Renal Sympathetic Nerve Activity Responses Research Articles

Effects of intrarenal pelvic infusion of tumour necrosis factor-α and interleukin 1-β on reno-renal reflexes in anaesthetised rats.

Reno-renal reflexes are disturbed in cardiovascular and hypertensive conditions when elevated levels of pro-inflammatory mediators/cytokines are present within the kidney. We hypothesised that exogenously administered inflammatory cytokines tumour necrosis factor alpha (TNF-α) and interleukin (IL)-1β modulate the renal sympatho-excitatory response to chemical stimulation of renal pelvic sensory nerves. In anaesthetised rats, intrarenal pelvic infusions of vehicle [0.9% sodium chloride (NaCl)], TNF-α (500 and 1000 ng/kg) and IL-1β (1000 ng/kg) were maintained for 30 min before chemical activation of renal pelvic sensory receptors was performed using randomized intrarenal pelvic infusions of hypertonic NaCl, potassium chloride (KCl), bradykinin, adenosine and capsaicin. The increase in renal sympathetic nerve activity (RSNA) in response to intrarenal pelvic hypertonic NaCl was enhanced during intrapelvic TNF-α (1000 ng/kg) and IL-1β infusions by almost 800% above vehicle with minimal changes in mean arterial pressure (MAP) and heart rate (HR). Similarly, the RSNA response to intrarenal pelvic adenosine in the presence of TNF-α (500 ng/kg), but not IL-1β, was almost 200% above vehicle but neither MAP nor HR were changed. There was a blunted sympatho-excitatory response to intrapelvic bradykinin in the presence of TNF-α (1000 ng/kg), but not IL-1β, by almost 80% below vehicle, again without effect on either MAP or HR. The renal sympatho-excitatory response to renal pelvic chemoreceptor stimulation is modulated by exogenous TNF-α and IL-1β. This suggests that inflammatory mediators within the kidney can play a significant role in modulating the renal afferent nerve-mediated sympatho-excitatory response.

The Effects of Volatile Anesthetics on Renal Sympathetic and Phrenic Nerve Activity during Acute Intermittent Hypoxia in Rats.

Coordinated activation of sympathetic and respiratory nervous systems is crucial in responses to noxious stimuli such as intermittent hypoxia. Acute intermittent hypoxia (AIH) is a valuable model for studying obstructive sleep apnea (OSA) pathophysiology, and stimulation of breathing during AIH is known to elicit long-term changes in respiratory and sympathetic functions. The aim of this study was to record the renal sympathetic nerve activity (RSNA) and phrenic nerve activity (PNA) during the AIH protocol in rats exposed to monoanesthesia with sevoflurane or isoflurane. Adult male Sprague-Dawley rats (n = 24; weight: 280-360 g) were selected and randomly divided into three groups: two experimental groups (sevoflurane group, n = 6; isoflurane group, n = 6) and a control group (urethane group, n = 12). The AIH protocol was identical in all studied groups and consisted in delivering five 3 min-long hypoxic episodes (fraction of inspired oxygen, FiO2 = 0.09), separated by 3 min recovery intervals at FiO2 = 0.5. Volatile anesthetics, isoflurane and sevoflurane, blunted the RSNA response to AIH in comparison to urethane anesthesia. Additionally, the PNA response to acute intermittent hypoxia was preserved, indicating that the respiratory system might be more robust than the sympathetic system response during exposure to acute intermittent hypoxia.

Inhibition and potentiation of the exercise pressor reflex by pharmacological modulation of TRPC6 in male rats.

We determined the role played by the transient receptor potential canonical 6 (TRPC6) channel in evoking the mechanical component of the exercise pressor reflex in male decerebrated Sprague-Dawley rats. TRPC6 channels were identified by quadruple-labelled (DiI, TRPC6, neurofilament-200 and peripherin) immunohistochemistry in dorsal root ganglion (DRG) cells innervating the triceps surae muscles (n=12). The exercise pressor reflex was evoked by statically contracting the triceps surae muscles before and after injection of the TRPC6 antagonist BI-749327 (n=11; 12μgkg-1 ) or SAR7334 (n=11; 7μgkg-1 ) or the TRPC6 positive modulator C20 (n=11; 18μgkg-1 ). Similar experiments were conducted while the muscles were passively stretched (n=8-12), a manoeuvre that isolated the mechanical component of the reflex. Blood pressure, tension, renal sympathetic nerve activity (RSNA) and blood flow were recorded. Of the DRG cells innervating the triceps surae muscles, 85% stained positive for the TRPC6 antigen, and 45% of those cells co-expressed neurofilament-200. Both TRPC6 antagonists decreased the reflex pressor responses to static contraction (-32 to -42%; P < 0.05) and to passive stretch (-35 to -52%; P < 0.05), whereas C20 increased these responses (55-65%; P < 0.05). In addition, BI-749327 decreased the peak and integrated RSNA responses to both static contraction (-39 to -43%; P < 0.05) and passive stretch (-56 to -62%; P < 0.05), whereas C20 increased the RSNA to passive stretch only. The onset latency of the decrease or increase in RSNA occurred within 2s of the onset of the manoeuvres (P < 0.05). Collectively, our results show that TRPC6 plays a key role in evoking the mechanical component of the exercise pressor reflex. KEY POINTS: The exercise pressor reflex plays a key role in the sympathetic and haemodynamic responses to exercise. This reflex is composed of two components, namely the mechanoreflex and the metaboreflex. The receptors responsible for evoking the mechanoreflex are poorly documented. A good candidate for this function is the transient receptor potential canonical 6 (TRPC6) channel, which is activated by mechanical stimuli and expressed in dorsal root ganglia of rats. Using two TRPC6 antagonists and one positive modulator, we investigated the role played by TRPC6 in evoking the mechanoreflex in decerebrated rats. Blocking TRPC6 decreased the renal sympathetic and the pressor responses to both contraction and stretch, the latter being a manoeuvre that isolates the mechanoreflex. In contrast, the positive modulator increased the pressor reflex to contraction and stretch, in addition to the sympathetic response to stretch. Our results provide strong support for a role played by the TRPC6 channel in evoking the mechanoreflex.

Inactivation of the paraventricular nucleus attenuates the cardiogenic sympathetic afferent reflex in the spontaneously hypertensive rat.

Myocardial ischemia causes the release of bradykinin, which stimulates cardiac afferents, causing sympathetic excitation and chest pain. Glutamatergic activation of the paraventricular hypothalamic nucleus (PVN) in the spontaneously hypertensive rat (SHR) drives elevated basal sympathetic activity. Thus, we tested the hypothesis that inactivation of the PVN attenuates the elevated reflex response to epicardial bradykinin in the SHR and that ionotropic PVN glutamate receptors mediate the elevated reflex. We recorded the arterial pressure and renal sympathetic nerve activity (RSNA) response to epicardial bradykinin application in anesthetized SHR and Wistar Kyoto (WKY) rats before and after PVN microinjection of GABA A agonist muscimol or ionotropic glutamate receptor antagonist kynurenic acid. Muscimol significantly decreased the arterial pressure response to bradykinin from 180.4 ± 5.8 to 119.5 ± 6.9 mmHg in the SHR and from 111.8 ± 7.0 to 84.2 ± 8.3 mmHg in the WKY and the RSNA response from 186.2 ± 7.1 to 142.7 ± 7.3% of baseline in the SHR and from 201.0 ± 11.5 to 160.2 ± 9.3% of baseline in the WKY. Kynurenic acid significantly decreased the arterial pressure response in the SHR from 164.5 ± 5.0 to 126.2 ± 7.7 mmHg and the RSNA response from 189.9 ± 13.7to 168.5 ± 12.7% of baseline but had no effect in the WKY. These results suggest that tonic PVN activity is critical for the full manifestation of the CSAR in both the WKY and SHR. Glutamatergic PVN activity contributes to the augmented CSAR observed in the SHR.

Neural discharge of muscle afferents and pressor response to mechanical stimulation are associated with muscle deformation velocity in rats.

Skeletal muscle reflexes play a crucial role in determining the magnitude of the cardiovascular response to exercise. However, evidence supporting an association between the magnitude of the pressor response and the velocity of muscle deformation has remained to be elucidated. Thus, we investigated the impact of different muscle deformation rates on the neural discharge of muscle afferents and pressor and sympathetic responses in Sprague-Dawley rats. In an ex vivo muscle-nerve preparation, action potentials elicited by sinusoidal mechanical stimuli (137 mN) at different frequencies (0.01, 0.05, 0.1, 0.2, and 0.25 Hz) were recorded in mechanosensitive group III and IV fibers. The afferent response magnitude to sine-wave stimulation significantly varied at different frequencies (ANOVA, P = 0.01). Specifically, as compared with 0.01 Hz (0.83 ± 0.96 spikes/s), the response magnitudes were significantly greater at 0.20 Hz (4.07 ± 5.04 spikes/s, P = 0.031) and 0.25 Hz (4.91 ± 5.30 spikes/s, P = 0.014). In an in vivo decerebrated rat preparation, renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) responses to passive stretch (1 kg) of hindlimb skeletal muscle at different velocities of loading (slow, medium, and fast) were measured. Pressor responses to passive stretch were significantly associated with the velocity of muscle deformation (ANOVA, P < 0.001). The MAP response to fast stretch (Δ 56 ± 12 mmHg) was greater than slow (Δ 33 ± 11 mmHg, P = 0.006) or medium (Δ 30 ± 11 mmHg, P < 0.001) stretch. Likewise, the RSNA response was related to deformation velocity (ANOVA, P = 0.024). These findings suggest that the muscle neural afferent discharge and the cardiovascular response to mechanical stimulation are associated with muscle deformation velocity.

Sex dependent attenuating effects of capsaicin administration on the mechanoreflex in healthy rats.

Stimulation of mechanically sensitive channels on the sensory endings of group III and IV thin fiber muscle afferents activates the mechanoreflex which contributes to reflex increases in sympathetic nerve activity (SNA) and blood pressure during exercise. Accumulating evidence suggests that activation of the non-selective cation channel transient receptor potential vanilloid-1 (TRPV1) on the sensory endings of thin fiber afferents with capsaicin may attenuate mechanosensation. However, no study has investigated the effect of capsaicin on the mechanoreflex. We tested the hypothesis that in male and female decerebrate, unanesthetized rats, the injection of capsaicin (0.05 µg) into the arterial supply of the hindlimb reduces the pressor and renal SNA (RSNA) response to 30s of 1 Hz rhythmic hindlimb muscle stretch (a model of isolated mechanoreflex activation). In male rats (n=8), capsaicin injection significantly reduced the integrated blood pressure (blood pressure index or BPI; pre: 363±78, post: 211±88 mmHg×s; P=0.023) and RSNA (∫ΔRSNA; pre: 687±206, post: 216±80 a.u., P=0.049) response to hindlimb muscle stretch. In female rats (n=8), capsaicin injection had no significant effect on the pressor (BPI; pre: 277±67; post: 207±77 mmHg×s; P=0.343) or RSNA (∫ΔRSNA pre: 697±123; post: 440±183 a.u.; P=0.307) response to hindlimb muscle stretch. The data suggest that the injection of capsaicin into the hindlimb arterial supply to stimulate TRPV1 on the sensory endings of thin fiber muscle afferents attenuates the mechanoreflex in healthy male, but not female, rats. The findings may carry important implications for chronic conditions in which an exaggerated mechanoreflex contributes to aberrant sympathoexcitation during exercise.

ARTERIAL BAROREFLEX FUNCTION IS IMPAIRED DURING THE RECOVERY FROM ACUTE LUNG INJURY IN A BLEOMYCIN-INDUCED RAT LUNG INJURY MODEL

Objective: Acute lung injury (ALI) and its clinical correlate, the acute respiratory distress syndrome (ARDS), results due, in part, to disruption of the normal capillary endothelial barrier and invokes perturbations of ventilatory control. ALI is associated with autonomic dysfunction and cardiac arrhythmias. In a recent study we suggested that rats were susceptible to cardiac arrhythmia even during the recovery from ALI, in part, due to abnormalities in cardiac sympathetic ganglia. On the other hand, if arterial baroreflex function is altered during the recovery of ALI remains unknown. The primary goal of this study was to examine baroreflex function during the recovery stage from ALI in a bleomycin (Bleo)-induced rat ALI model. Design and method: ALI was induced in male SD rats (200-250g) using a single intra-tracheal injection of Bleo (2.5 mg/kg). 4 weeks post Bleo (n = 7) or saline (sham, n = 6), rats were anesthetized with urethane (800 mg/kg ip) and alpha-chloralose (40 mg/kg ip) to measure baroreflex sensitivity. Baroreflex curves were generated by measuring the heart rate (HR) and renal sympathetic nerve activity (RSNA) responses to decreases and increases in mean blood pressure (MAP) by intravenous administration of nitroglycerin (25 μg) followed by phenylephrine (10 μg). Differences between sham and Bleo rats were determined by one-way ANOVA followed by the Tukey post hoc test. Results: Compared to sham rats, there was a significant decrease in the RSNA (117.4±11.6 vs. 77.5±8.9 %baseline, P = 0.018) or HR (147.5±9.7 vs. 75.1±11.3 bpm, P&lt;0.001) range in Bleo rats. Minimum RSNA or HR was also decreased in bleo rats compared to sham rats. There was no significant difference in either the slope coefficient or the blood pressure at the midpoint of the range (BP50) between the two groups. There was a significant decrease in peak slope [or maximum gain (Gainmax) in Bleo rats (MAP-RSNA Gainmax: 3.11±0.27 vs. 1.74±0.31, p = 0.017; MAP-HR Gainmax: 3.57±0.31 vs. 1.21±0.14, p&lt;0.001). Conclusions: Our data demonstrate impaired baroreflex function during the recovery state from ALI. Autonomic dysfunction may also contribute to increased cardiovascular risk during the recovery stage from ALI.

Sympathoexcitation and pressor responses induced by acetate, an ethanol metabolite in the hypothalamic paraventricular nucleus involves activation of NMDA receptors in rats

Alcohol abuse plays a crucial role in the prevalence of cardiovascular and neuronal degenerated diseases. However, the central mechanism that mediates these effects are not fully understood. It has been well know that autonomic hypothalamic paraventricular nucleus (PVN) play an important role in regulating sympathetic outflow and arterial blood pressure (ABP). Moreover, it has been demonstrated that the increased glutamatergic activity among PVN neurons contributed to the sympathoexcitation and development of hypertension. Furthermore, we have reported that ethanol and acetate increase sympathetic outflow and arterial pressure, which may involve the activation of NMDA receptors in autonomic central nucleus of amygdala. Combing with the fact that autonomic PVN neurons are abundantly expressed NMDA receptors, we hypothesize that acetate, the metabolic products from alcohol, activates the PVN neurons through the increase in glutamatergic activity and contributes to the sympathoexcitation and development of chronic diseases, such as hypertension. In anesthetized rats, the effect of acetate microinjected in the PVN on the renal sympathetic nerve activity (RSNA) and arterial blood pressure (ABP) was determined. The PVN acetate microinjection increased sympathoexcitatiory and pressor response in a dose dependent dose-dependent (1.5mm, 0.5mm, 2.0mm, 7.5mm) manner, and 2mm of acetate showed a minimum dose evoked a maximal response. To determine the role of acetate-stimulated glutamatergic receptor activation in driving sympathoexcitatory and pressor responses, either 2mm acetate or pre-treatment of Kynurenic acid (KYN, 7.2mm) ionotropic excitatory amino acid receptor blocker, or D-2-amino-5-phosphopentanoate (AP5, 3.0mm), a N-methyl-D-aspartate (NMDA) receptor antagonist, followed by 2mm acetate were microinjected into the PVN of male Sprague Dawley rats (300-500g, n= 6-9/ group). RSNA and ABP responses were compared among vehicle (saline) and 2mm acetate, pre-treatment of KYN and 2mm acetate, or pre-treatment AP5 and 2mm acetate protocols. 2mm acetate significantly increased RSNA (60-70% baseline, p &lt; 0.001) and mean arterial pressure (MAP, 10-12mmHg, p &lt; 0.05). Non-selective glutamatergic receptor antagonist, KYN significantly blocked the sympathoexcitatory (RSNA, p &lt; 0.05) and pressor response (MAP, p &lt; 0.05) evoked by 2mm acetate in the PVN. Furthermore, selective NMDA receptor antagonist, AP5 significantly attenuates the sympathoexcitatory response induced by PVN 2mm acetate (RSNA, P &lt; 0.05 and MAP, p &lt; 0.01). These data indicate acetate can increase glutamatergic activity and excitability of pre-sympathetic PVN neurons via activation of local NMDA receptors. The metabolism of ethanol to acetate following by alcohol consumption may contribute, in part, to the development of neurogenic hypertension and/or other cardiovascular diseases associated with increased sympathetic outflow. R15HL145655, R15HL150703 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Exercise augments small conductance Ca2+ -activated potassium channel (SK) function in the paraventricular nucleus (PVN) of Sprague Dawley rats to reduce sympathetic outflow

Elevated sympathetic outflow is a key feature of cardiovascular disease (CVD) that worsens disease progression. Our lab has shown that SK channels expressed in the PVN play a crucial role in regulating neuronal activity and sympathetic outflow, and that SK channels become dysfunctional in rats fed a high salt diet. Exercise has been shown to be an effective treatment for reducing sympathoexcitation in CVD including hypertension and heart failure, but the underlying mechanisms are not fully understood. We hypothesized that aerobic exercise would upregulate SK channel function in the PVN to reduce sympathetic nerve activity (SNA). To test this, 5–6 week old Sprague Dawley rats were randomly divided into sedentary (SED) and exercise (EXT) two groups and fed a 0.4% NaCl normal salt diet. Following acclimation, EXT groups ran on a motorized treadmill 5 days/week for 8-10 weeks. Conscious blood pressure was measured weekly via tail plethysmography. After 8-10 weeks, animals were anesthetized and underwent in vivo surgery to record the renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) following PVN microinjection of the SK channel blocker, apamin (0.25mM, 60nL/side). The data showed that the RSNA response to PVN apamin was significantly enhanced in EXT rats compared with SED rats (320.8 ± 174.6 % baseline, n=9 vs 184.8 ± 143.1 % baseline, n=9; p = 0.02). The corresponding ABP response to apamin was not significantly different in EXT rats compared with SED rats (20.40 ± 9.98 mmHg, n=9 vs 25.27 ± 9.97 mmHg, n=8; p = 0.1658). Our data indicates exercise enhances PVN SK channel function to reduce sympathetic outflow. This improvement of SK channel function may be one mechanism by which exercise reduces SNA in CVD including hypertension and heart failure. Support: 1R15HL145655 (Chen); 1R15 HL150703 (Shan); MTU Health Research Institute. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Antagonism of TRPV4 channels partially reduces mechanotransduction in rat skeletal muscle afferents.

Mechanical distortion of working skeletal muscle induces sympathoexcitation via thin fibre afferents, a reflex response known as the skeletal muscle mechanoreflex. However, to date, the receptor ion channels responsible for mechanotransduction in skeletal muscle remain largely undetermined. Transient receptor potential vanilloid 4 (TRPV4) is known to sense mechanical stimuli such as shear stress or osmotic pressure in various organs. It is hypothesized that TRPV4 in thin-fibre primary afferents innervating skeletal muscle is involved in mechanotransduction. Fluorescence immunostaining revealed that 20.1 ± 10.1% of TRPV4 positive neurons were small dorsal root ganglion (DRG) neurons that were DiI-labelled, and among them 9.5 ± 6.1% of TRPV4 co-localized with the C-fibre marker peripherin. In vitro whole-cell patch clamp recordings from cultured rat DRG neurons demonstrated that mechanically activated current amplitude was significantly attenuated after the application of the TRPV4 antagonist HC067047 compared to control (P=0.004). Such reductions were also observed in single-fibre recordings from a muscle-nerve ex vivo preparation where HC067047significantly decreased afferent discharge to mechanical stimulation (P=0.007). Likewise, in an in vivo decerebrate rat preparation, the renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) responses to passive stretch of hindlimb muscle were significantly reduced by intra-arterial injection of HC067047 (ΔRSNA: P=0.019, ΔMAP: P=0.002). The findings suggest that TRPV4 plays an important role in mechanotransduction contributing to the cardiovascular responses evoked by the skeletal muscle mechanoreflex during exercise. KEY POINTS: Although a mechanical stimulus to skeletal muscle reflexively activates the sympathetic nervous system, the receptors responsible for mechanotransduction in skeletal muscle thin fibre afferents have not been fully identified. Evidence suggests that TRPV4 is a mechanosensitive channel that plays an important role in mechanotransduction within various organs. Immunocytochemical staining demonstrates that TRPV4 is expressed in group IV skeletal muscle afferents. In addition, we show that the TRPV4 antagonist HC067047 decreases the responsiveness of thin fibre afferents to mechanical stimulation at the muscle tissue level as well as at the level of dorsal root ganglion neurons. Moreover, we demonstrate that intra-arterial HC067047 injection attenuates the sympathetic and pressor responses to passive muscle stretch in decerebrate rats. These data suggest that antagonism of TRPV4 attenuates mechanotransduction in skeletal muscle afferents. The present study demonstrates a probable physiological role for TRPV4 in the regulation of mechanical sensation in somatosensory thin fibre muscle afferents.

Paraventricular Nucleus V1a Receptor Knockdown Blunts Neurocardiovascular Responses to Acute Stress in Male Rats after Chronic Mild Unpredictable Stress

Chronic stress and depression impart increased risk for adverse cardiovascular events. Autonomic dysregulation, particularly sympathoexcitation, has long been associated with poor cardiovascular outcomes. Vasopressin (AVP) receptors with the paraventricular nucleus (PVN), known as an integrating locus for hemodynamic and autonomic function, have been implicated in behavior and stress. The present studies were designed to test the hypothesis that knockdown of vasopressin V1aR within the PVN in male Sprague Dawley rats subjected to chronic mild unpredictable stress (CMS) would result in lower resting hemodynamics and renal sympathetic nerve activity (RSNA) and mitigate the responses to acute stressors. Male rats underwent CMS for 4 weeks; controls were housed in standard caging. Twenty days into the paradigm, the PVN was injected with either small interfering RNA (siRNA) directed against V1aR or scrambled RNA (scrRNA). Arterial pressure, heart rate and RSNA were ascertained by telemetry with the animals in their home cages. Pretreatment with siRNA to V1aR prevented the increase in arterial pressure to PVN microinjection with exogenous AVP. Basal mean arterial pressure (MAP) was significantly higher in scrRNA-treated but not in siRNA-treated CMS rats vs control rats. Paradoxically, basal RSNA was approximately two-fold higher in siRNA-treated CMS rats. Acute emotional stress delivered as 15-sec air-jet resulted in greater peak and duration of the MAP and RSNA responses in scrRNA-treated CMS rats vs control; siRNA treatment inhibited the responses. The 15-sec exposure to ammonia to test the nasopharyngeal reflex, whose circuitry does not include the PVN, produced similar increases in arterial pressure, heart rate, and RSNA in controls and both groups of CMS rats. Thus, CMS increases arterial pressure and predisposes to greater hemodynamic and RSNA responses to acute emotional stress. The higher basal RSNA in siRNA-treated rats may be due to functional and/or anatomical neuroplasticity occurring during more protracted inhibition of V1aR PVN signaling. Vasopressinergic signaling via V1aR in PVN modulates the cardiovascular and sympathetic responses to both the chronic and acute stress.

Impact of Selective Renal Afferent Denervation on Oxidative Stress and Vascular Remodeling in Spontaneously Hypertensive Rats.

Oxidative stress and sustained sympathetic over-activity contribute to the pathogenesis of hypertension. Catheter-based renal denervation has been used as a strategy for treatment of resistant hypertension, which interrupts both afferent and efferent renal fibers. However, it is unknown whether selective renal afferent denervation (RAD) may play beneficial roles in attenuating oxidative stress and sympathetic activity in hypertension. This study investigated the impact of selective RAD on hypertension and vascular remodeling. Nine-week-old normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) were subjected to selective renal afferent denervation (RAD) with 33 mM of capsaicin for 15 min. Treatment with the vehicle of capsaicin was used as a control. The selective denervation was confirmed by the reduced calcitonin gene-related peptide expression and the undamaged renal sympathetic nerve activity response to the stimulation of adipose white tissue. Selective RAD reduced plasma norepinephrine levels, improved heart rate variability (HRV) and attenuated hypertension in SHR.It reduced NADPH oxidase (NOX) expression and activity, and superoxide production in the hypothalamic paraventricular nucleus (PVN), aorta and mesenteric artery of SHR. Moreover, the selective RAD attenuated the vascular remodeling of the aorta and mesenteric artery of SHR. These results indicate that selective removal of renal afferents attenuates sympathetic activity, oxidative stress, vascular remodeling and hypertension in SHR. The attenuated superoxide signaling in the PVN is involved in the attenuation of sympathetic activity in SHR, and the reduced sympathetic activity at least partially contributes to the attenuation of vascular oxidative stress and remodeling in the arteries of hypertensive rats.

Intramuscular insulin administration potentiates sympathetic and pressor responses to capsaicin in rats

Insulin centrally activates the sympathetic nervous system. However, little is known about the effects of insulin on peripheral primary sensory neurons. Transient receptor potential vanilloid 1 (TRPV1), of which capsaicin (CAP) is an agonist, is widely expressed in small dorsal root ganglia (DRG) neurons subserving skeletal muscle thin‐fiber afferents expressing sensory nociceptors and/or metabolically sensitive skeletal muscle receptors. Evidence suggests that insulin increases the sensitivity of these sensory receptors as well as translocation of TRPV1 from cytosol to plasma membrane in DRG neurons. We therefore hypothesized that insulin potentiates the responsiveness of thin fiber muscle afferents to TRPV1 activation.PurposeThe aim of this investigation was 1) to examine whether TRPV1 and insulin receptors (IR) are co‐localized in small‐diameter (&lt; 30 µm) DRG neurons subserving thin‐fiber afferents (C‐fibers or A‐delta fibers), and 2) to determine the effects of insulin on renal sympathetic nerve activity (RSNA) and mean arterial blood pressure (MAP) responses to stimulation of TRPV1 by injection of CAP into the arterial supply of the hindlimb in decerebrated rats.MethodsL4‐L6 DRGs were harvested from Sprague‐Dawley rats (n=3, 19 weeks, body weight: 518 ± 6 g). The percentage of neurons that expressed IR and/or isolectin B4 binding (IB4, a C‐fiber neuronal marker) in TRPV1 positive small DRGs neurons were assessed. RSNA and MAP responses to intra‐arterial administration of CAP (0.3 µg/100 µL) were measured in decerebrate Sprague‐Dawley rats (10 ± 1 weeks, body weight: 330 ± 43 g) before and after intramuscular injection of either insulin (5 U/mL, 30 µL) or saline (vehicle control, 30 µL). To avoid insulin/saline spill over into the systemic circulation, a vascular occluder placed around the abdominal aorta was inflated immediately prior to intramuscular administration of insulin or saline.ResultsImmunohistochemical staining demonstrated that 22 % of TRPV1 positive DRG neurons expressed IR and IB4 binding. Intramuscular insulin administration did not change blood glucose as well as plasma insulin levels assessed from the jugular vein. The RSNA response to CAP after insulin administration was significantly greater than post injection of vehicle (vehicle: 25 ± 24, n=9, vs. insulin: 54 ± 32 %, n=8, P=0.04). While the MAP response to CAP was not significantly changed by vehicle injection (before: 26 ± 13 vs. after: 24 ± 12 mmHg, n=18), insulin administration significantly increased MAP responsiveness (before: 24 ± 1 vs. after: 29 ± 15 mmHg, n=22, P=0.01).ConclusionThe data demonstrate that 1) TRPV1 and IR co‐localize on small DRG neurons and 2) intramuscular insulin administration significantly augments sympathetic and pressor responses to chemical stimulation of skeletal muscle afferents. These findings suggest that insulin potentiates TRPV1 responsiveness at the muscle tissue level, possibly contributing to insulin‐induced sympathoexcitation.

Renal Sympathetic Nerve Activity Responses to Hypoxia and Hypercapnia in Rats Exposed to Chronic Intermittent Hypoxia

Increased responsiveness of the carotid bodies to acute hypoxia was shown in animals exposed to chronic intermittent hypoxia (CIH). The kidney is sensitive to hypoxia and decreased cortical partial pressure of oxygen (PO2) levels was revealed in rats exposed to moderate CIH. We hypothesized that the response of the renal sympathetic nerve activity (RSNA) to acute hypoxia and hypercapnia is enhanced in rats exposed to CIH. Wistar rats were exposed to 96 cycles of CIH (6% O2, 8 hr/day, n=6) or to normoxia (sham, 21% O2, n=9) for 14 days. On day 15, rats were anesthetized (induction: urethane, α‐chloralose and sodium pentobarbitone mixture i.p. (416, 27 and 33mg/kg), maintenance: urethane and α‐chloralose mixture i.v. (62 and 4mg/kg)) to record cardiovascular parameters and RSNA. Rats were provided with supplementary oxygen to maintain PO2 levels &gt; 84 mmHg. During hypoxia, rats breathed 10% O2for two minutes. This was followed with a hypercapnic challenge during which rats breathed 10% CO2 for 5 minutes. Following a 90‐minute recovery period, capsaizepine (CPZ, 5µg/ml/hr), a TRPV1 blocker, was infused into the right kidney. Then, hypoxic and hypercapnic challenges were repeated following the intra‐renal infusion of CPZ. Data are expressed as mean±SD and were analyzed using t‐test or ANOVA, where relevant. CIH rats were hypertensive (130±9 vs. 118±9mmHg, p=0.039) with comparable basal HR (425±38 vs. 407±34bpm), and RSNA (% of max, 33±12 vs. 25±12) compared with sham rats. In response to acute hypoxia, RSNA was increased to an equivalent extent in CIH‐exposed and sham rats (Maximum change in RSNA of baseline, 128±23 vs. 125±16%; RSNA (% of max), 36±7 vs. 44±22%). Additionally, the increase in HR in response to acute hypoxia was similar between CIH‐exposed and sham rats (∆HR, +42±29 vs. +30±27bpm). Mean arterial blood pressure (MAP) was similarly decreased in CIH‐exposed and sham rats during acute hypoxia (Maximum change in MAP, ‐33±24 vs. ‐27±17mmHg). The decrease in PaO2 levels during hypoxia was comparable between exposure groups (∆PaO2, ‐48±15 vs. ‐47±10mmHg). Hypercapnia induced a comparable increase in the RSNA in CIH‐exposed and sham rats (Maximum change in RSNA of baseline, +121±15 vs. +113±8%). The analysis of absolute RSNA when normalized to maximum RSNA showed that CIH‐exposed rats had increased RSNA compared with sham rats before and after exposure to hypercapnia (RSNA (% of max), 37±8 vs. 26±10%, p=0.037). The increase in PaCO2 levels during hypercapnia was similar between CIH and sham groups (∆PaCO2, +24±8 vs. +19±6mmHg). Intra‐renal CPZ did not alter the increase in RSNA and HR in response to hypoxia and hypercapnia in CIH‐exposed and sham rats. Protein expression of TRPV1 receptors in kidney tissue was equivalent in both exposure groups (1.88±0.23 vs. 1.68±0.26ng/mg). Our results indicate that the responsiveness of RSNA to acute hypoxia and hypercapnia is not altered following exposure to moderate CIH sufficient to increase RSNA and blood pressure.

Protein Kinase C Epsilon Contributes to the Exaggerated Mechanoreflex in Rats with Heart Failure

BackgroundDuring exercise, skeletal muscle contraction stimulates mechanically sensitive channels on the sensory endings of thin fiber muscle afferents. Stimulation of these mechanically sensitive channels initiates a reflex, termed the mechanoreflex, which contributes importantly to reflex increases in sympathetic nerve activity (SNA) and mean arterial blood pressure (MAP). In patients and animals with heart failure with reduced ejection fraction (HF‐rEF), mechanoreflex activation contributes to exaggerated increases in SNA and is linked to decreased exercise tolerance and increased mortality. The exaggerated mechanoreflex in HF‐rEF is partially attributed to an increased responsiveness of thin fiber skeletal muscle afferents to mechanical stimulation. Several lines of evidence demonstrate that signaling within sensory neurons mediated by the non‐traditional protein kinase c epsilon (PKCε) isoform augments mechanically activated channel function and produces mechanical hyperalgesia. Whether PKCε‐mediated signaling within sensory neurons contributes the exaggerated mechanoreflex in HF‐rEF is unknown.Purpose &amp; HypothesisWe determined the role played by PKCε within sensory endings of thin fiber muscle afferents in evoking the mechanoreflex in rats with HF‐rEF and healthy control rats. We tested the hypothesis that injection of the selective PKCε inhibitor PKCε141 (44μg) into the arterial supply of the hindlimb would reduce the reflex increase in renal SNA (RSNA) and MAP evoked during 30 seconds of 1 Hz dynamic hindlimb muscle stretch (a model of mechanoreflex activation) in HF‐rEF rats, but not in healthy control rats.MethodsExperiments were performed on Sprague‐Dawley rats subjected to coronary artery ligation surgery to produce myocardial infarction and HF‐rEF or a sham ligation. At least six weeks after the initial surgery, in decerebrate, unanesthetized rats we compared (Student’s t‐tests) the mechanoreflex evoked by hindlimb skeletal muscle stretch before and after injection of PKCε141 into the arterial supply of the hindlimb.ResultsEjection fraction (determined via echocardiography) was significantly reduced in HF‐rEF rats (48±1%) compared to sham rats (80±2%, P&lt;0.01). Intra‐arterial PKCε141 injection reduced the pressor and RSNA response to hindlimb muscle stretch in HF‐rEF rats (n=6, 4M/2F, peak ΔMAP pre: 34±8; post: 19±7 mmHg; P=0.04; peak ΔRSNA pre: 121±32; post: 70±24%; P=0.04), but not SHAM rats (n=4, 3M/1F, peak ΔMAP pre: 14±4; post: 14±3 mmHg; P=0.87; peak ΔRSNA pre: 62±32; post: 58±26%; P=0.80) rats. In control experiments (n=3 HF‐rEF rats, 2M/1F), injection of PKCε141 into the jugular vein had no effect on the pressor or RSNA response to muscle stretch demonstrating a lack of central nervous system effects.ConclusionsThese data suggest that that PKCε signaling within the sensory neuron endings contributes to the exaggerated mechanoreflex in HF‐rEF and enhance the understanding of the sympathetic and cardiovascular adjustments to exercise in the ~6 million Americans with HF‐rEF.

Exercise Training Upregulates SK+ Channel Function in the Hypothalamic Paraventricular Nucleus (PVN) of Animals

Increased sympathetic outflow is a hallmark of cardiovascular disease (CVD). Previously, our lab demonstrated that small conductance Ca2+ activated K channels (SK+) expressed in the PVN play a crucial role in regulating sympathetic outflow, and SK+ currents were significantly decreased in rats with salt‐sensitive hypertension (SSH). Since SK+ channels among autonomic PVN neurons play a crucial role in regulating sympathetic outflow in SSH, antihypertensive therapies targeted at upregulating SK+ channel expression in the PVN should be pursued. Exercise is a powerful, non‐pharmacological intervention to treat CVD, however, the interaction between SK+ channels among the hypothalamic PVN and exercise is unknown. The objective is to determine the role of exercise in regulating SK+ channels function and sympathetic outflow as well as arterial blood pressure (ABP) in animals. We hypothesize that exercise upregulates SK+ channel function in the PVN and attenuates sympathetic outflow. To test this hypothesis, Sprague Dawley (SD) rats are divided into sedentary (SED) and exercise (EXT) groups based on their willingness to run on a motorized treadmill. Exercise intensity is determined through a maximum exercise capacity test and rats run at 70% of their exercise capacity speed for 30 minutes 5 days/week for 8‐10 weeks. Then rats are anesthetized and renal sympathetic nerve activity (RSNA) and ABP are recorded following bilateral PVN injections of apamin, a selective SK+ channel blocker (0.25mM, 60nL/side). We found that the RSNA response to PVN apamin trended toward significance in EXT rats compared with SED rats (342.1 ± 164.2 % baseline, n=6 vs 120.9 ± 25.1 % baseline, n=3; p = 0.0598). The corresponding ABP response to apamin was not significantly different in EXT rats compared with SED rats (16.5 ± 5.7 mmHg, n=6 vs 24.6 ± 7.2, n=3; p = 0.1039). Collectively, these results indicate exercise activates PVN SK+ channels function and reduces sympathetic outflow. The improvement of channel function or increase in expression may contribute to the beneficial effects of exercise on reducing sympathetic outflow in patients with CVD, including hypertension.

Exposure to Repeated Cold Stress Influences Sympathetic and Cardiovascular Responses to Muscle Stretch in Decerebrated Rats

Chronic stress is known to cause adverse physical and mental effects such as pain, chronic fatigue, and depression, and it is strongly related to many diseases and syndromes (e.g., fibromyalgia, chronic fatigue syndrome, and post‐traumatic stress disorder). Evidence suggests that exercise and physical activity in leisure time are effective in improving stress‐induced symptoms. Repeated cold stress (RCS), in which an animal is repeatedly exposed to alternating room and low temperatures, is one of the chronic stress models that induces chronic pain and depression. Mechanical hyperalgesia through thin muscle afferent fibers has also been reported to occur from RCS exposure. Since the muscle afferents have a dual modulatory function in nociception and cardiovascular reflex, we hypothesized that RCS augments the mechanical component of the exercise pressor reflex (the skeletal muscle mechanoreflex), i.e., skeletal muscle afferents‐mediated increases sympathetic nerve activity (SNA) and arterial blood pressure (AP).[Purpose]The purpose of this study was to clarify the impacts of RCS on sympathetic and cardiovascular responses to stimulation of the skeletal muscle mechanoreflex in decerebrated rats.[Methods]Male Sprague‐Dawley rats (body weight: 410 ± 15 g, age: 12 weeks) were exposed to RCS using a homemade automated RCS device. The rats were alternately moved to room temperature (22°C) and cold temperature (4°C) compartments at 30‐min intervals for 5 days. To assess the skeletal muscle mechanoreflex function, we measured mean AP (MAP), heart rate (HR), and renal SNA (RSNA) responses to 30‐s static passive stretching of the hindlimb muscles by in vivo recording from unanesthetized decerebrated rats. Stretching was performed by tracing the maximum tension curve obtained from 30‐s electrical stimulation. Changes (Δ) from baseline to peak values of the measured parameters between control and RCS rats were compared.[Results]Peak tension during the Achilles tendon stretch from baseline was not significantly different between groups (control: Δ929 ± 63 g [n = 6] vs RCS: Δ765 ± 66 g [n = 4], p = 0.17). Importantly, the peak RSNA response in RCS rats (Δ236.3% and Δ242.7%, n = 2) tended to be greater than that in control rats (Δ37.1% and Δ23.9%, n = 2). Moreover, RCS had a significantly (p &lt; 0.05) greater HR response (RCS: Δ3.7 ± 1.0 bpm [n = 4] vs control: Δ0.6 ± 0.5 bpm [n = 6]), and MAP response tended (P = 0.07) to be higher in the RCS group (Δ23 ± 6 mmHg, n = 4) than in the control group (Δ8 ± 4 mmHg, n = 6).[Conclusions]Our preliminary data demonstrate that RCS augments the skeletal muscle mechanoreflex. These results suggest that chronic stress can potentially cause exaggerated cardiovascular responses during physical activity.

Dapagliflozin Attenuates Sympathetic and Pressor Responses to Stress in Young Prehypertensive Spontaneously Hypertensive Rats

Sodium‐glucose cotransporter 2 inhibitor (SGLT2i) is a class of anti‐diabetic medications approved for lowering blood glucose in type 2 diabetes mellitus (T2DM) and reducing cardiovascular risks in heart failure patients with or without T2DM. SGLT2i is also known to reduce blood pressure (BP) in hypertensive patients with T2DM. Mechanisms underlying these benefits of SGLT2i are unclear, but sympathetic overactivation is known to play a major role in the pathogenesis and progression of hypertension and heart failure. Whether SGLT2i reduces sympathetic nerve activity (SNA) and BP in a non‐diabetic prehypertension model is undetermined. Therefore, we conducted studies to determine effects of a SGLT2i, dapagliflozin (DAPA), on conscious BP as well as SNA in prehypertensive spontaneously hypertensive rats (SHRs) during physical stress. Accordingly, we randomly assigned 4‐week‐old non‐diabetic SHRs to either a normal powdered food treated (control) group or a normal powdered food containing DAPA treated (0.5mg/kg/day) group. After 4 weeks on the diet, we measured conscious BP in both groups of rats using radiotelemetry. We also assessed changes in mean arterial pressure (MAP) and renal SNA (RSNA) in response to hindlimb muscle contraction (ie, activation of the exercise pressor reflex, EPR), passive muscle stretch (ie, activation of the mechanically sensitive component of the EPR, known as the mechanoreflex), and intra‐arterial capsaicin injection (ie, activation of the metabolically sensitive component of the EPR, known as the metaboreflex) in a separate group of decerebrate control and DAPA SHRs. We found that fasting blood glucose levels were not significantly different between DAPA and control groups (65±27 vs. 78±20 mg/dL). Heart weight and heart weight‐to‐tibial length was significantly (all P&lt;0.01) decreased by 19% and 16%, respectively in DAPA rats compared to control rats. We also found that daytime/nighttime MAP were markedly lower in DAPA SHRs than control SHRs (daytime: 120±5 vs. 130±6 mmHg, nighttime: 123±6 vs. 132±6 mmHg, P&lt;0.05). A significant reduction in HR in DAPA rats was also observed compared to control rats (daytime: 299±15 vs. 337±20 bpm, nighttime: 320±12 vs. 357±12 bpm, P&lt;0.01). Similarly, the rises in MAP and RSNA during EPR activation were significantly attenuated in DAPA SHRs compared to control SHRs (ΔMAP: 10±5 vs. 29±15 mmHg, ΔRSNA: 43±17 vs. 96±48 %, P&lt;0.05). The pressor and sympathetic responses to mechanoreflex stimulation were also markedly lower in DAPA than control (ΔMAP: 10±7 vs. 25±14 mmHg, ΔRSNA: 31±17 vs. 68±39 %, P&lt;0.05). There was a tendency for MAP and RSNA responses to metaboreflex activation to be lower in the DAPA group, but the difference did not reach statistical significance (ΔMAP: 38±18 vs. 58±24 mmHg, P=0.07, ΔRSNA: 58±29 vs. 95±45 %, P=0.08). In conclusion, our data demonstrate a novel role for SGLT2i in reducing resting BP as well as the activity of skeletal muscle reflexes, independent of blood glucose. Our study may have important clinical implications for preventing hypertension and related cardiovascular damage in young prehypertensive individuals.

Insulin potentiates the response to capsaicin in dorsal root ganglion neurons in vitro and muscle afferents ex vivo in normal healthy rodents.

Systemic insulin administration evokes sympathoexcitatory actions, but the mechanisms underlying these observations are unknown. We reported that insulin sensitizes the response of thin-fibre primary afferents, as well as the dorsal root ganglion (DRG) that subserves them, to mechanical stimuli. However, little is known about the effects of insulin on primary neuronal responses to chemical stimuli. TRPV1, whose agonist is capsaicin (CAP), is widely expressed on chemically sensitive metaboreceptors and/or nociceptors. The aim of this investigation was to determine the effects of insulin on CAP-activated currents in small DRG neurons and CAP-induced action potentials in thin-fibre muscle afferents of normal healthy rodents. Additionally, we investigated whether insulin potentiates sympathetic nerve activity (SNA) responses to CAP. In whole-cell patch-clamp recordings from cultured mice DRG neurons in vitro, the fold change in CAP-activated current from pre- to post-application of insulin (n=13) was significantly (P<0.05) higher than with a vehicle control (n=14). Similar results were observed in single-fibre recording experiments ex vivo as insulin potentiated CAP-induced action potentials compared to vehicle controls (n=9 per group, P<0.05). Furthermore, insulin receptor blockade with GSK1838705 significantly suppressed the insulin-induced augmentation in CAP-activated currents (n=13) as well as the response magnitude of CAP-induced action potentials (n=9). Likewise, the renal SNA response to CAP after intramuscular injection of insulin (n=8) was significantly (P<0.05) greater compared to vehicle (n=9). The findings suggest that insulin potentiates TRPV1 responsiveness to CAP at the DRG and muscle tissue levels, possibly contributing to the augmentation in sympathoexcitation during activities such as physical exercise. KEY POINTS: Evidence suggests insulin centrally activates the sympathetic nervous system, and a chemical stimulus to tissues activates the sympathetic nervous system via thin fibre muscle afferents. Insulin is reported to modulate putative chemical-sensitive channels in the dorsal root ganglion neurons of these afferents. In the present study, it is demonstrated that insulin potentiates the responsiveness of thin fibre afferents to capsaicin at muscle tissue levels as well as at the level of dorsal root ganglion neurons. In addition, it is demonstrated that insulin augments the sympathetic nerve activity response to capsaicin in vivo. These data suggest that sympathoexcitation is peripherally mediated via insulin-induced chemical sensitization. The present study proposes a possible physiological role of insulin in the regulation of chemical sensitivity in somatosensory thin fibre muscle afferents.

In silico trial of baroreflex activation therapy for the treatment of obesity-induced hypertension.

Clinical trials evaluating the efficacy of chronic electrical stimulation of the carotid baroreflex for the treatment of hypertension (HTN) are ongoing. However, the mechanisms by which this device lowers blood pressure (BP) are unclear, and it is uncertain which patients are most likely to receive clinical benefit. Mathematical modeling provides the ability to analyze complicated interrelated effects across multiple physiological systems. Our current model HumMod is a large physiological simulator that has been used previously to investigate mechanisms responsible for BP lowering during baroreflex activation therapy (BAT). First, we used HumMod to create a virtual population in which model parameters (n = 335) were randomly varied, resulting in unique models (n = 6092) that we define as a virtual population. This population was calibrated using data from hypertensive obese dogs (n = 6) subjected to BAT. The resultant calibrated virtual population (n = 60) was based on tuning model parameters to match the experimental population in 3 key variables: BP, glomerular filtration rate, and plasma renin activity, both before and after BAT. In the calibrated population, responses of these 3 key variables to chronic BAT were statistically similar to experimental findings. Moreover, blocking suppression of renal sympathetic nerve activity (RSNA) and/or increased secretion of atrial natriuretic peptide (ANP) during BAT markedly blunted the antihypertensive response in the virtual population. These data suggest that in obesity-mediated HTN, RSNA and ANP responses are key factors that contribute to BP lowering during BAT. This modeling approach may be of value in predicting BAT responses in future clinical studies.