Electrical carotid sinus nerve stimulation attenuates experimental colitis induced by acetic acid in rats.

AIMS: The carotid bodies are sensors that detect physiological signals and convey them to the central nervous system, where the stimuli are processed inducing reflexes through efferent pathways. Recent studies have demonstrated that electrical stimulation of the carotid sinus nerve (CSN) triggers the anti-inflammatory reflex under different conditions. However, whether this electrical stimulation attenuates colitis was never examined. This study aimed to evaluate if the electrical CSN stimulation attenuates the experimental colitis induced by intrarectal administration of acetic acid in rats. METHODS: Electrodes were implanted around the CSN to stimulate the CSN, and a catheter was inserted into the left femoral artery to record the arterial pressure. The observation of hypotensive responses confirmed the effectiveness of the electrical CNS stimulation. This maneuver was followed by a 4 % acetic acid or saline administered intrarectally. After 24 h, colons were segmented into distal and proximal parts for macroscopy, histological and biochemical assessment. KEY FINDINGS: As expected, the electrical CSN stimulation was effective in decreasing arterial pressure in saline and colitis rats. Moreover, electrical CSN stimulation effectively reduced colonic tissue lesions, colitis scores, and histopathologic parameters associated with colitis. In addition, the CSN stimulation also reduced the colonic mucosa pro-inflammatory cytokine interleukin-1 beta, and increased the anti-inflammatory interleukin-10, in rats submitted to colitis. SIGNIFICANCE: These findings indicated that electrical CSN stimulation breaks the vicious cycle of local colon inflammation in colitis, which might contribute to its better outcome.

Autonomic Regulation of Inflammation in Conscious Animals.

Bioelectronic medicine is a novel field in modern medicine based on the specific neuronal stimulation to control organ function, cardiovascular, and immune homeostasis. However, most studies addressing neuromodulation of the immune system have been conducted on anesthetized animals, which can affect the nervous system and neuromodulation. Here, we review recent studies involving conscious experimental rodents (rats and mice) to better understand the functional organization of neural control of immune homeostasis. We highlight typical experimental models of cardiovascular regulation, such as electrical activation of the aortic depressor nerve or the carotid sinus nerve, bilateral carotid occlusion, the Bezold-Jarisch reflex, and intravenous administration of the bacterial endotoxin lipopolysaccharide. These models have been used to investigate the relationship between neuromodulation of the cardiovascular and immune systems in conscious rodents (rats and mice). These studies provide critical information about the neuromodulation of the immune system, particularly the role of the autonomic nervous system, i.e., the sympathetic and parasympathetic branches acting both centrally (hypothalamus, nucleus ambiguus, nucleus tractus solitarius, caudal ventrolateral medulla, and rostral ventrolateral medulla), and peripherally (particularly spleen and adrenal medulla). Overall, the studies in conscious experimental models have certainly highlighted to the reader how the methodological approaches used to investigate cardiovascular reflexes in conscious rodents (rats and mice) can also be valuable for investigating the neural mechanisms involved in inflammatory responses. The reviewed studies have clinical implications for future therapeutic approaches of bioelectronic modulation of the nervous system to control organ function and physiological homeostasis in conscious physiology.

Short-term effect of ligature-induced periodontitis on cardiovascular variability and inflammatory response in spontaneously hypertensive rats.

BACKGROUND: We previously reported that periodontal disease (PD) induces high arterial pressure variability (APV) consistent with sympathetic overactivity and elicits myocardial inflammation in Balb/c mice. However, it is unknown whether PD can change APV and heart rate variability (HRV) in spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. This study aimed to evaluate the hemodynamic level, HRV, and APV associating with myocardial inflammation and plasma concentrations of oxide nitric (NO) in SHR and WKY rats with PD. METHODS: Three weeks after bilateral ligation of the first mandibular molar, or Sham operation, the rats received catheters into the femoral artery and had their arterial pressure (AP) recorded the following day. Subsequently, plasma, heart, and jaw were collected. The NO was quantified by the chemiluminescence method in plasma, and the myocardial IL-1ß concentrations were evaluated by ELISA. In the jaw was evaluated linear alveolar bone loss induced by PD. RESULTS: The linear alveolar bone loss in jaws of SHR with PD was higher than in all other groups. AP and heart rate were higher in SHR than in their WKY counterparts. SHR with PD showed lower AP than control SHR. HRV and APV were different between SHR and WKY rats; however, no differences in these parameters were found between the animals with PD and their control counterparts. Plasma NO and myocardial IL-1ß concentrations were higher in SHR with PD as compared to control WKY. A significant correlation was found between linear alveolar bone loss and plasma NO and myocardial IL-1ß concentrations. CONCLUSION: Our results demonstrated that short-term PD lowered the AP in SHR, which might be due to the higher levels of plasma NO. Even though PD did not affect either HRV or APV, it did induce myocardial inflammation, which can determine cardiovascular dysfunction in long-term PD.

The Bezold-Jarisch Reflex and The Inflammatory Response Modulation in Unanesthetized Endotoxemic Rats.

Evidence indicates that the activation of the parasympathetic branch of the autonomic nervous system may be effective in treating inflammatory diseases. Previously, we have described that baroreflex activation displays anti-inflammatory properties. Analogous to the baroreflex, the Bezold-Jarisch reflex also promotes parasympathetic activation with simultaneous inhibition of the sympathetic system. Thus, the present study aimed to evaluate whether the activation of the Bezold-Jarisch reflex would also have the ability to reduce inflammation in unanesthetized rats. We used lipopolysaccharide (LPS) injection (5mg/kg, i.p.) to induce systemic inflammation in male Wistar Hannover rats and phenylbiguanide (PBG) administration (5µg/kg, i.v.) to activate the Bezold-Jarisch reflex. Spleen, heart, hypothalamus, and blood samples were collected to determine the levels of cytokines. Compared to baseline, PBG reduced the arterial pressure (115±2 vs. 88±5mmHg) and heart rate (380±7 vs. 114±26bpm), immediately after its administration, confirming the activation of the parasympathetic system and inhibition of the sympathetic system. From the immunological point of view, the activation of the Bezold-Jarisch reflex decreased the plasma levels of TNF (LPS: 775±209 vs. PBG + LPS: 248±30pg/ml) and IL-6 levels in the spleen (LPS: 39±6 vs. PBG + LPS: 24±4pg/mg of tissue). However, it did not change the other cytokines in the plasma or the other tissues evaluated. These findings confirm that the activation of the Bezold-Jarisch reflex can modulate inflammation and support the understanding that the cardiovascular reflexes regulate the immune system.

Physiological Sympathetic Activation Reduces Systemic Inflammation: Role of Baroreflex and Chemoreflex.

Baroreflex and chemoreflex act through the autonomic nervous system, which is involved with the neural regulation of inflammation. The present study reports the effects of reflex physiological sympathetic activation in endotoxemic rats using bilateral carotid occlusion (BCO), a physiological approach involving the baroreflex and chemoreflex mechanisms and the influence of the baroreceptors and peripheral chemoreceptors in the cardiovascular and systemic inflammatory responses. After lipopolysaccharide (LPS) administration, the arterial pressure was recorded during 360 min in unanesthetized rats, and serial blood samples were collected to analyze the plasma cytokine levels. BCO elicited the reflex activation of the sympathetic nervous system, providing the following outcomes: (I) increased the power of the low-frequency band in the spectrum of the systolic arterial pressure during the BCO period; (II) reduced the levels of pro-inflammatory cytokines in plasma, including the tumor necrosis factor (TNF) and the interleukin (IL)-1ß; (III) increased the plasma levels of anti-inflammatory cytokine IL-10, 90 min after LPS administration. Moreover, selective baroreceptor or chemoreceptor denervation deactivated mechanosensitive and chemical sensors, respectively, and decreased the release of the LPS-induced cytokine but did not alter the BCO modulatory effects. These results show, for the first time, that physiological reflex activation of the sympathetic circuit decreases the inflammatory response in endotoxemic rats and suggest a novel function for the baroreceptors as immunosensors during the systemic inflammation.

Carotid sinus nerve stimulation attenuates alveolar bone loss and inflammation in experimental periodontitis.

Baroreceptor and chemoreceptor reflexes modulate inflammatory responses. However, whether these reflexes attenuate periodontal diseases has been poorly examined. Thus, the present study determined the effects of electrical activation of the carotid sinus nerve (CSN) in rats with periodontitis. We hypothesized that activation of the baro and chemoreflexes attenuates alveolar bone loss and the associated inflammatory processes. Electrodes were implanted around the CSN, and bilateral ligation of the first mandibular molar was performed to, respectively, stimulate the CNS and induce periodontitis. The CSN was stimulated daily for 10 min, during nine days, in unanesthetized animals. On the eighth day, a catheter was inserted into the left femoral artery and, in the next day, the arterial pressure was recorded. Effectiveness of the CNS electrical stimulation was confirmed by hypotensive responses, which was followed by the collection of a blood sample, gingival tissue, and jaw. Long-term (9 days) electrical stimulation of the CSN attenuated bone loss and the histological damage around the first molar. In addition, the CSN stimulation also reduced the gingival and plasma pro-inflammatory cytokines induced by periodontitis. Thus, CSN stimulation has a protective effect on the development of periodontal disease mitigating alveolar bone loss and inflammatory processes.

Electrical stimulation of the carotid sinus lowers arterial pressure and improves heart rate variability in L-NAME hypertensive conscious rats.

We evaluated the effects of long-term (48 h) electrical stimulation of the carotid sinus (CS) in hypertensive rats. L-NAME-treated (10 days) Wistar rats were implanted with a catheter in the femoral artery and a miniaturized electrical stimulator attached to electrodes positioned around the left CS, encompassing the CS nerve. One day after implantation, arterial pressure (AP) was directly recorded in conscious animals for 60 min. Square pulses (1 ms, 3 V, 30 Hz) were applied intermittently (20/20 s ON/OFF) to the CS for 48 h. After the end of stimulation, AP was recorded again. Nonstimulated rats (control group) and rats without electrodes around the CS (sham-operated) were also studied. Next, the animals were decapitated, and segments of mesenteric resistance arteries were removed to study vascular function. After the stimulation period, AP was 16 ± 5 mmHg lower in the stimulated group, whereas sham-operated and control rats showed similar AP between the first and second recording periods. Heart rate variability (HRV) evaluated using time and frequency domain tools and a nonlinear approach (symbolic analysis) suggested that hypertensive rats with electrodes around the CS, stimulated or not, exhibited a shift in cardiac sympathovagal balance towards parasympathetic tone. The relaxation response to acetylcholine in endothelium-intact mesenteric arteries was enhanced in rats that underwent CS stimulation for 48 h. In conclusion, long-term CS stimulation is effective in reducing AP levels, improving HRV and increasing mesenteric vascular relaxation in L-NAME hypertensive rats. Moreover, only the presence of electrodes around the CS is effective in eliciting changes in HRV similar to those observed in stimulated rats.

Time Course of Hemodynamic Responses to Different Doses of Lipopolysaccharide in Unanesthetized Male Rats.

Lipopolysaccharide (LPS) administration is a well-known method to induce systemic inflammation widely used for investigating new therapeutic strategies for sepsis treatment, which is characterized by clinical manifestations such as tachycardia and hypotension. However, there are different doses of LPS used in several studies, and the hemodynamic responses were not always well characterized. Thus, the present study aimed to evaluate the arterial pressure, heart rate, heart rate variability, and baroreflex function from rats, over time, to different doses of LPS. Femoral artery and vein catheters were inserted into anesthetized Wistar-Hannover male rats for arterial pressure recording and LPS administration, respectively. On the next day, the arterial pressure was recorded before and after (90, 180, and 360 min) LPS injection (0.06, 20, 30, and 40 mg/kg). All doses of LPS tested increased the heart rate and decreased baroreflex sensitivity over time. In addition, while LPS administration of 20, 30, and 40 mg/kg increased the mean arterial pressure over time, 0.06 mg/kg decreased the mean arterial pressure at 360 min, as compared to baseline values. Furthermore, high doses of LPS decreased the power of the HF band of the cardiac interval spectrum over time, and the higher dose increased the power of the LF band. Our data indicate that high doses of LPS promote hypertensive response over time, while a low dose decreases arterial pressure. Moreover, the changes in heart rate variability and baroreflex function elicited by LPS may be not associated with arterial pressure response produced by the endotoxemia.

Cortical stimulation in conscious rats controls joint inflammation.

The neuronal control of the immune system is fundamental to the development of new therapeutic strategies for inflammatory disorders. Recent studies reported that afferent vagal stimulation attenuates peripheral inflammation by activating specific sympathetic central and peripheral networks, but only few subcortical brain areas were investigated. In the present study, we report that afferent vagal stimulation also activates specific cortical areas, as the parietal and cingulate cortex. Since these cortical structures innervate sympathetic-related areas, we investigate whether electrical stimulation of parietal cortex can attenuate knee joint inflammation in non-anesthetized rats. Our results show that cortical stimulation in rats increased sympathetic activity and improved joint inflammatory parameters, such as local neutrophil infiltration and pro-inflammatory cytokine levels, without causing behavioral disturbance, brain epileptiform activity or neural damage. In addition, we superposed the areas activated by afferent vagal or cortical stimulation to map common central structures to depict a brain immunological homunculus that can allow novel therapeutic approaches against inflammatory joint diseases, such as rheumatoid arthritis.

Nonlinearities of heart rate variability in animal models of impaired cardiac control: contribution of different time scales.

Heart rate variability (HRV) has been extensively explored by traditional linear approaches (e.g., spectral analysis); however, several studies have pointed to the presence of nonlinear features in HRV, suggesting that linear tools might fail to account for the complexity of the HRV dynamics. Even though the prevalent notion is that HRV is nonlinear, the actual presence of nonlinear features is rarely verified. In this study, the presence of nonlinear dynamics was checked as a function of time scales in three experimental models of rats with different impairment of the cardiac control: namely, rats with heart failure (HF), spontaneously hypertensive rats (SHRs), and sinoaortic denervated (SAD) rats. Multiscale entropy (MSE) and refined MSE (RMSE) were chosen as the discriminating statistic for the surrogate test utilized to detect nonlinearity. Nonlinear dynamics is less present in HF animals at both short and long time scales compared with controls. A similar finding was found in SHR only at short time scales. SAD increased the presence of nonlinear dynamics exclusively at short time scales. Those findings suggest that a working baroreflex contributes to linearize HRV and to reduce the likelihood to observe nonlinear components of the cardiac control at short time scales. In addition, an increased sympathetic modulation seems to be a source of nonlinear dynamics at long time scales. Testing nonlinear dynamics as a function of the time scales can provide a characterization of the cardiac control complementary to more traditional markers in time, frequency, and information domains.NEW & NOTEWORTHY Although heart rate variability (HRV) dynamics is widely assumed to be nonlinear, nonlinearity tests are rarely used to check this hypothesis. By adopting multiscale entropy (MSE) and refined MSE (RMSE) as the discriminating statistic for the nonlinearity test, we show that nonlinear dynamics varies with time scale and the type of cardiac dysfunction. Moreover, as complexity metrics and nonlinearities provide complementary information, we strongly recommend using the test for nonlinearity as an additional index to characterize HRV.

Modulation of experimental arthritis by vagal sensory and central brain stimulation.

Articular inflammation is a major clinical burden in multiple inflammatory diseases, especially in rheumatoid arthritis. Biological anti-rheumatic drug therapies are expensive and increase the risk of systemic immunosuppression, infections, and malignancies. Here, we report that vagus nerve stimulation controls arthritic joint inflammation by inducing local regulation of innate immune response. Most of the previous studies of neuromodulation focused on vagal regulation of inflammation via the efferent peripheral pathway toward the viscera. Here, we report that vagal stimulation modulates arthritic joint inflammation through a novel "afferent" pathway mediated by the locus coeruleus (LC) of the central nervous system. Afferent vagal stimulation activates two sympatho-excitatory brain areas: the paraventricular hypothalamic nucleus (PVN) and the LC. The integrity of the LC, but not that of the PVN, is critical for vagal control of arthritic joint inflammation. Afferent vagal stimulation suppresses articular inflammation in the ipsilateral, but not in the contralateral knee to the hemispheric LC lesion. Central stimulation is followed by subsequent activation of joint sympathetic nerve terminals inducing articular norepinephrine release. Selective adrenergic beta-blockers prevent the effects of articular norepinephrine and thereby abrogate vagal control of arthritic joint inflammation. These results reveals a novel neuro-immune brain map with afferent vagal signals controlling side-specific articular inflammation through specific inflammatory-processing brain centers and joint sympathetic innervations.

Multiscale entropy analysis of heart rate variability in heart failure, hypertensive, and sinoaortic-denervated rats: classical and refined approaches.

The analysis of heart rate variability (HRV) by nonlinear methods has been gaining increasing interest due to their ability to quantify the complexity of cardiovascular regulation. In this study, multiscale entropy (MSE) and refined MSE (RMSE) were applied to track the complexity of HRV as a function of time scale in three pathological conscious animal models: rats with heart failure (HF), spontaneously hypertensive rats (SHR), and rats with sinoaortic denervation (SAD). Results showed that HF did not change HRV complexity, although there was a tendency to decrease the entropy in HF animals. On the other hand, SHR group was characterized by reduced complexity at long time scales, whereas SAD animals exhibited a smaller short- and long-term irregularity. We propose that short time scales (1 to 4), accounting for fast oscillations, are more related to vagal and respiratory control, whereas long time scales (5 to 20), accounting for slow oscillations, are more related to sympathetic control. The increased sympathetic modulation is probably the main reason for the lower entropy observed at high scales for both SHR and SAD groups, acting as a negative factor for the cardiovascular complexity. This study highlights the contribution of the multiscale complexity analysis of HRV for understanding the physiological mechanisms involved in cardiovascular regulation.

Renal nerve ultrastructural alterations in short term and long term experimental diabetes.

BACKGROUND: Despite the evidence that renal hemodynamics is impaired in experimental diabetes, associated with glomeruli structural alterations, renal nerves were not yet investigated in experimental models of diabetes and the contribution of nerve alterations to the diabetic nephropathy remains to be investigated. We aimed to determine if ultrastructural morphometric parameters of the renal nerves are affected by short term and/or long term experimental diabetes and if insulin treatment reverses these alterations. Left renal nerves were evaluated 15 days or 12 weeks (N = 10 in each group) after induction of diabetes, with a single injection of streptozotocin (STZ). Control rats (N = 10 in each group) were injected with vehicle (citrate buffer). Treated animals (N = 10 in each group) received a single subcutaneous injection of insulin on a daily basis. Arterial pressure, together with the renal nerves activity, was recorded 15 days (short-term) or 12 weeks (long-term) after STZ injection. After the recordings, the renal nerves were dissected, prepared for light and transmission electron microscopy, and fascicle and fibers morphometry were carried out with computer software. RESULTS: The major diabetic alteration on the renal nerves was a small myelinated fibers loss since their number was smaller on chronic diabetic animals, the average morphometric parameters of the myelinated fibers were larger on chronic diabetic animals and distribution histograms of fiber diameter was significantly shifted to the right on chronic diabetic animals. These alterations began early, after 15 days of diabetes induction, associated with a severe mitochondrial damage, and were not prevented by conventional insulin treatment. CONCLUSIONS: The experimental diabetes, induced by a single intravenous injection of STZ, in adult male Wistar rats, caused small fiber loss in the renal nerves, probably due to the early mitochondrial damage. Conventional treatment with insulin was able to correct the weight gain and metabolic changes in diabetic animals, without, however, correcting and / or preventing damage to the thin fibers caused by STZ-induced diabetes. The kidney innervation is impaired in this diabetic model suggesting that alterations of the renal nerves may play a role in the development of the diabetic nephropathy.