Almorexant had minimal effect on locomotor activity in BPN/3J mice (P=0.4).

Figure 2.
Figure 2.

Line graphs show hourly averages of mean arterial pressure (MAP), heart rate (HR), and locomotor activity (Activity) during the dark period in BPN/3J (left, gray, n=7) and BPH/2J mice (right, black, n=7) treated with vehicle (closed circles) and almorexant at doses of 30 (open squares) and 100 mg/kg (IP; open triangles). The vertical dotted line represents the time of administration. The hatched box highlights the 5-hour response to be compared with the preceding 1-hour control period. The black and white horizontal bars on the bottom axis highlight the dark and light periods, respectively. Histograms show mean change from the 1-hour control period ± SEM in mice treated with vehicle (IP; filled bars), almorexant at 30 (hatched) or 100 mg/kg (IP; unfilled). Statistics represent effect compared with vehicle in each strain; *P<0.05; ***P<0.001.

During the 5 hours after vehicle administration (25% cyclodextrin, IP), there was no difference from the 1-hour control period in MAP (P=0.38) and HR (P=0.26) in either strain. Although there was no effect of vehicle on locomotor activity level in BPN/3J mice, there was a reduction of 29% from baseline in BPH/2J mice (P<0.001; Figure 2).

Effect of Almorexant Administered Orally During the Dark Period

In the dark period, MAP, HR, and locomotor activity during the 1-hour control period before treatments were greater in BPH/2J mice than in BPN/3J mice (Pstrain<0.001).

During the 5 hours after administration, almorexant (100 and 300 mg/kg, PO) reduced MAP by 5% (P=0.01) and 8% (P=0.001) in BPH/2J mice, which was greater than the effect of vehicle (P<0.001; Figure 3). In contrast, almorexant did not reduce MAP from baseline in BPN/3J mice at either 100 or 300 mg/kg PO (P>0.62). In the period 6 to 10 hours after almorexant administration (300 mg/kg, PO), there was a reduction in MAP compared with the 1-hour control period in BPH/2J mice (P=0.007) and this was greater than the effect of vehicle (P=0.003; Table S3). In contrast, during the 6- to 10-hour period post almorexant (300 mg/kg, PO) administration, MAP was comparable with the 1-hour control period in BPN/3J mice (P=0.87), and this was comparable with the effect of vehicle (P=1.0). Almorexant (300 mg/kg) reduced HR by 9% from baseline in BPH/2J mice (P=0.017) which was greater than the effect of vehicle (P=0.001), whereas the 100 mg/kg dose had minimal effect on HR in BPH/2J mice (P=0.28). Neither the 100 nor 300 mg/kg dose of almorexant had an effect on HR in BPN/3J mice (P>0.30). Almorexant (100 and 300 mg/kg, PO) also reduced locomotor activity from baseline in BPH/2J mice by 51% (P<0.001) and 69% (P=0.001), respectively, which was greater than the effect of vehicle (P<0.001). In contrast, almorexant (100 and 300 mg/kg, PO) had minimal effect on locomotor activity in BPN/3J mice (P>0.15)

Figure 3.
Figure 3.

Line graphs show hourly averages of mean arterial pressure (MAP), heart rate (HR), and locomotor activity (Activity) during the dark period in BPN/3J (left, gray, n=7) and BPH/2J mice (right, black, n=7) treated with vehicle (closed circles) and almorexant at doses of 100 (open squares) and 300 mg/kg PO (open triangles). The vertical dotted line represents the time of administration. The hatched box highlights the 5-hour response to be compared with the preceding 1-hour control period. The black and white horizontal bars on the bottom axis highlight the dark and light periods, respectively. Histograms show mean change from the 1-hour control period ± SEM in mice treated with vehicle PO (filled bars), almorexant at 100 (hatched) or 300 mg/kg PO (unfilled). Statistics represent effect compared with vehicle in each strain; *P<0.05; **P<0.01; ***P<0.001.

Methocel, the vehicle used for PO delivery of almorexant, had no effect on MAP or locomotor activity in BPH/2J or BPN/3J mice during the 5 hours after gavage treatment (P>0.13; Figure 3). Methocel had no effect on HR in BPH/2J mice but increased HR modestly from baseline in BPN/3J mice (P=0.01).

Effect of Intraperitoneal Administered Almorexant Administered During the Light Period

During the light period, MAP and locomotor activity were comparable between strains (P>0.091), whereas HR was greater in BPH/2J than in BPN/3J mice (P=0.035; Figure 4).

Figure 4.
Figure 4.

Line graphs show hourly averages of mean arterial pressure (MAP), heart rate (HR), and locomotor activity (Activity) during the light period in BPN/3J (left, gray, n=5) and BPH/2J mice (right, black, n=5) treated with vehicle (closed circles) and almorexant at doses of 100 mg/kg (IP; open squares). The vertical dotted line represents the time of administration. The hatched box highlights the 5-hour response to be compared with the preceding 1-hour control period. The black and white horizontal bars on the bottom axis highlight the dark and light periods, respectively. Histograms show mean change from the 1-hour control period ± SEM in mice treated with vehicle (IP; filled bars) and almorexant at 100 mg/kg (IP; hatched). Statistics represent effect compared with vehicle in each strain; *P<0.05; ***P<0.001.

In the 5 hours after injection of almorexant (100 mg/kg, IP) during the light period, there was no change in MAP from baseline in either BPN/3J or BPH/2J mice (P>0.24; Figure 4). The effect of almorexant on MAP was comparable with the effect of vehicle in BPN/3J mice (P=0.14), but different to vehicle in BPH/2J mice (P<0.001). During the light period, almorexant increased HR from baseline by 10% in BPH/2J mice (P=0.035) and this was greater than the effect of vehicle (P=0.012). In contrast, almorexant had minimal effect in HR in BPN/3J mice (P=0.21), which was comparable with vehicle (P=0.15). Almorexant did not change locomotor activity compared with baseline in either BPN/3J or BPH/2J mice (P>0.23) during the light period. The effect of almorexant on locomotor activity was comparable with vehicle in BPN/3J mice (P=0.62) but different in BPH/2J mice (P<0.001).

Intraperitoneal injection of vehicle (25% cyclodextrin), raised MAP from baseline by 12% in BPH/2J mice (P<0.001) and 8% in BPN/3J mice (P=0.031; Figure 4). HR was also raised from baseline after vehicle injections by 16% BPN/3J (P=0.017) and 21% in BPH/2J mice (P=0.001). Administration of vehicle also raised locomotor activity by 2.6-fold in BPH/2J mice (P=0.001) but had little effect on locomotor activity in BPN/3J mice (P=0.78).

Cardiovascular Variability and Cardiac Baroreflex Sensitivity

During the dark period, after vehicle administration, mid-frequency (MF) MAP power was 2.3-fold greater in BPH/2J than BPN/3J mice (Pstrain<0.001), whereas MF HR power (Pstrain=0.10) was similar between strains (Figure 5; Table S1). Baroreflex gain was 40% lower in vehicle-treated BPH/2J compared with BPN/3J mice (Pstrain=0.004). Low frequency MAP power and high frequency were also greater in BPH/2J than in BPN/3J mice (Pstrain<0.001; Table S1).

Figure 5.
Figure 5.

Left, Average mid frequency (MF, 0.3–0.5 Hz) mean arterial pressure (MAP) power (mm Hg)2; middle, MF heart rate (HR) power (bpm)2; and right, baroreflex gain from cross-spectral analysis (gain, bpm/mm Hg) during the dark period. Measurements are after treatment with vehicle (Veh, filled bars) and almorexant (Almo, 100 mg/kg, IP, unfilled bars) in BPN/3J (BPN, gray) and BPH/2J mice (BPH, black). Values are mean±SEM. Effect of almorexant compared with vehicle in each strain; ***P<0.001.

After almorexant (100 mg/kg, IP) administration, MF MAP power was 70% lower compared with vehicle-treated BPH/2J mice (P<0.001), whereas no effect of almorexant was observed in BPN/3J mice (P=0.7; Figure 5; Table S1). Thus, post almorexant values for MF MAP power were comparable between BPN/3J and BPH/2J mice (P=0.50). After almorexant treatment, there was no difference in MF HR power in BPN/3J (P=0.42) or BPH/2J mice (P=0.23) compared with vehicle. After almorexant treatment, baroreflex gain was comparable with vehicle treatment in BPN/3J mice (P=0.59). In contrast, baroreflex gain was 90% greater in BPH/2J mice after almorexant treatment compared with vehicle (P<0.001).

Low frequency MAP power and high frequency were also reduced in BPH/2J mice treated with almorexant compared with vehicle (Ptreat=0.001; Table S1).

Cardiovascular Response to Angiotensin-Converting Enzyme Inhibition and Ganglion Blockade After Almorexant Administration

Administration of enalaprilat (1.5 mg/kg, IP) reduced MAP from the 30-minute baseline period in BPH/2J (−11.8±2.4 mm Hg; P<0.001; n=5), but not BPN/3J mice (+1.3±2.9 mm Hg; n=5; Pstrain=0.02; Figure 6). After administration of almorexant (100 mg/kg, IP), enalaprilat did not reduce MAP from the 30-minute baseline in BPH/2J mice (−1.2±2.7 mm Hg; n=3), effectively abolishing the depressor response when compared with BPH/2J mice not treated with almorexant (P=0.041). In contrast, after almorexant treatment, the response to enalaprilat in BPN/3J mice (−3.3±3.6 mm Hg) was not different compared with mice not treated with almorexant (P=0.68).

Figure 6.
Figure 6.

Mean arterial pressure (MAP) response to administration of enalaprilat (top) and pentolinium after pretreatment with enalaprilat (bottom) in BPN/3J (gray, left, n=3–5) and BPH/2J mice (black, right, n=3–5). Filled circles and bars represent untreated mice (U) and unfilled circles and bars represent mice administered with almorexant (A, 100 mg/kg, IP) during the dark period (6 hours after administration). Each point represents the mean value averaged across a 5-minute period. The dashed vertical reference line represents the time-point of administration of the drug. Shaded area represents the period analyzed for comparison of the effect of treatment. Bar graphs represent mean±SEM change in MAP in response to enalaprilat (top) and the subsequent response to pentolinium (bottom) after enalaprilat pretreatment. Significance refers to effect of almorexant compared with untreated mice; *P<0.05.

Injection of pentolinium (5 mg/kg, IP; after enalaprilat pretreatment) caused marked depressor responses during the dark period in mice not treated with almorexant, which were greater in BPH/2J mice compared with BPN/3J mice (BPH/2J: −54±6 mm Hg versus BPN/3J: −32±4; P=0.007; n=5/group; Figure 6). After administration of almorexant (100 mg/kg, IP), the depressor responses to pentolinium were attenuated by 40% in BPH/2J mice compared with the response in mice not treated with almorexant (n=3; P=0.018). In comparison, the depressor response to pentolinium in BPN/3J mice (n=3) administered almorexant was only 25% lower than in mice that were not treated with almorexant and that effect did not reach statistical significance (P=0.35).

BP-Activity Relation

Vehicle-treated BPH/2J mice had a similar relation between MAP and log locomotor activity in terms of slope to BPN/2J (BPN/3J: 7.1±1.3 versus BPH/2J: 7.3±1.0 mm Hg per log unit activity; P=0.9), whereas the intercept tended to be greater in BPH/2J (135±4 mm Hg) compared with BPN/3J mice (119±3; P=0.061; Figure 7). Almorexant treatment reduced the slope and intercept of the BP-activity relation in BPH/2J mice to 3.5±1.1 mm Hg per log unit activity (P=0.05) and 122±4 mm Hg, respectively (P<0.001). In contrast, almorexant had no effect on slope or intercept in BPN/3J mice (P=0.90).

Figure 7.
Figure 7.

Left, Average relation between log-locomotor activity and mean arterial pressure (MAP, delayed by 6 seconds) in BPN/3J (gray lines; n=5) and BPH/2J mice (black lines; n=5) during the 10-hour after treatment (5-hour light and 5-hour dark) with vehicle (solid lines) or almorexant (100 mg/kg, IP; dashed lines). Right, Example of the relation between log-locomotor activity and MAP from a single BPH mouse before (Pre, gray symbols) and after treatment with almorexant (Post, black symbols).

Orexin Neuron Number and Distribution

Neurons immunoreactive for orexin were present in the dorsal hypothalamus of both strains, as expected (Figure 8A). Total counts of orexin neurons per hemisection were higher in the BPH/2J than in the BPN/3J mice (BPH/2J: 107±4 versus BPN/3J: 84±3; 29% more; P=0.001; t test; Figure 8C).

Figure 8.
Figure 8.

Photomicrographs (A) and plot (B) showing examples of orexin neuron distribution in 30 µm-thick transverse sections of the hypothalamus (one side) in BPH/2J and BPN/3J mice. Histograms (C–E) comparing the distribution of orexin neurons in BPN/3J and BPH/2J mice across different hypothalamic zones, as defined in (B). Values are shown as means±SEM; *P<0.05; **P<0.01; ***P<0.001, t test or Bonferoni post hoc analysis. 3 V indicates third ventricle; DMH, dorsomedial hypothalamus; f, fornix; Hyp, hypothalamus; LHA, lateral hypothalamic area; LHyp, lateral part of the hypothalamus; LPeF, lateral part of the perifornical hypothalamus; MHyp, medial part of the hypothalamus; MPeF, medial part of the perifornical hypothalamus; opt, optic tract; and PeF, perifornical hypothalamus.

Dividing the hypothalamus into medial and lateral zones respective to the fornix (medial part of the hypothalamus [MHyp] and lateral part of the hypothalamus [LHyp], respectively; Figure 8B) revealed that the strain difference was in the LHyp, not in the MHyp, and that most of the orexin neurons were in LHyp (Figure 8D). Thus, BPH/2J mice had 41% more orexin neurons in LHyp than BPN/3J mice (73±3 versus 51±2, respectively), whereas the counts in MHyp were equivalent (34±2 versus 32±2, respectively). These main effects for strain, laterality, and interaction were F1,28=28, F1,28=171, and F1,28=19, respectively (all P<0.001).

The MHyp and LHyp were further divided into dorsomedial hypothalamus and medial part of the perifornical area, medially and lateral part of the perifornical area (LPeF) and lateral hypothalamic area (LHA), laterally (Figure 8B). It revealed that the strain difference in LHyp was in both LPeF and the most lateral LHA (Figure 8E). Thus BPH/2J mice had 58% more orexin neurons in LHA than BPN/3J mice (38±2 versus 24±1) and 27% more in LPeF (35±2 versus 27±1), whereas the counts in MPeF and dorsomedial hypothalamus were again equivalent in both strains (27±2 versus 25±6 and 7.6±1 versus 6.5±1, respectively). These main effects and interaction were F1, 56=37, F3, 56=141, and F3, 56=10.0, respectively (all P<0.001; Figure 8E).

Discussion

The main finding from the present study was that systemic administration of the dual orexin receptor antagonist, almorexant, markedly lowered BP in BPH/2J hypertensive mice but not BPN/3J mice during the dark period, when hypertension is at its greatest. Importantly, the most effective dose of almorexant was capable of reducing BP in BPH/2J mice to levels comparable with normotensive BPN/3J mice, effectively abolishing the hypertension, suggesting an overactive orexinergic system does indeed contribute to the hypertension. This hypotensive effect of almorexant was also accompanied by decreases in 2 indirect measures of SNS activity, including MF MAP power as well as the depressor response to pentolinium. Finally, immunohistochemical staining for orexin revealed a greater number of orexin neurons in the BPH/2J mice relative to the BPN/3J mice, supporting a greater contribution from the orexinergic system. Thus, these findings suggest that an overactive orexinergic system likely contributes to the exaggerated activity of the SNS and the hypertension apparent during the dark period in BPH/2J mice.

Contribution of Orexin to BPH/2J Mice Hypertension

The present findings show that regardless of route, almorexant had hypotensive effects in BPH/2J mice with greater effects observed after intraperitoneal than oral administration. However, oral administration seemed to maintain the BP reduction for a longer duration when compared with the effect of vehicle. These effects of almorexant are consistent with the hypotensive effects observed in the stress-induced hypertensive rat model,12 as well as SHR, another genetic model of hypertension.11,13 Furthermore, it is apparent from the present study and studies using normotensive Wistar-Kyoto rats11,13 that orexin does not seem to be critical for maintenance of basal BP in normotensive animals. However, the reported hypotensive effects of orexin receptor antagonism have been observed in SHR in both the conscious (sleep and wake) and anesthetised state, suggesting that the orexinergic system may be constantly overactive in SHR regardless of the state of arousal of the animal.11,13 Although the present study does not report the sleep/wake state of the mice, we did assess the effect of almorexant during the light and dark period of the 24-hour light cycle, with the light period representing a time when mice are predominantly sleeping or resting and the dark period when mice are predominantly awake and active. Interestingly, almorexant does not lower BP from baseline in BPH/2J mice during the light period, which contrasts findings in conscious SHR where an almost equal hypotensive effect was observed during both the light and dark periods.11

The hypotensive effect of orexin receptor inhibition in BPH/2J mice during the dark period, but not the light period, is consistent with the elevated expression of orexin precursor mRNA (hcrt) in the hypothalamus of BPH/2J mice being enhanced specifically during the dark period.6 Because the hypotensive effectiveness of almorexant seems to mirror hypothalamic hcrt mRNA levels during the light and dark periods, this could suggest that the level of orexin being produced is driving overactivity of the orexinergic system in this model. Furthermore, the diurnal differences in the hypotensive effect of almorexant suggests that the orexinergic system is not tonically overactive in BPH/2J mice as seems to be the case in SHR11 but rather is dynamically controlled and enhanced at times when orexinergic neurons are usually most active.20 This would be primarily during the dark period but also in response to external stimuli during the light period as is observed (for example, in the first hour after almorexant injection during the light period). It may contribute to the exaggerated cardiovascular stress reactivity which is characteristic of BPH/2J mice.7

The idea of a stronger orexinergic drive in BPH/2J mice is further supported by the finding of >29% orexin neurons in these mice compared with the BPN/3J mice. It is also consistent with previous work in the SHR showing 25% to 48% more orexin neurons compared with normotensive Wistar-Kyoto rats.14,15 However, the distribution of these extra orexin neurons differs between the 2 models of hypertension. In the BPH/2J mice, extra orexin neurons were only in the lateral hypothalamus (LPeF and LHA), whereas in the SHR they were primarily in the medial hypothalamus (dorsomedial hypothalamus, MPeF) and to some extent laterally as well (LPeF), but not in the most lateral hypothalamus (LHA).14,15 This is interesting, given the difference in orexinergic drive between SHR and BPH/2J as revealed by almorexant (ie, tonic control versus dynamic control, respectively). The results suggest that medial orexin neurons would exert a more tonic drive than lateral orexin ones which would be recruited in phase with dark period activities when the animal is awake and interacting with its environment (eg, foraging, communicating). We and others have proposed that medial orexin neurons are more important for stress-related arousal than laterally placed neurons which are more important for reward-related arousal.14,21 It may be that medially placed neurons also have a more tonic role to play than lateral orexin neurons. Thus, an upregulation of medially placed orexin neurons would drive BP up chronically throughout the cycle (eg, SHR), whereas with an upregulation of lateral orexin neurons, BP would only be driven up in relation to the active period of the cycle (eg, BPH/2J mice).

Sympathetic and Renin–Angiotensin System Influence

It is widely reported that orexin is capable of increasing SNS activity.8,9 Indeed, the present study shows that almorexant reduced MF MAP power, an indirect indicator of SNS activity in BPH/2J, but not BPN/3J mice. Furthermore, although almorexant seemed to attenuate the depressor response to pentolinium in both strains, only BPH/2J mice showed a significant reduction. Together, these findings suggest that greater orexinergic signaling contributes to the enhanced SNS activity characteristic of BPH/2J mice. Interestingly, high- and low- frequency power of MAP were reduced with almorexant treatment, suggesting an overall reduction in BP variability. Much like the present study, Li et al11 report that the hypotensive effect of systemic administration of almorexant in SHR was accompanied by a decrease in indirect measures of SNS activity, including spectral analysis of BP in the sympathetic frequency band and decreases in plasma norepinephrine levels. From the present findings, we cannot determine exactly how orexin is activating the SNS. One possibility is that orexin is directly influencing the SNS via activation of the brain stem premotor sympathetic regulatory brain regions within the rostral ventrolateral medulla, similar to that which is reported in SHR.13,15 Orexin can activate ≈88% of adrenergic neurons within the rostral ventrolateral medulla of rats,22 and it is these adrenergic bulbospinal neurons within the rostral ventrolateral medulla which are well known to influence sympathetic vasomotor tone. In addition, orexinergic neurons may directly activate the SNS via the sympathetic preganglionic neurons in the spinal cord.23 Alternatively, on the basis of the dynamic nature of the effect of almorexant in BPH/2J mice, it is possible that orexin indirectly influences the SNS via effects on higher forebrain regions involved in arousal.

The overactivity of the SNS in BPH/2J mice is also associated with augmented activity of the renin–angiotensin system, particularly during the dark period of the light cycle.3 Interestingly, the present findings show that almorexant abolished the depressor response to enalaprilat in BPH/2J mice, such that the response resembled that of normotensive BPN/3J mice. Thus, not only does orexin seem to contribute to the overactive SNS activity but also it likely contributes to the resulting overactivity of the peripheral renin–angiotensin system in BPH/2J mice. We reported that renal renin mRNA is elevated during the dark period in BPH/2J mice and their levels strongly correlate with the depressor response to pentolinium.3 Therefore, it is likely that this effect of almorexant on renin–angiotensin system activity in BPH/2J mice is because of a reduction in renal sympathetic nerve activity.

Effect of Almorexant on HR

Almorexant also produces bradycardic effects in BPH/2J mice during the dark period, which is consistent with the effect of almorexant observed in SHR, although unlike SHR this effect was not observed during the light period.11 Interestingly, the bradycardic effect of almorexant in BPH/2J mice is associated with an increase in the high frequency HR power (Table S1) which is an indicator of vagal activity.24 Furthermore, the rapid and pronounced bradycardic effects of intraperitoneal almorexant in BPH/2J mice are similar to the acute effects observed after intraperitoneal administration of rilmenidine, another centrally acting sympatholytic agent, which was shown to produce hypotensive effects in BPH/2J mice predominantly via a vagal excitatory effect rather than its sympatholytic effect.25 Indeed, orexin is capable of increasing HR by indirect vagal inhibition.26 However, it is unclear from the present findings, how much of the bradycardic effect of almorexant in BPH/2J mice is because of a decrease in SNS activity compared with an increase in vagal activity.

Effect of Sleep/Locomotion

Orexin can increase wakefulness and locomotor activity,27 whereas inhibition with almorexant is known to promote sleep and reduce locomotor activity, likely via inhibition of the orexin receptor type 2.28 It is clear from the present results that almorexant reduces the heightened locomotor activity in BPH/2J mice, which could reflect the effect of orexin on neuronal regulation of motor activity29 or promotion of sleep,30 both of which can influence BP.31,32 Thus, an important question is whether the hypotensive effect of almorexant in BPH/2J mice is dependent on reductions in locomotor activity. The relation between BP and locomotor activity after treatment with almorexant shows that almorexant has a prominent hypotensive effect at high-activity levels and this hypotensive effect is reduced at lower-activity levels, suggesting that some of the effect of orexin on BP is likely activity dependent. During periods of no locomotor activity, 50% of the hypotensive effect remains. The reduction in the slope of the activity-BP relation after almorexant also suggests that at least part of the effect of activity on BP in BPH/2J mice is mediated by orexin but interestingly this does not seem to be the case in normotensive BPN/3J mice. Importantly, the orexinergic system is known to affect a wide range of functions, including energy homeostasis,33 sleep wakefulness,30 arousal, and stress,16 which can ultimately affect locomotor activity. Thus, without further assessment of the effect of almorexant on these parameters, it is difficult to determine how much of the hypotensive effects observed in BPH/2J mice are indirectly associated with these factors compared with direct inhibition of the SNS. Also, such side effects may limit the use of this drug in patients.

Perspectives

The results of the present study demonstrate that greater activity of the central orexinergic system contributes to the marked hypertension apparent during the dark period in the BPH/2J model of hypertension. This study provides important verification of the contribution of orexin in a second genetic model of hypertension. Thus, it is possible that augmentation of the orexinergic system in some forms of hypertension may be genetically determined. However, stress is another common characteristic of the hypertensive models that respond to orexin inhibition, namely BPH/2J mice and SHR. Both have exaggerated cardiovascular reactivity to stress34,35 and stress-induced hypertensive rats, where hypertension is induced by chronic exposure to stressful stimuli.12 Thus, it would be interesting to know whether patients with stress-related forms of hypertension also have overactive orexinergic systems. Interestingly, patients with panic disorder, a condition which is more prevalent in patients with hypertension,36 display greater orexin in the cerebrospinal fluid than control patients.37 Thus, it may be that enhanced central orexinergic drive is another common contributing factor to panic disorder and certain forms of hypertension alike.

Acknowledgments

We thank Francois Jenck and Michel Steiner from Actelion Pharmaceuticals for the gift of almorexant.

Sources of Funding

This work was supported by grants from the National Health and Medical Research Council of Australia (NHMRC; project grants APP1065714 and APP1024891) and in part by the Victorian Government’s Operational Infrastructure Support Program. Investigators were supported by a NHMRC/National Heart Foundation Postdoctoral Fellowship (1012881 to P.J. Davern), NHMRC Early Career Fellowship (1091688 To K.L. Jackson), and NHMRC Principal Research Fellowship (1002186 to G.A. Head).

Disclosures

None.

Footnotes

  • Received December 23, 2015.
  • Revision received January 11, 2016.
  • Accepted February 14, 2016.

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Novelty and Significance

What Is New?

  • The greater hypotensive effect of almorexant treatment demonstrates that orexin contributes to the neurogenic hypertension in BPH/2J mice.

  • Orexin neurons are more abundant in BPH/2J mice and are preferentially located in the lateral hypothalamus.

What Is Relevant?

  • The present study demonstrates a considerable contribution of orexin to the hypertension in BPH/2J mice, which is consistent with findings in spontaneously hypertensive rats, another genetic model of hypertension.

Summary

The present study demonstrates a greater contribution of orexin to the sympathetically mediated hypertension in BPH/2J mice, which is associated with a greater abundance of orexinergic neurons in the lateral hypothalamus.

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  • Contribution of Orexin to the Neurogenic Hypertension in BPH/2J MiceNovelty and Significance
    Kristy L. Jackson, Bruno W. Dampney, John-Luis Moretti, Emily R. Stevenson, Pamela J. Davern, Pascal Carrive and Geoffrey A. Head
    Hypertension. 2016;67:959-969, originally published March 14, 2016
    https://doi.org/10.1161/HYPERTENSIONAHA.115.07053
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