NEUROMODULATION OF BARORECEPTOR REFLEX

Abstract

Modulation of neural activity of a subject's aortic depressor nerve (ADN) and/or carotid sinus nerve (CSN) can modulate baroreceptor reflex function, thereby providing ways of treating or preventing disorders associated with malfunction or loss of the baroreceptor reflex.

Claims

1. A system for modulating neural activity in a subject's aortic depressor nerve (ADN) and/or carotid sinus nerve (CSN), the system comprising: at least one neural interfacing element having at least one electrode configured to be in signaling contact with the ADN and/or CSN that is being modulated, and a signal generator comprising at least one voltage or current source configured to generate at least one signal to be applied to the ADN and/or CSN that is being modulated via the at least one electrode to modulate the neural activity of the ADN and/or CSN that is being modulated to produce a change in a physiological parameter in the subject, wherein the amplitude of the at least one signal is ≤0.4 mA; wherein the change in the physiological parameter is one or more of the group consisting of: a decrease in mean arterial pressure, a decrease in heart rate, an increase in minute ventilation, an improvement in regularity of a heart rhythm, an improvement in heart conduction, an increase in heart contractility, a decrease in vascular resistance, an increase in cardiac output, an increase in blood flow, an increase in minute ventilation, an increase in a hemodynamic response, a decrease in a chronotropic evoked response, a decrease in a dromotropic evoked response, a decrease in a lusitropic evoked response, a decrease in an inotropic evoked response, and a decrease in pain perception; wherein the signal generator is controlled to deliver to the ADN and/or CSN that is being modulated the at least one signal having a total intensity, the total intensity being below a predetermined threshold, the predetermined threshold defined as the total intensity of a signal required to be received by the ADN and/or CSN to produce a ≤30 mmHg drop in the mean arterial blood pressure.

2. The system of claim 1, wherein the at least one signal is configured to be applied to the ADN, and the at least one electrode is suitable for placement on or around the ADN.

3. The system of claim 1, wherein the at least one signal is configured to be applied to the CSN, wherein the at least one electrode is suitable for placement on or around the CSN.

4. The system of claim 1, wherein the at least one signal includes a first signal and a second signal, the first signal is configured to be applied to the ADN and the second signal is configured to be applied to the CSN, wherein the at least one electrode includes a first electrode and a second electrode, the first electrode is suitable for placement on or around the ADN and the second electrode is suitable for placement on or around the CSN, wherein the first signal is to be applied via the first electrode and the second signal is to be applied via the second electrode.

5. The system of claim 1, wherein the at least one signal is configured to be applied to the ADN and/or CSN unilaterally or bilaterally.

6. The system of claim 4, wherein the at least one signal is configured to be applied to the ADN and the CSN ipsilaterally.

7. The system of claim 1, wherein the predetermined threshold is ≤30 μAs.

8. The system of claim 1, wherein the total intensity of the signal is between 0.1 TINT and 0.9 TINT, where TINT is the predetermined threshold.

9. The system of claim 1, wherein the at least one signal has a predetermined duty cycle of ≤65%.

10. The system of claim 1, wherein the at least one signal has a pulse width of ≤1 ms.

11. The system of claim 1, wherein the frequency of the at least one signal is ≤70 Hz.

12. The system of claim 1, wherein the at least one signal is applied in a (ONy−OFFz)n pattern where n>1, y>0, and z>0, and the at least one signal is applied for: (a) ≤20 s, or (b) ≤30 min at any given time up to 12 times a day.

13. The system of claim 1, wherein the signal generator is controlled to deliver the at least one signal during a specific time of a day.

14. The system of claim 1, further comprising a detector configured to: detect one or more signals indicative of one or more second physiological parameters; determine from the one or more signals the one or more second physiological parameters; determine the one or more second physiological parameters indicative of worsening of the second physiological parameter; and cause the at least one signal to be applied to the ADN and/or CSN via the at least one electrode, wherein the second physiological parameter is one or more of the group consisting of: systemic arterial blood pressure, heart rate, heart rhythm, electrical conduction in the heart and heart contractility, vascular resistance, cardiac output, rate of blood flow, minute ventilation, and pain perception.

15. The system of claim 14, further comprising a memory arranged to store data pertaining to the second physiological parameters indicative of a disorder associated with malfunction or loss of the baroreceptor reflex, wherein determining the one or more second physiological parameters indicative of worsening of the second physiological parameter comprises comparing the one or more second physiological parameters with the data.

16. The system of claim 14, wherein one of the second physiological parameters is the arterial blood pressure, wherein the detector comprises a pressure sensor.

17. A method of treating or preventing a disorder associated with malfunction or loss of a baroreceptor reflex in a subject by reversibly modulating neural activity of a subject's aortic depressor nerve (ADN) and/or carotid sinus nerve (CSN), comprising: implanting in the subject the system of claim 1; positioning the neural interfacing element in signaling contact with the ADN and/or CSN; and optionally activating the system to provide at least one signal wherein the at least one signal is applied to the ADN and/or the CSN.

18. The method of claim 17, wherein the method is for treating or preventing a cardiovascular disorder and a disorder associated therewith, or a cardiorespiratory and a disorder associated therewith.

19. A method for treating or preventing a disorder associated with malfunction or loss of a baroreceptor reflex, comprising: applying a signal to a subject's aortic depressor nerve (ADN) and/or carotid sinus nerve (CSN) via at least one neural interfacing element having at least one electrode in signaling contact with the ADN and/or CSN, such that the signal reversibly modulates neural activity of the ADN and/or CSN to produce a change in a physiological parameter in the subject: wherein the change in the physiological parameter is one or more of the group consisting of: a decrease in mean arterial pressure, a decrease in heart rate, an increase in minute ventilation, an improvement in the regularity of the a heart rhythm, an improvement in heart conduction, an increase in heart contractility, a decrease in vascular resistance, an increase in cardiac output, an increase in blood flow, an increase in minute ventilation, an increase in a hemodynamic response, a decrease in a chronotropic evoked response, a decrease in a dromotropic evoked response, a decrease in a lusitropic evoked response, a decrease in an inotropic evoked response, and a decrease in pain perception, wherein the total intensity of the signal received by the ADN and/or CSN that is being modulated is below a predetermined threshold, the predetermined threshold defined as the total intensity of a signal required to be received by the ADN and/or CSN to produce a ≤30 mmHg drop in the mean arterial blood pressure: and wherein the amplitude of the at least one signal is ≤0.4 mA.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0271] FIG. 1 is a schematic diagram of aortic and carotid baroreceptor nerve terminals and nerve trunks [1]. This diagram illustrates the relative anatomical positions of aortic and carotid baroreceptors nerve terminals, their nerve fibers and their somata regions. Aortic baroreceptor nerve terminals are located in the aortic arch. The afferent nerve trunk is the aortic depressor nerve. Soma are in the nodose ganglia (NG). Carotid baroreceptors are positioned in the internal carotid artery next to the carotid bifurcation. Its afferent nerve is the carotid sinus nerve. The soma are located within the petrosal ganglia (PG).

[0272] FIG. 2 is a block diagram illustrating elements of a system for performing electrical modulation in ADN and/or CSN according to the present disclosure.

[0273] FIGS. 3A and 3B show the circadian rhythms in mean arterial blood pressure (MAP; FIG. 3A) and in heart rate (HR; FIG. 3B) in conscious freely-moving adult (16-week old) Wistar-Kyoto rats (WKY) and Spontaneously Hypertensive rats (SHR). The data are presented as the mean±SEM. There were 18 rats in each group.

[0274] FIGS. 4A and 4B show percentage changes where FIG. 4A shows mean arterial blood pressure (MAP) and FIG. 4B shows heart rate (HR) elicited by the electrical stimulation (3V, 1 mA, 2-ms pulse length for 5 sec) of the left aortic depressor nerve in freely-moving 16-week old Wistar Kyoto (WKY) rats and Spontaneously Hypertensive rats (SHR). The data are presented as mean±SEM. There were 12 rats in each group. *P<0.05, significant change from Pre. †P<0.05, 2.5 Hz versus 1 Hz.

[0275] FIGS. 5A and 5B show the percentage changes where FIG. 5A shows mean arterial blood pressure (MAP) and FIG. 5B shows heart rate (HR) elicited by electrical stimulation (3V, 1 mA, 2 ms pulse length for 5 sec) of left carotid sinus nerve in conscious 16-week old Wistar Kyoto (WKY) rats and Spontaneously Hypertensive rats (SHR). The data are presented as mean±SEM. There were 12 rats in each group. *P<0.05, significant change from Pre. †P<0.05, 2.5 Hz versus 1 Hz.

[0276] FIGS. 6A and 6B show the percentage changes in minute ventilation (MV) elicited by electrical stimulation (3V, 1 mA, 2-ms pulse length for 5 sec) where FIG. 6A is of the left aortic depressor nerve and FIG. 6B is of the left carotid sinus nerve in freely-moving 16-week old Wistar Kyoto (WKY) rats and Spontaneously Hypertensive rats (SHR). The data are presented as mean±SEM. There were 12 rats in each group. *P<0.05, significant change from Pre. †P<0.05, 2.5 Hz versus 1 Hz.

[0277] FIGS. 7A and 7B show the circadian rhythms in mean arterial blood pressure (MAP; FIG. 7A) and heart rate (HR; FIG. 7B) in freely-moving 16-week old Spontaneously Hypertensive rats (SHR), which received sham electrical stimulations (ES) of the aortic depressor nerve (SHR—sham) or actual episodes of 1 Hz electrical stimulation (for each period of ES, 12 episodes of stimulation at 3V, 1 mA, 2-ms pulse length for 5 sec, each episode separated by 1 min). The data are presented as mean±SEM. There were 12 rats in each group.

[0278] FIG. 8A shows the western blot analyses of Enac protein in nodose ganglia of 16 week old WKY and SHR. Data are mean±SEM. There were 18 rats in each group in A, and 12 rats in each group in B and C. *P<0.05, Stimulation versus Control.

[0279] FIGS. 8B and 8C show the western blot analyses of Enac protein in aortic arches of 16 week old WKY and SHR. Data are mean±SEM. There were 12 rats in each group. *P<0.05, Stimulation versus Control.

[0280] FIGS. 9A-9C show the baseline mean arterial blood pressures (MAP) in Spontaneously Hypertensive rats (SHR) immediately before they received episodes of electrical stimulation of the right ADN on days 7, 14 and 21 post-surgery. There were 4 male SHR in the group. The data are presented as mean±SEM. *P<0.05, Day 14 or Day 21 versus Day 7. †P<0.05, Day 21 versus Day 14 (FIGS. 9B and 9C).

[0281] FIGS. 10A-10C show the falls in mean arterial blood pressure (MAP) in Spontaneously Hypertensive rats (SHR) elicited by electrical stimulation of the right ADN on days 7, 14 and 21 post-surgery. There were 4 male SHR in the group. The data are presented as mean±SEM. *P<0.05, Day 14 or Day 21 versus Day 7. †P<0.05, Day 21 versus Day 14.

[0282] FIGS. 11A-11C show the time-course of decreases in mean arterial blood pressure (MAP) in Spontaneously Hypertensive rats (SHR) elicited by electrical stimulation of the right ADN on days 7, 14 and 21 post-surgery. There were 4 male SHR in the group. The data are presented as mean±SEM. *P<0.05, Day 14 or Day 21 versus Day 7. †P<0.05, Day 21 versus Day 14.

[0283] FIGS. 12A and 12B shows the body weights of the rats during the experiment. The data are presented as mean±SEM. *P<0.05, Day 14 or Day 21 versus Day 7. †P<0.05, Day 21 versus Day 14.

[0284] FIGS. 13A-13C show the effects of electrical stimulation of one aortic depressor nerve (ADN-S) on frequency of breathing and disordered breathing Index (DBI) values of freely-moving sham-operated Sprague-Dawley rats. There were 9 rats in each group. The data is presented as mean±SEM. *P<0.05, significant response. †P<0.05, ADN-S versus Sham-stimulation. Stimulation immediately post H-H challenge.

[0285] FIGS. 14A-14C show the effects of electrical stimulation of one aortic depressor nerve (ADN-S) on frequency of breathing and disordered breathing Index (DBI) values of freely-moving sham-operated Sprague-Dawley rats. There were 9 rats in each group. The data is presented as mean±SEM. *P<0.05, significant response. †P<0.05, ADN-S versus Sham-stimulation. Simulation at 5 min post H-H challenge.

[0286] FIG. 15 shows the mean arterial blood pressure (MAP) values of sham-operated Sprague-Dawley rats and those with bilateral aortic depressor nerve transection (ADNX) during the light and dark cycles. There were 10 rats in each group. The data is presented as mean±SEM.

[0287] FIG. 16 shows the mean arterial blood pressure (MAP) values of sham-operated Sprague-Dawley rats and those with bilateral aortic depressor nerve transection (ADNX) during the light and dark cycles. There were 10 rats in each group. The data is presented as mean±SEM.

[0288] FIG. 17 shows the disordered breathing index (DBI) values of sham-operated Sprague-Dawley rats and those with bilateral aortic depressor nerve transection (ADNX) during the light and dark cycles. There were 10 rats in each group. The data is presented as mean±SEM.

[0289] FIGS. 18A and 18B show a sample data trace showing blood pressure (BP), heart rate (HR), femoral blood flow (FBF) and mesenteric blood flow (MBF) responses to right aortic depressor nerve stimulation in urethane-anesthetized male Sprague Dawley (SD) rats. The stimulations were performed using bipolar silver stimulating electrodes (1-20 Hz, 0.4 mA, 0.2 ms for 20 s).

[0290] FIG. 19 shows a sample data trace showing blood pressure (BP), heart rate (HR) and femoral blood flow (FBF) responses to left aortic depressor nerve stimulation in urethane-anesthetized male and female Sprague Dawley (SD) rats. Stimulations were performed using bipolar silver stimulating electrodes (1-20 Hz, 0.4 mA, 0.2 ms for 20 s).

[0291] FIG. 20 shows the mean arterial blood pressure (MAP) responses elicited by electrical stimulation (1-20 Hz, 0.4 mA, 0.2 ms for 20 s) of the left or the right aortic depressor nerve (ADN) in urethane-anesthetized male and female Sprague-Dawley rats. Stimulation was performed using bipolar silver stimulating electrodes. Data presented as mean±SEM (n=6 rats in each group).

[0292] FIG. 21 shows the effect of modifying pulse width, amplitude and frequency of left aortic depressor nerve (ADN) stimulation on changes in peak mean arterial pressure (MAP) in anesthetized male spontaneously hypertensive rats (SHR) (n=4).

[0293] FIGS. 22A-22B2 show the mean arterial pressure (MAP) responses where FIG. 22A, FIGS. 22A1 and 22A2 are to low (5 Hz) frequency (FIGS. 22B, 22B1 and 22B2 are to high (15 Hz) frequency), continuous (20 s) versus intermittent (5 s on/3 s off and 5 s on/3 s off for 20 s) left aortic depressor nerve (ADN) stimulation (0.4 mA, 0.2 ms) in 25-26 weeks old male SHR (n=8), FIGS. 22A & 22B show time course analysis calculated as 5 s bins and plotted as 40 s baseline and 80 s after stimulation; FIGS. 22A1 & 22B1 show peak changes in MAP relative to baseline; and FIGS. 22A2 & 2B2 show differences in peak changes evoked by intermittent versus continuous stimulation. *P≤0.05.

[0294] FIGS. 23A-23B2 show the heart rate (HR) responses where FIGS. 23A, 23A1 and 23A2 are to low (5 Hz) frequency (FIGS. 23B, 23B1 and 23B2 are to high (15 Hz) frequency), continuous (20 s) versus intermittent (5 s on/3 s off and 5 s on/3 s off for 20 s) left aortic depressor nerve (ADN) stimulation (0.4 mA, 0.2 ms) in 25-26 weeks old male SHR (n=8), FIGS. 23A & 23B show time course analysis calculated as 5 s bins and plotted as 40 s baseline and 80 s after stimulation; FIGS. 23A1 & 23B1 show peak changes in HR relative to baseline; and FIGS. 23A2 & 23B2 show differences in peak changes evoked by intermittent versus continuous stimulation. *P≤0.05.

[0295] FIGS. 24A-24B2 show he femoral vascular resistance (FVR) responses where FIGS. 24A, 24A1 and 24A2 are to low (5 Hz) frequency (FIGS. 24B, 24B1 and 24B2 are to high (15 Hz) frequency), continuous (20 s) versus intermittent (5 s on/3 s off and 5 s on/3 s off for 20 s) left aortic depressor nerve (ADN) stimulation (0.4 mA, 0.2 ms) in 25-26 weeks old male SHR (n=8); FIGS. 24A & 24B show time course analysis calculated as 5 s bins and plotted as 40 s baseline and 80 s after stimulation; FIGS. 24A1 & 24B1 show peak changes in FVR relative to baseline; and FIGS. 24A2 & 24B2 show differences in peak changes evoked by intermittent versus continuous stimulation. *P≤0.05.

[0296] FIGS. 25A-25B2 show the mesenteric vascular resistance (MVR) responses where FIGS. 25A, 25A1 and 25A2 are to low (5 Hz) frequency (FIGS. 25B, 25B1 and 25B2 are to high (15 Hz) frequency), continuous (20 s) versus intermittent (5 s on/3 s off and 5 s on/3 s off for 20 s) left aortic depressor nerve (ADN) stimulation (0.4 mA, 0.2 ms) in 25-26 weeks old male SHR (n=8). FIGS. 25A & 25B show time course analysis calculated as 5 s bins and plotted as 40 s baseline and 80 s after stimulation; FIGS. 25A1 & 25B1 show peak changes in MVR relative to baseline; and FIGS. 25A2 & 25B2 show differences in peak changes evoked by intermittent versus continuous stimulation. *P≤0.05.

[0297] FIG. 26 shows the percent changes in mean arterial blood pressure (MAP) elicited by a 30 second burst of electrical stimulation (0.2, 0.5 or 1.0 ms, 5 Hz, 1 mA) of the left (L), right (R) or both (LR) cervical sympathetic chains (CSC, left panels) or L, R or LR superior cervical ganglia (SCG, right panels). The CSC and SCG studies were done in different rats (n=6 per group). Data are shown as mean±SEM.

[0298] FIG. 27 shows the change in mean arterial pressure (MAP), heart rate (HR), mesenteric blood flow (MBF) and femoral blood flow (FBF) upon unilateral left ADN stimulation (1, 2.5, 5, 10, 20 and 40 Hz, 0.4 mA, 0.2 ms for 20 s) in sodium pentobarbital-anaesthetized male spontaneously hypertensive rats.

[0299] FIGS. 28A-28D show the frequency dependent reductions where FIG. 28A is mean arterial pressure (MAP), FIG. 28B is heart rate (HR), FIG. 28C is mesenteric blood flow (MBF) and FIG. 28D is femoral blood flow (FBF) upon left or right unilateral or bilateral ADN stimulation in male spontaneously hypertensive rats. Mean data ±S.E.M of 6-9 animals. .sup.aP≤0.05, left vs. right ADN, .sup.bP≤0.05, left vs. bilateral ADN and .sup.cP≤0.05, right vs. bilateral ADN analyzed by 2-way ANOVA followed by Tukey's posthoc.

[0300] FIG. 29 shows mean arterial pressure (MAP), heart rate (HR), mesenteric blood flow (MBF) and femoral blood flow (FBF) upon unilateral left ADN stimulation (1, 2.5, 5, 10, 20 and 40 Hz, 0.4 mA, 0.2 ms for 20 s) in urethane-anaesthetized male Sprague Dawley rats.

[0301] FIGS. 30A-30D show frequency dependent reductions where FIG. 30A is mean arterial pressure (MAP), FIG. 30B is heart rate (HR), FIG. 30C mesenteric blood flow (MBF) and FIG. 30D is femoral blood flow (FBF) upon left or right unilateral or bilateral ADN stimulation in male Sprague Dawley rats. Mean data ±S.E.M of 3-5 animals. .sup.bP≤0.05, left vs. bilateral ADN analyzed by 2-way ANOVA followed by Tukey's post hoc.

[0302] FIGS. 31A-31D show representative stained (methylene blue, toluidine blue and hematoxylin) vaginal smears collected from female spontaneously hypertensive rats (SHR) illustrating all 4 stages of oestrus cycle female spontaneously hypertensive rats.

[0303] FIG. 32 shows mean arterial pressure (MAP), heart rate (HR), mesenteric blood flow (MBF) and femoral blood flow (FBF) upon unilateral left ADN stimulation (1, 2.5, 5, 10, 20 and 40 Hz, 0.4 mA, 0.2 ms for 20 s) in sodium pentobarbital-anaesthetized female spontaneously hypertensive rats.

[0304] FIGS. 33A-33D show frequency dependent reductions where FIG. 33A is mean arterial pressure (MAP), FIG. 33B is heart rate (HR), FIG. 33C is mesenteric blood flow (MBF) and FIG. 33D is femoral blood flow (FBF) upon left or right unilateral or bilateral ADN stimulation in female spontaneously hypertensive rats. Mean data ±S.E.M of 5-8 animals. .sup.cP≤0.05, right vs. bilateral ADN analyzed by 2-way ANOVA followed by Tukey's post hoc.

[0305] FIGS. 34A-34D show representative stained (methylene blue, toluidine blue and hematoxylin) vaginal smears collected from female spontaneously hypertensive rats (SHR) illustrating all 4 stages of oestrus cycle in Sprague Dawley rats.

[0306] FIG. 35 shows mean arterial pressure (MAP), heart rate (HR), mesenteric blood flow (MBF) and femoral blood flow (FBF) upon unilateral left ADN stimulation (1, 2.5, 5, 10, 20 and 40 Hz, 0.4 mA, 0.2 ms for 20 s) in urethane-anaesthetized female Sprague Dawley (SD) rats.

[0307] FIGS. 36A-36D show frequency dependent reductions where FIG. 36A is mean arterial pressure (MAP), FIG. 36B is heart rate (HR), FIG. 36C is mesenteric blood flow (MBF) and FIG. 36D femoral blood flow (FBF) upon left or right unilateral or bilateral ADN stimulation in female Sprague Dawley (SD) rats. Mean data ±S.E.M of 6-7 animals. .sup.aP≤0.05, left vs. right; .sup.bP≤0.05, left vs. bilateral and ADN .sup.cP≤0.05, right vs. bilateral ADN analysed by 2-way ANOVA followed by Tukey's posthoc.

MODES FOR CARRYING OUT THE PRESENT DISCLOSURE

[0308] Study 1

[0309] This study investigated whether electrical stimulation of aortic depressor nerves (ADN) in freely-moving Spontaneously Hypertensive rats (SHR) can be a potential therapeutic modality from multiple perspectives including physiology and biochemistry.

[0310] Introduction

[0311] Baroreceptor afferents emanating from the aortic arch travel within the aortic depressor nerve (ADN) whereas baroafferents emanating from the carotid sinus travel in the carotid sinus nerve (CSN), which also carries chemoafferents from the carotid body [76,77]. In the rat, the ADN has a pure population of baroreceptor afferents3-7 and the electrical stimulation of this nerve is being used to evaluate neural/hemodynamic processes in normotensive and hypertensive rats [78,79,80,81,82].

[0312] Baroreceptor afferent sensitivity and baroreceptor reflex-mediated changes in heart rate and sympathetic nerve activity are impaired in adult spontaneously hypertensive rats (SHR) [83,84,85,86,87]. The deficit in baroreflex function lies in the mechanosensitive regions of the peripheral terminals imbedded in vascular smooth muscle [83-85,87]. Electrical stimulation (ES) of baroafferent fibers in the ADN of SHR bypasses the site of impaired baroreceptor mechano-sensory transduction and provides data about the central processing of the afferent input and the properties of central and efferent components of the baroreflex [81,82]. ES allows for precise control of afferent signals transmitted to the nucleus of the tractus solitaries [81,82].

[0313] This study investigated ES of ADN and CSN at low frequencies in SHR.

[0314] Results

[0315] Circadian Rhythms in MAP and Heart Rate

[0316] Actual levels of MAP and heart rate of conscious normotensive 16-week old Wistar-Kyoto rats (WKY) and Spontaneously Hypertensive rats (SHR) during the consecutive day-night cycles are shown in FIGS. 3A and 3B. As reported by others [88,89,90,91], MAP and heart rate of WKY and SHR displayed a diurnal rhythm with MAP values being consistently higher during the dark phases and MAP values of SHR being consistently higher than those of the WKY.

[0317] Cardiovascular Responses Elicited by ADN Stimulation

[0318] Salgado and his colleagues [81,82] employed a relatively high stimulus intensity (1 mA, 2 ms pulses) to activate all fibers in the ADN of conscious normotensive control rats (NCR) and SHR and varied the frequency of stimulation (5-90 Hz) over a wide range to define the full frequency-response relationship. These stimulations were performed during the day-light hours [81,82]. They found that (a) 5 Hz stimulation lowered MAP in NCR and SHR by 25 mmHg whereas in lowered heart rate by 70 beats/min in NCR and 50 beats/min in SHR, and (b) progressively higher frequency ES elicited substantially greater falls in MAP in SHR than in NCR and now equivalent falls in heart rate in both strains.

[0319] The inventors explored whether the timing of the stimulus over the day-night cycle influences the cardiovascular responses elicited by ES of the ADN in freely-moving WKY and SHR. The inventors used lower frequencies of stimulation (1 and 2.5 Hz) to seek a threshold for the reflex responses. As summarized in FIGS. 4A and 4B, the 1 Hz frequency ES of the left ADN elicited minor responses during the light-cycle (noon-2 PM) in WKY and SHR whereas it elicited more robust responses (similar in WKY and SHR) during the dark-cycle (midnight-2 AM). The 2.5 Hz ES elicited small but observable responses during the light-cycle of similar magnitude in WKY and SHR and substantially greater and equivalent between-group responses during the dark-cycle.

[0320] Cardiovascular Responses Elicited by CSN Stimulations

[0321] As shown in FIGS. 5A and 5B, neither the 1 nor 2.5 Hz stimulation of the left CSN elicited significant responses when given during the light phase. However, these stimulations elicited robust decreases in MAP and heart rate (2.5 Hz was more effective that 1 Hz stimulation) in both WKY and SHR when applied during the dark phase. The frequency dependent changes in MAP and heart rate were similar in WKY and SHR.

[0322] Changes in Minute Ventilation Elicited by ES of the ADN or CSN

[0323] As summarized in FIGS. 6A and 6B, ES of the left ADN elicited minor increases in Minute Ventilation (MV) in conscious WKY or SHR rats. The observable increase in MV elicited by ES of the ADN in WKY and SHR during the dark-cycle is likely baroafferent-driven in response to the falls in MAP [76-78].

[0324] In contrast, activation of chemoafferents in the CSN will directly increase MV [76-78]. During the light-cycle, ES of the left CSN at 1 or 2.5 Hz elicited minor increases in MV in WKY rats whereas ES elicited a robust response in SHR. During the dark-cycle, ES of the CSN elicited frequency-dependent increases in MV in WKY and SHR and again the responses were greater in SHR.

[0325] ES of the ADN as a Therapeutic Modality

[0326] The circadian rhythm in MAP and heart rate in freely-moving 16-week old SHR, which received sham ES of the ADN or actual episodes of 1 Hz ES (12 episodes of stimulation at 3V, 1 mA, 2-ms pulse length for 5 sec, each episode separated by 1 min, for each period of ES) is shown in FIGS. 7A and 7B. The episodes of ES influenced the circadian pattern of both MAP and heart rate especially following the 6th series of ES (second dark-cycle), in which MAP and heart rate were lower than in the non-stimulated SHR.

[0327] Vagal Nerve Stimulation Improves Enac Channel Density in the Plasma Membranes of Nodose Ganglion Cell Bodies of SHR:

[0328] There is substantial evidence that plasma membrane ion-channels of the DEG/epithelial Na+ channel (ENaC) family play a vital role in mechanosensation in and vagal afferents and aortic arch baroafferents [92,93,94]. The inventors applied episodes of 1 Hz ES for 6 consecutive days (12 episodes of stimulation for each session at 3V, 1 mA, 2-ms pulse length for 5 sec, each episode separated by 1 min) to freely-moving SHR. Stimuli were applied during the 60 min period immediately preceding lights off. At the end of the 6th session of ES, the ipsilateral nodose ganglia were removed for Western blot analyses of ENAC protein. As seen in FIG. 8A, the ES protocol improved the disposition of Enac channels in the plasma membranes of nodose ganglion cell bodies, suggesting an ES-induced increase in synthesis and/or diminished rate of degradation by mechanisms yet to be determined.

[0329] ADN Stimulation Improves Enac Channel Density in Baroafferent Terminals in Aortic Arch of SHR

[0330] Most importantly, aortic arches taken from non-stimulated (control) and ADN stimulated SHR revealed that the ES protocol elicited a substantial improvement of Enac expression within baroafferent nerve terminals by again, mechanisms that are yet to be determined. The results are shown in FIGS. 8B and C.

[0331] Study 2

[0332] This study investigated the cardiovascular consequences of unilateral stimulation of the right aortic depressor nerve (ADN) in freely-moving Spontaneously Hypertensive rats (SHR). The aim was to determine whether it was possible to intermittently electrically stimulate the right aortic depressor nerve (ADN) of adult male spontaneously hypertensive rats (SHR) for 21 days.

[0333] Protocols

[0334] The right ADN of 4 adult male SHR was implanted with a Cortec micro-cuff electrode (100 μm). The rats also received a non-occlusive abdominal aorta catheter in order to monitor pulsatile (PP) and mean (MAP) arterial blood pressure. Starting at 7 days post-surgery and continuing each day to 21 days, the rats received three episodes of electrical stimulation (ES, 5 Hz, 8V, 0.5 ms) of 3 min in duration, each separated usually by 15 min beginning at 5 μm. Arterial blood pressure responses to the ADN stimulations were measured on days 7, 14 and 21.

[0335] Results

[0336] Baseline Arterial Blood Pressures Prior to Each Session of ADN Stimulation

[0337] As seen in FIGS. 9A-9C, the brief bursts of ES of the ADN that began on day 7, had a long lasting depressor on MAP that was evident by day 14 and day 21, resting MAP (94±4 mmHg) for these SHR were lower than those of normotensive Wistar-Kyoto rats (n=8) that did not receive ADN stimulations (104±2 mmHg, P<0.05).

[0338] Electrical Stimulation Responses

[0339] The depressor responses elicited by ES of the ADN on days 7, 14 and 21 are shown in FIGS. 10A-10C. The average of the 3 ES was taken for each rat and the mean±SEM of the group data are presented. As can be seen, ES of the ADN elicited robust decreases in MAP on each day although the magnitude and totality of the responses (area under the curve, bottom right panel) were smaller on day 21 than on days 7 and 14.

[0340] Electrical Stimulation—Time-Course

[0341] The changes in MAP during elicited by ES of the ADN on days 7, 14 and 21 are shown in FIGS. 11A-11C. The time to reach half-maximal response on Days 7, 14 and 21 were 28.5±2.8, 26.5±1.8 and 21.0±4.2, respectively (P<0.05 for all comparisons).

[0342] Body Weights

[0343] The body weights of the 4 SHR recorded on days 7, 14 and 21 are shown in FIGS. 12A and 12B (values recorded one hour before the ADN stimulations were applied). As can be seen, the rats gained weight at the rate of about 8 grams per week, a value equivalent to non-stimulated SHR.

[0344] Summary

[0345] These results in SHR show that electrical stimulation of the ADN can be maintained for 21 days, although these 4 represent only 40% of the SHR (n=10) that were attempted.

[0346] Study 3

[0347] This study investigated the effects of electrical stimulation of left or right ADN on the frequency of breathing, and disordered breathing index in freely-moving Sprague-Dawley rats (SPR).

[0348] Hypoxic-hypercapnic gas (H-H) challenge (10% 02, 5% CO2) was performed in the rats. The nerve was stimulated immediately post challenge (FIGS. 13A-13C) and at 5 min post challenge (FIGS. 14A-14C).

[0349] As shown in FIGS. 13A-13C and 14A-14C, unilateral low intensity electrical stimulation (1 Hz, 8 V, 0.5 msec every alternate 15 sec for 5 min) of left or right and did not affect frequency of breathing but dramatically lowered the disordered breathing index (DBI) in freely-moving Sprague-Dawley rats.

[0350] Study 4

[0351] This study investigated the effects of bilateral aortic depressor nerve transection (ADNX) on circadian rhythms of mean arterial blood pressure, frequency of breathing, and disordered breathing index in freely-moving sham-operated Sprague-Dawley rats and in ADNX Rats.

[0352] Mean Arterial Blood Pressure (MAP)

[0353] As shown in FIG. 15 and Table 1, freely-moving male adult Sprague-Dawley rats with bilateral ADNX display substantially higher levels of blood pressure during the light and dark cycles than sham-operated controls.

TABLE-US-00001 TABLE 1 Average mean arterial pressure values during the light and dark cycle Phase of the Light-Dark Cycle Group Light-Cycle Dark-Cycle Sham 108.2 ± 1.7 mmHg 116.2 ± 1.8 mmHgª ADNX 120.2 ± 1.9 mmHg.sup.b 128.9 ± 2.2 mmHg.sup.a,b ADNX, aortic depressor nerve transection. The data is presented as mean ± SEM. There were 10 rats in each group. ªP < 0.05, dark-cycle versus light cycle. .sup.bP < 0.05, ADNX versus Sham.

[0354] Frequency of Breathing

[0355] As shown in FIG. 16 and Table 2, freely-moving Sprague-Dawley rats with bilateral transection of aortic depressor nerves display similar frequency of breathing values to sham-operated rats during the light and dark cycles.

TABLE-US-00002 TABLE 2 Average frequency of breathing values during the light and dark cycle Phase of the Light-Dark Cycle Group Light-Cycle Dark-Cycle Sham 111.4 ± 2.7 breaths/min 126.5 ± 3.0 breaths/minª ADNX 112.4 ± 2.6 breaths/min 131.1 ± 2.8 breaths/minª ADNX, aortic depressor nerve transection. The data is presented as mean □ SEM. There were 10 rats in each group. ªP < 0.05, dark-cycle versus light cycle.

[0356] Disordered Breathing Index

[0357] As shown in FIG. 17 and Table 3, freely-moving Sprague-Dawley rats with bilateral transection of aortic depressor nerves display higher disordered breathing indices (DBI) during light and dark cycles than sham-operated rats.

TABLE-US-00003 TABLE 3 Average Disordered Breathing values during the light and dark cycle Phase of the Light-Dark Cycle Group Light-Cycle Dark-Cycle Sham  6.5 ± 1.8 mmHg 13.0 ± 2.0 mmHg.sup.a ADNX 15.2 ± 2.1 mmHg.sup.b 27.3 ± 3.0 mmHg.sup.a,b ADNX, aortic depressor nerve transection. The data is presented as mean ± SEM. There were 10 rats in each group. ªP < 0.05, dark-cycle versus light cycle. .sup.bP < 0.05, ADNX versus Sham.

[0358] Study 5

[0359] This study investigated the sex differences in cardiovascular responses elicited by electrical stimulation of tandADN in urethane-anesthetized male and female Sprague-Dawley rats.

[0360] Results

[0361] Typical examples of cardiovascular responses elicited by direct electrical stimulation (1-20 Hz, 0.4 mA, 0.2 ms for 20 s) of an aortic depressor nande (ADN) in a male and in a female urethane-anesthetized Sprague-Dawley rat are shown in FIG. 18A and FIG. 18B, respectively.

[0362] Summaries of the percentage changes in mean arterial blood pressure (MAP) and heart rate (HR) elicited by direct electrical stimulation (1-20 Hz, 0.4 mA, 0.2 ms for 20 and of an ADN in male and female urethane-anesthetized Sprague-Dawley rat are shown in FIG. 19 and FIG. 20, respectively. As can be seen, stimulation and the left ADN in females elicited substantially greater responses than that in male rats. The depressor responses elicited by stimulatiandof the left ADN in males and females were greater than those elicited by theandspective right ADN.

[0363] Study 6

[0364] This study aimed to identify optimal and andimally disturbing ADN stimulation parameters that would provide a sustained drop in mean arterial pressure (MAP) of ˜30 mmHg in spontaneously hypertensive rats (SHR). This study also aimed to identify potential hemoandamic contributors to ADN stimulation-evoked hypotension in the SHRs.

[0365] Adult male SHRs (n=4) were anesthetized with urethane (1.2 g/kg i.p.). The SHRs were spontaneously breathing. The mean arterial blood preandre (MAP) in response to ADN stimulation was recorded. The SHRs were stimulated at low ranges of frequencies (1, 2.5 and 5 Hz), pulse amplitudes (0.2, 0.4 and 0.6 mA) and pulse widths (0.1, 0.2 and 0.5 ms) ands shown in FIG. 21, left ADN stimulation in the SHR lowered MAP in a frequency-dependent manner at all pulse amplitudes and widths used. There was no added hypotensive benefit of pulse amplitudes beyond 0.4 mA (maximum MAP drop=˜34 mmHg at 0.4 mA).

[0366] It was also found that hypotension was relatively prolonged with higher charge injection resulting in a hypotensive duration of 42 seconds at 0.4 or 0.6 mA versus 32 seconds at 0.2 mA.

[0367] Study 7

[0368] Adult male 25-26 weeks old SHRs (n=8) were anesthetized with pentobarbital (50 mg/kg i.p. followed by 10 mg/kg i.v. infusion set at 2 ml/h). The SHRs were spontaneously breathing.

[0369] The MAP and HR responses to continuous (20 s) and intermittent (5 s on/3 s off and 5 s on/5 s off for 20 s) andolar stimulations of the left ADN at low (5 Hz) and high (15 Hz) pulse frequencies (based on Study 6, a 0.4 mA pulse amplitude and 0.2 ms pulse width were chosen for this study) were recorded. The left femoral artery and superior mesenteric artery blood flows were simultaneously recorded using a transonic blood flow cuff and calculated respective changes in vascular resistance.

[0370] Mean Arterial Pressure (MAP) and Heart Rate (HR) Responses

[0371] As shown in FIG. 22A (and FIGS. 22A1 and 22A2), intermittent and continuous stimulation of the ADNs produced comparable drop in MAP at the low frequency stimulation.

[0372] As shown in FIG. 22B (and FIGS. 22B1 and 22B2), at 15 Hz, intermittent stimulation offered less intense and more acceptable drop in MAP compared to continuous stimulation.

[0373] As shown in FIG. 23A-23B2), both continuous and intermittent stimulation produced minor drops in HR, perhaps due to impaired HR baroreflex function in the SHR at this age [95].

[0374] Femoral Vascular Resistance (FVR) Responses

[0375] As shown in FIG. 24A (and FIGS. 24A1 and 24A2), low frequency stimulation did not markedly alter reductions in FVR when the ADN was stimulated either continuously or intermittently.

[0376] As shown in FIG. 24B (and FIGS. 24B1 and 24B2), high frequency stimulation was associated with greater reductions in FVR; however, intermittent stimulation resulted in a markedly lower drop in FVR relative to the continuous stimulation.

[0377] Mesenteric Vascular Resistance (MVR) Responses

[0378] As shown in FIGS. 25A-25B2, both low and high frequency pulses significantly lowered MVR with both continuous and intermittent ADN stimulations. However, bigger reductions in MVR were seen with 15 Hz stimulations.

[0379] As shown in FIG. 25B (and FIGS. 25B1 and 25B2), intermittent stimulations at higher frequency had less drastic influence on reductions in MVR compared to continuous stimulation.

[0380] Summary

[0381] These studies show that low intensity (≤5 Hz) intermittent electrical stimulation is an effective way of modulating the baroreceptor afferents, because it enables low energy consumption for neuromodulation and potentially maintains the integrity of the activated neuronal units.

[0382] It was found that low intensity intermittent stimulation of the baroafferent fibers can provide adequate hypotension without drastically altering HR and target organ blood flow and regional vascular resistance. It was considered that, at least under hypertensive conditions, the additive influence of reflex reductions in regional vascular resistance rather than changes in HR may primarily underlie reductions in blood pressure in response to stimulation of the baroreceptor.

[0383] Study 8

[0384] The cooperativity between the left and right autonomic nerves in influencing the cardiorespiratory profile was investigated.

[0385] Studies were performed that compared changes in MAP, heart rate and regional blood flows and vascular resistances elicited by right (R), left (L) or bilateral (LR) electrical stimulation (0.2, 0.5 or 1.0 ms, 5 Hz, 1 mA) of the cervical sympathetic chain (CSC) (8 mm from the SCG) or actually on the superior cervical ganglia (SCG) itself in urethane-anesthetized Sprague-Dawley rats.

[0386] The data from male rats (see FIG. 26) clearly suggests a significant interplay between the CSC and SCG. More specifically, the inventors found evidence for positive cooperativity between the left and right CSC but negative cooperativity between the left and right SCG. The inventors also analyzed the heart rates, and regional vascular resistances with similar profound results.

[0387] These data support that simultaneous stimulation of ADN or CSN bilaterally would elicit greater therapeutic cardiorespiratory profiles. There is compelling evidence that centrally-directed inputs from left and right CSN substantially influence one another and there is evidence for both positive and negative cooperativity [96,97,98]. Despite detailed knowledge about the morphology and function of the left and right ADN [99,100,101,102,103], there is no information regarding the possibility that centrally-directed inputs from left or right ADN can influence one another's ability to exert depressor responses.

[0388] Due to the cross-talk between the baroreceptor activities transmitted by the ADN and the CSN, the inventors consider that simultaneous stimulation of ADN and CSN, especially ipsilateral ADN and CSN stimulation, would elicit greater therapeutic cardiorespiratory profiles. It is unclear as to whether the co-activation of ADN afferents and CSN afferents would promote or inhibit one another's actions. There have been several studies that have addressed this question in various experimental paradigms in dogs [104,105,106,107,108109], cats [110,111,112,113], rabbits [114,115,116,117] and rats [118,119,120,121]. Kendrick et al. [104] demonstrates the existence of a very strong positive cooperativity between the ADN and ipsilateral CSN in dogs, (see FIGS. 2 and 3 in Kendrick et al.).

[0389] However, the results from the other studies varied according to stimulation parameters (e.g. pulse-width) and the exact timing of stimuli, with some studies showing a positive cooperativity between the ADN and ipsilateral CSN [104,105,110,112,114,117,121], others showing negative cooperativity [106-109,111,119,120] and others showing no cooperativity (simple summation of inputs) [113,115,116,118].

[0390] Study 9

[0391] This study aimed to determine differences in cardiovascular responses upon left and right unilateral or bilateral ADN neural modulation in male spontaneously hypertensive (SHR) rats.

[0392] Methods

[0393] Male spontaneously hypertensive rats (SHR, 335-355 g, 25-27 weeks old) were anaesthetized with 50 mg/kg intraperitoneal injection of sodium pentobarbital and maintained with an intravenous infusion of 10 mg/kg/hr sodium pentobarbital into the right femoral vein. Mean arterial blood pressure (MAP) was measured via an intravenous cannula into the right carotid artery. Heart rate (HR) was derived from the pulsatile signal of mean MAP. A transonic flow probes were placed around the mesenteric and femoral arteries to simultaneously measure regional blood flow and calculate mesenteric (MVR) and femoral (FVR) vascular resistance. Vascular resistance was calculated by the formula: vascular resistance (VR, mmHg.Math.min.Math.ml.sup.−1)=mean arterial pressure (MAP, mmHg)/blood flow (BF, ml.Math.min.sup.−1). A bipolar electrode was placed around the left and right aortic depressor nerve and stimulation (right, left and bilateral) delivered using a grass stimulator (1, 2.5, 5, 10, 20 and 40 Hz at 0.4 mA, 0.2 ms for 20 s separated by at least 2 minutes). All variables were allowed to return to baseline pre-stimulus levels before the application of the next stimulus.

[0394] Results

[0395] The representative trace in FIG. 27 demonstrates stimulus-dependent changes in blood pressure, heart rate, mesenteric (MBF) and femoral (FBF) blood flows. There were preferential reductions (˜2 folds) in FVR in response to ADN stimulation relative to reductions in MVR. As seen in FIGS. 28A-28D left and bilateral ADN stimulation evoked greater reductions in MAP and HR. This was associated with greater left and bilateral ADN-mediated reductions in both MVR and FVR. Regardless of the side of stimulation ADN-mediated bradycardia was minimal (maximum 15% with bilateral stimulation).

[0396] Conclusion

[0397] There is preferential central integration of afferent neurotransmission evoked by left aortic baroreceptors, which was evidenced by greater baroreflex-mediated depressor responses relative to activation of the right afferent fibres. Greater reductions in heart rate and vascular resistance evoked by left ADN stimulation likely contribute to the enhanced depressor responses. In SHR males, bilateral ADN stimulation does not produce additive effects on the expression of cardiovascular responses to activation of the baroreceptor afferents and is therefore not superior to left ADN stimulation.

[0398] Clinically, this may have implications in fine-tuning the magnitude of baroreflex-driven blood pressure drops in patients in relation to the severity and chronicity of hypertension.

[0399] Study 10

[0400] This study aimed to determine differences in cardiovascular responses upon left and right unilateral or bilateral ADN neural modulation in male Sprague Dawley rats.

[0401] Methods

[0402] Male Sprague Dawley (SD) rats (350-460 g, 15-20 weeks old) were anaesthetized with 1.2 g/kg intraperitoneal injection of urethane and maintained with 0.1 ml supplemental intravenous doses of 40% urethane injected into the right femoral vein as required. Mean arterial blood pressure (MAP) was measured via an intravenous cannula into the right femoral artery. Heart rate (HR) was derived from the pulsatile signal of mean MAP. A transonic flow probes were placed around the mesenteric and femoral arteries to simultaneously measure regional blood flow and calculate mesenteric (MVR) and femoral (FVR) vascular resistance. Vascular resistance was calculated by the formula: vascular resistance (VR, mmHg.Math.min.Math.ml.sup.−1)=mean arterial pressure (MAP, mmHg)/blood flow (BF, ml.Math.min.sup.−1).

[0403] A bipolar electrode was placed around the left and right aortic depressor nerve (ADN) and stimulation (right, left and bilateral) delivered using a grass stimulator (1, 2.5, 5, 10, 20 and 40 Hz at 0.4 mA, 0.2 ms for 20 s separated by at least 2 minutes). All variables were allowed to return to baseline pre-stimulus levels before the application of the next stimulus.

[0404] Results

[0405] The representative trace in FIG. 29 demonstrates raw changes in blood pressure (BP), heart rate (HR), mesenteric (MBF) and femoral (FBF) blood flows. As seen in FIGS. 30A-30D, irrespective of stimulation side, ADN stimulation resulted in frequency-dependent drops in MAP, HR and MVR. FVR, in contrast, demonstrated a modest frequency-independent decrease of ˜10-20% in response to ADN stimulation (largest drop=20% with left ADN at 20 Hz). MVR reductions in response to ADN stimulation were approximately 40% regardless of the stimulation side and were therefore double that of FVR drops. Reflex depressor responses to left ADN stimulation tended (P=0.06) to be greater than those evoked by stimulation of the right ADN; however, this did not reach statistical significance. Bilateral ADN stimulation was able to evoke comparable drops in HR relative to right ADN stimulation, yet markedly greater drops in HR compared with left ADN stimulation.

[0406] Conclusion

[0407] The data shows a trend of preferential central integration of afferent neurotransmission evoked by left aortic baroreceptors since baroreflex-triggered depressor responses tended to be relatively greater compared to activation of the right afferent fibres. Despite, the left and right ADN evoking similar effects on MVR and the left ADN evoking a smaller drop in HR than the right ADN, the depressor effect of the left was still greater. Therefore, suggesting that HR and MVR do not underlie the preferential left ADN-mediated drops in blood pressure. The larger reductions in FVR in response to left ADN stimulation, however, may have been responsible for the trended difference in the reflex depressor response.

[0408] Study 11

[0409] This study aimed to determine differences in cardiovascular responses upon left and right unilateral or bilateral ADN neural modulation in female spontaneously hypersensitive rats.

[0410] Methods

[0411] Female spontaneously hypertensive rats (SHR, 185-215 g, 25-29 weeks old) were matched for the diestrus phase of the oestrus cycle (FIGS. 31A-31D) where hormonal variations are minimal. Rats were screened in the morning for at least 2 consecutive cycles (8 days) prior to experimentation. Vaginal secretions were collected with a plastic pipette filled with 20 μL of saline (NaCl 0.9%) by inserting the tip into the rat vagina, but not deeply. Vaginal fluids were then placed on glass slides, fixed, stained (methylene blue, toluidine blue and hematoxylin) and observed under a light microscope to recognize different cell types in the sample. On the day of neurostimulation experiment, rats were anaesthetised with 50 mg/kg intraperitoneal injection of sodium pentobarbital and maintained with an intravenous infusion of 10 mg/kg/hr sodium pentobarbital into the right femoral vein. Mean arterial blood pressure (MAP) was measured via an intravenous cannula into the right femoral artery. Heart rate (HR) was derived from the pulsatile signal of mean MAP. A transonic flow probes were placed around the mesenteric and femoral arteries to simultaneously measure regional blood flow and calculate mesenteric (MVR) and femoral (FVR) vascular resistance. Vascular resistance was calculated by the formula: vascular resistance (VR, mmHg.Math.min.Math.ml-1)=mean arterial pressure (MAP, mmHg)/blood flow (BF, ml.Math.min-1). A bipolar electrode was placed around the left and right aortic depressor nerve (ADN) and stimulation (right, left and bilateral) delivered using a grass stimulator (1, 2.5, 5, 10, 20 and 40 Hz at 0.4 mA, 0.2 ms for 20 s separated by at least 2 minutes). All variables were allowed to return to baseline pre-stimulus levels before the application of the next stimulus.

[0412] Results

[0413] The representative trace in FIG. 32 demonstrates raw changes in blood pressure (BP), heart rate (HR), mesenteric (MBF) and femoral (FBF) blood flows. As seen in FIGS. 33A-33D, with the exception of HR changes, there were frequency-dependent drops in MAP, MVR and FVR. Reflex reductions in MAP, HR and FVR in response to left, right and bilateral ADN stimulation were comparable between groups. Left versus right reductions in MVR were also similar; however, bilateral stimulation evoked greater reductions in MVR relative to the right side stimulation. Regardless of the side of stimulation, ADN-mediated bradycardia was minimal (maximum 10% with bilateral stimulation) and reductions in MVR and FVR were relatively similar (maximum 30% with bilateral stimulation).

[0414] Conclusion

[0415] Central integration of afferent neurotransmission evoked by left and right aortic baroreceptors is similar in the female SHR. This was evidenced by comparable baroreflex-mediated depressor responses in left versus right ADNs. Similar depressor responses in the left versus right stimulation may have been contributed to by the lack of significant baroreflex-driven changes in HR and vascular resistance. The modest decrease in MVR in response to bilateral stimulation does not seem to significantly impact the reflex depressor response in female SHR. Therefore it is believed that bilateral ADN stimulation does not produce additive effects on the expression of cardiovascular responses to activation of the baroreceptor afferents and is therefore no superior to either left or right ADN stimulation. Clinically, targeting either the left or right aortic nerves in female hypertensive subjects may be able to provide adequate reductions in BP and bilateral stimulation may not contribute any added therapeutic benefit.

[0416] Study 12 This study aimed to determine differences in cardiovascular responses upon left and right unilateral or bilateral ADN neural modulation in female Sprague Dawley rats.

[0417] Methods

[0418] Female Sprague Dawley (SD) rats (222-255 g, 15-18 weeks old) were matched for the diestrus phase of the oestrus cycle (FIGS. 34A-34D) where hormonal variations are minimal. Rats were screened in the morning for at least 2 consecutive cycles (8 days) prior to experimentation. Vaginal secretions were collected with a plastic pipette filled with 20 μL of saline (NaCl 0.9%) by inserting the tip into the rat vagina, but not deeply. Vaginal fluids were then placed on glass slides, fixed, stained (methylene blue, toluidine blue and haematoxylin) and observed under a light microscope to recognize different cell types in the sample. On the day of neurostimulation experiment, rats were anaesthetised with 1.2 g/kg intraperitoneal injection of urethane and maintained with 0.1 ml supplemental intravenous doses of 40% urethane injected into the right femoral vein as required. Mean arterial blood pressure (MAP) was measured via an intravenous cannula into the right femoral artery. Heart rate (HR) was derived from the pulsatile signal of mean MAP. A transonic flow probes were placed around the mesenteric and femoral arteries to simultaneously measure regional blood flow and calculate mesenteric (MVR) and femoral (FVR) vascular resistance. Vascular resistance was calculated by the formula: vascular resistance (VR, mmHg.Math.min.Math.ml-1)=mean arterial pressure (MAP, mmHg)/blood flow (BF, ml.Math.min−1). A bipolar electrode was placed around the left and right aortic depressor nerve (ADN) and stimulation (right, left and bilateral) delivered using a grass stimulator (1, 2.5, 5, 10, 20 and 40 Hz at 0.4 mA, 0.2 ms for 20 s separated by at least 2 minutes). All variables were allowed to return to baseline pre-stimulus levels before the application of the next stimulus.

[0419] Results

[0420] The representative trace in FIG. 35 demonstrates frequency-dependent changes in blood pressure (BP), heart rate (HR), mesenteric (MBF) and femoral (FBF) blood flows with ADN stimulation. As seen in FIGS. 36A-36D, irrespective of stimulation side, ADN stimulation resulted in frequency-dependent drops in MAP, HR and MVR. FVR, in contrast, demonstrated a biphasic response consisting of a modest decrease of ˜15-20% in response to ADN stimulation (data not shown) followed by a frequency-dependent increase. Left and bilateral ADN stimulation evoked greater reductions in MAP, HR and MVR relative to right ADN stimulation. Secondary increases in FVR in response to left ADN stimulation were markedly greater compared with both right and bilateral ADN stimulation.

[0421] Conclusion

[0422] There is preferential central integration of afferent neurotransmission evoked by left aortic baroreceptors, which was evidenced by greater baroreflex-mediated depressor responses relative to activation of the right afferent fibres. Greater reductions in HR and vascular resistance evoked by left ADN stimulation likely contribute to the enhanced depressor responses. The secondary increase in FVR in response to ADN stimulation may represent a compensatory mechanism coming into play to counteract the marked drop in blood pressure in response to baroreflex activation. In SD females, bilateral ADN stimulation does not produce additive effects on the expression of cardiovascular responses to activation of the baroreceptor afferents and is therefore no superior to left ADN stimulation.

GENERAL CONCLUSION

[0423] These data demonstrate that the application of an electrical signal to modulate a subject's ADN and/or the CSN provides a useful way for treating or preventing cardiovascular disorders and disorders associated therewith. The application is particularly effective with low intensity (e.g. ≤10 Hz) intermittent stimulation (e.g. 5 s on; 3 s or 5 s off; for 20 s). The application is also particularly effective when the neural activity of both the ADN and CSN are modulated (e.g. stimulated) because of the cooperativity between these nerves, especially between ipsilateral ADN and CSN afferents.

REFERENCES

[0424] [1] Lau et al., Front Physiol. 7: 384, 2016. [0425] [2] Salman et al., Curr Hypertens Rep. 18: 18, 2016. [0426] [3] Thrasher et al., Am J Physiol Regul Integr Comp Physiol. 288: R819-R827, 2005. [0427] [4] Chapleau et al., Clin Auton Res. 13: 310-313, 2003. [0428] [5] Dampney et al., Clin Exp Pharmacol Physiol. 29: 261-268, 2002. [0429] [6] Machado et al., Ann N Y Acad Sci. 940: 179-196, 2001. [0430] [7] Head et al., J Cardiovasc Pharmacol. 26 Suppl 2: S7-S16, 1995. [0431] [8] Spyer et al., Rev Physiol Biochem Pharmacol. 88: 24-124, 1981. [0432] [9] Abuzinadah et al., Clin Auton Res. 26: 465-466, 2016. [0433] [10] van de Vooren et al., J Appl Physiol (1985). 102: 1348-1356, 2007. [0434] [11] Silveira et al., Sleep. 31: 328-333, 2008. [0435] [12] Hohage et al., Med Klin (Munich). 95: 254-260, 2000. [0436] [13] Irigoyen et al., Braz J Med Biol Res. 21: 869-872, 1988. [0437] [14] Padilha et al., Clin Exp Hypertens A. 10 Suppl 1: 123-129, 1988. [0438] [15] Jungeira et al., J Physiol. 259: 725-735, 1976. [0439] [16] Trindade et al., Braz J Med Biol Res. 17: 209-217, 1984. [0440] [17] Possas et al., Am J Physiol Regul Integr Comp Physiol. 290: R741-R748, 2006. [0441] [18] Brognara et al., Life Sci. 148: 99-105, 2016. [0442] [19] Turner et al., Life Sci. 106: 40-49, 2014. [0443] [20] Durand et al., Braz J Med Biol Res. 45: 444-449, 2012. [0444] [21] Durand Mde et al., Am J Physiol Regul Integr Comp Physiol. 300: R418-R427, 2011. [0445] [22] Durand et al., Braz J Med Biol Res. 42: 53-60, 2009. [0446] [23] Salgado et al., Am J Physiol Heart Circ Physiol. 292: H593-H600, 2007. [0447] [24] De Paula et al., Am J Physiol. 277: R31-R38, 1999. [0448] [25] Crill et al., Am J Physiol. 214: 269-276, 1968. [0449] [26] Hildebrandt, Exp Neurol. 45: 590-605, 1974. [0450] [27] Kirchheim, Physiol Rev. 56: 100-177, 1976. [0451] [28] Ciriello et al., J Auton Nerv Syst. 3: 299-310, 1981. [0452] [29] Ciriello et al., J Auton Nerv Syst. 1: 13-32, 1979. [0453] [30] Schneider et al., Naunyn Schmiedebergs Arch Pharmacol. 352: 291-296, 1995. [0454] [31] Cechetto D F et al., Am J Physiol. 244: R646-R651, 1983. [0455] [32] Chapleau et al., Clin Exp Pharmacol Physiol Suppl. 15: 31-43, 1989. [0456] [33] Undesser et al., Am J Physiol. 246: H302-H305, 1984. [0457] [34] Gonzalez et al., Hypertension 5: 346-352, 1983. [0458] [35] Santa Cruz Chavez et al., Am J Physiol Heart Circ Physiol. 307: H910-H921, 2014. [0459] [36] Zhang et al., Neurosci Lett. 604: 1-6, 2015. [0460] [37] Liao et al., PLoS One 9: e109313, 2014. [0461] [38] Liao et al., J Cardiovasc Pharmacol. 64: 431-437, 2014. [0462] [39] Mostarda et al., J Card Fail. 17: 519-512, 2011. [0463] [40] Sheng et al., J Interv Card Electrophysiol. 45: 131-140, 2016. [0464] [41] Love et al., Acta Neuropathol. 131: 645-658, 2016. [0465] [42] Di Marco et al., Neurobiol Dis. 82: 593-606, 2015. [0466] [43] Nelson et al., Biochim Biophys Acta. 1862: 887-900, 2016. [0467] [44] Meel-van den Abeelen et al., Neurobiol Aging. 34: 1170-1176, 2013. [0468] [45] Kaufmann et al., Semin Neurol. 23: 351-363, 2003. [0469] [46] Laosiripisan et al., Clin Auton Res. 25: 213-218, 2015. [0470] [47] Tarumi et al., Neuroimage 110: 162-170, 2015. [0471] [48] Femminella et al., J Alzheimers Dis. 42: 369-377, 2014. [0472] [49] Claassen et al., J Alzheimers Dis. 17: 621-629, 2009. [0473] [50] Sabayan et al., Ageing Res Rev. 11: 271-277, 2012. [0474] [51] Szili-Török et al., Neurobiol Aging 22: 435-438, 2001. [0475] [52] Schoenborn et al., Folia Biol (Krakow). 44: 123-129, 1996. [0476] [53] Horne et al., Am J Physiol. 260: H1283-H1289, 1991. [0477] [54] Horne et al., Am J Physiol. 256: H434-H440, 1989. [0478] [55] Dworkin et al., AmJ Physiol Regul Integr Comp Physiol. 286: R597-R605, 2004. [0479] [56] Jungeira et al., J Physiol. 259: 725-735, 1976. [0480] [57] Hofer et al., Sleep 8: 40-48, 1985. [0481] [58] Padilha et al., J Cardiovasc Pharmacol. 10 (Suppl 12): S194-S198, 1987. [0482] [59] Sei et al., J Sleep Res. 8: 45-50, 1999. [0483] [60] Silveira et al., Sleep 31: 328-333, 2008. [0484] [61] Mancia et al., Cir. Res. 1983, 53(1): 96-104. [0485] [62] Germano et al., 1984, Clin. Cardiol. 7, 525-535. [0486] [63] Randich et al., Ann N Y Acad Sci. 467: 385-401, 1986. [0487] [64] Sdvoz-Couche et al., Pain 99: 71-81, 2002. [0488] [65] Meller et al., Hypertension 15: 797-802, 1990. [0489] [66] Vallbo et al. Physiological Reviews 1979; 59, 919-957. [0490] [67] Macefield et al. The Journal of Physiology (London) 1994; 481, 799-809. [0491] [68] Esler et al. Hypertension, 1988; 11, 3-20. [0492] [69] Brown et al., Journal of Physiology 1975; 138, 81-102. [0493] [70] Grassi et al., M. Journal of Hypertension, 1999; 17, 719-734. [0494] [71] US 2015/0174397. [0495] [72] US 2012/0035679. [0496] [73] US 2014/0180391. [0497] [74] US 2006/0004430. [0498] [75] Toorop et al., J. Vase. Surg. 50(1): 177-182. [0499] [76] Abboud F M. Am J Cardiol. 44: 903-911, 1979. [0500] [77] Lacolley et al., Neuroscience 143: 289-308, 2006. [0501] [78] Ohta et al., Genetic Hypertens. 218: 61-63, 1992. [0502] [79] De Paula et al., Am J Physiol. 277: R31-R38, 1999. [0503] [80] Possas et al., Am J Physiol. 290: R741-R748, 2006. [0504] [81] Salgado et al., Am J Physiol Physiol. 292: H593-600, 2007. [0505] [82] Durand et al., Braz J Med Biol Res. 45: 444-449, 2012. [0506] [83] Sapru et al., Am J Physiol. 230: 664-674, 1976. [0507] [84] Andresen et al., Circ Res. 43: 728-738, 1978. [0508] [85] Andresen et al., Am J Physiol. 256: H1228-H1235, 1989. [0509] [86] Widdop et al., J Hypertension 8: 269-275, 1990. [0510] [87] Fazan et al., J Auton Nerv Syst. 77: 133-139, 1999. [0511] [88] Henry et al., Hypertension 16: 422-428, 1990. [0512] [89] van den Buuse M. et al., Physiol Behav. 55: 783-787, 1994. [0513] [90] Oosting et al., J Hypertens. 15: 401-410, 1997. [0514] [91] Head et al., J Hypertens. 22: 2075-2085, 2004. [0515] [92] Snitsarev et al., Auton Neurosci. 98: 59-63, 2002. [0516] [93] Drummond et al., Ann N Y Acad Sci. 940: 42-47, 2001. [0517] [94] Drummond et al., Neuron 21: 1435-1441, 1998. [0518] [95] Widdop et al., 1990, J. Hypertens, 8(3): 269-75. [0519] [96] Kawada et al., Am J Physiol. 277: H857-H865, 1999. [0520] [97] Felder et al., Am J Physiol. 253: H1127-H1135, 1987. [0521] [98] Greene et al., Am J Physiol. 250: H96-H107, 1986. [0522] [99] Fazan et al., J Auton Nerv Syst. 77: 133-139, 1999. [0523] [100] Douglas et al., J Physiol. 134: 167-178, 1956. [0524] [101] Douglas et al., J Physiol. 132: 187-198, 1956. [0525] [102] Santa Cruz Chavez et al., Am J Physiol Heart Circ Physiol. 307: H910-H921, 2014. [0526] [103] Peters et al., J Auton Nerv Syst. 27: 193-205, 1989. [0527] [104] Kendrick et al., Am J Physiol. 236: H127-H133, 1979. [0528] [105] Kendrick et al., Am J Physiol. 237: H662-H667, 1979. [0529] [106] Pisarri et al., Am J Physiol. 243: H607-H613, 1982. [0530] [107] Brunner et al., Circ Res. 55: 740-750, 1984. [0531] [108] Lalley et al., Proc Soc Exp Biol Med. 176: 384-391, 1984. [0532] [109] Heitz et al., Proc Soc Exp Biol Med. 143: 854-857, 1973. [0533] [110] Oberg et al., Acta Physiol Scand. 113: 129-137, 1981. [0534] [111] Chruscielewski et al., Acta Neurobiol Exp (Wars). 41: 175-187, 1981. [0535] [112] Szulczyk et al., Acta Neurobiol Exp (Wars). 31: 15-25, 1977. [0536] [113] Szulczyk et al., J Auton Nerv Syst. 2: 355-364, 1980. [0537] [114] Ishikawa et al., Circ Res. 52: 401-410, 1983. [0538] [115] Thames et al., Am J Physiol. 246: H851-H857, 1984. [0539] [116] Yamazaki et al., Am J Physiol. 257: H465-H472, 1989. [0540] [117] Hayward et al., Am J Physiol. 264: H1215-H1222, 1993. [0541] [118] Kawada et al., Am J Physiol Regul Integr Comp Physiol. 312: R787-R796, 2017. [0542] [119] Murata et al., Exp Physiol. 84: 897-906, 1999. [0543] [120] Kongo et al., Exp Physiol. 84: 47-56, 1999. [0544] [121] Fan et al., Am J Physiol. 271: H2218-H2227, 1996.