Search

Abstract Background The involvement of the autonomic nervous system in the regulation of inflammation is an emerging concept with significant potential for clinical applications. Recent studies demonstrate that stimulating the vagus nerve activates the cholinergic anti-inflammatory pathway that inhibits pro-inflammatory cytokines and controls inflammation. The α7 nicotinic acetylcholine receptor (α7nAChR) on macrophages plays a key role in mediating cholinergic anti-inflammatory effects through a downstream intracellular mechanism involving inhibition of NF-κB signaling, which results in suppression of pro-inflammatory cytokine production. However, the role of the α7nAChR in the regulation of other aspects of the immune response, including the recruitment of monocytes/macrophages to the site of inflammation remained poorly understood. Results We observed an increased mortality in α7nAChR-deficient mice (compared with wild-type controls) in mice with endotoxemia, which was paralleled with a significant reduction in the number of monocyte-derived macrophages in the lungs. Corroborating these results, fluorescently labeled α7nAChR-deficient monocytes adoptively transferred to WT mice showed significantly diminished recruitment to the inflamed tissue. α7nAChR deficiency did not affect monocyte 2D transmigration across an endothelial monolayer, but it significantly decreased the migration of macrophages in a 3D fibrin matrix. In vitro analysis of major adhesive receptors (L-selectin, β1 and β2 integrins) and chemokine receptors (CCR2 and CCR5) revealed reduced expression of integrin αM and αX on α7nAChR-deficient macrophages. Decreased expression of αMβ2 was confirmed on fluorescently labeled, adoptively transferred α7nAChR-deficient macrophages in the lungs of endotoxemic mice, indicating a potential mechanism for α7nAChR-mediated migration. Conclusions We demonstrate a novel role for the α7nAChR in mediating macrophage recruitment to inflamed tissue, which indicates an important new aspect of the cholinergic regulation of immune responses and inflammation.

Abstract Background Acute pancreatitis is a common and serious inflammatory condition currently lacking disease modifying therapy. The cholinergic anti-inflammatory pathway (CAP) is a potent protective anti-inflammatory response activated by vagus nerve-dependent α7 nicotinic acetylcholine receptor (α7nAChR) signaling using splenic CD4+ T cells as an intermediate. Activating the CAP ameliorates experimental acute pancreatitis. Galantamine is an acetylcholinesterase inhibitor (AChEI) which amplifies the CAP via modulation of central muscarinic ACh receptors (mAChRs). However, as mAChRs also activate pancreatitis, it is currently unknown whether galantamine would be beneficial in acute pancreatitis. Methods The effect of galantamine (1–6 mg/kg-body weight) on caerulein-induced acute pancreatitis was evaluated in mice. Two hours following 6 hourly doses of caerulein (50 µg/kg-body weight), organ and serum analyses were performed with accompanying pancreatic histology. Experiments utilizing vagotomy, gene knock out (KO) technology and the use of nAChR antagonists were also performed. Results Galantamine attenuated pancreatic histologic injury which was mirrored by a reduction in serum amylase and pancreatic inflammatory cytokines and an increase the anti-inflammatory cytokine IL-10 in the serum. These beneficial effects were not altered by bilateral subdiaphragmatic vagotomy, KO of either choline acetyltransferase+ T cells or α7nAChR, or administration of the nAChR ganglionic blocker mecamylamine or the more selective α7nAChR antagonist methyllycaconitine. Conclusion Galantamine improves acute pancreatitis via a mechanism which does not involve previously established physiological and molecular components of the CAP. As galantamine is an approved drug in widespread clinical use with an excellent safety record, our findings are of interest for further evaluating the potential benefits of this drug in patients with acute pancreatitis.

Abstract Background The noradrenergic innervation of the spleen is implicated in the autonomic control of inflammation and has been the target of neurostimulation therapies for inflammatory diseases. However, there is no real-time marker of its successful activation, which hinders the development of anti-inflammatory neurostimulation therapies and mechanistic studies in anti-inflammatory neural circuits. Methods In mice, we performed fast-scan cyclic voltammetry (FSCV) in the spleen during intravenous injections of norepinephrine (NE), and during stimulation of the vagus, splanchnic, or splenic nerves. We defined the stimulus-elicited charge generated at the oxidation potential for NE (~ 0.88 V) as the “NE voltammetry signal” and quantified the dependence of the signal on NE dose and intensity of neurostimulation. We correlated the NE voltammetry signal with the anti-inflammatory effect of splenic nerve stimulation (SpNS) in a model of lipopolysaccharide- (LPS) induced endotoxemia, quantified as suppression of TNF release. Results The NE voltammetry signal is proportional to the estimated peak NE blood concentration, with 0.1 μg/mL detection threshold. In response to SpNS, the signal increases within seconds, returns to baseline minutes later, and is blocked by interventions that deplete NE or inhibit NE release. The signal is elicited by efferent, but not afferent, electrical or optogenetic vagus nerve stimulation, and by splanchnic nerve stimulation. The magnitude of the signal during SpNS is inversely correlated with subsequent TNF suppression in endotoxemia and explains 40% of the variance in TNF measurements. Conclusions FSCV in the spleen provides a marker for real-time monitoring of anti-inflammatory activation of the splenic innervation during autonomic stimulation.

Abstract Background There is emerging evidence that the nervous system regulates immune and metabolic alterations mediating Metabolic syndrome (MetS) pathogenesis via the vagus nerve. This study evaluated the effects of transcutaneous auricular vagus nerve stimulation (TAVNS) on key cardiovascular and inflammatory components of MetS. Methods We conducted an open label, randomized (2:1), two-arm, parallel-group controlled trial in MetS patients. Subjects in the treatment group (n = 20) received 30 min of TAVNS with a NEMOS® device placed on the cymba conchae of the left ear, once weekly. Patients in the control group (n = 10) received no stimulation. Hemodynamic, heart rate variability (HRV), biochemical parameters, and monocytes, progenitor endothelial cells, circulating endothelial cells, and endothelial micro particles were evaluated at randomization, after the first TAVNS treatment, and again after 8 weeks of follow-up. Results An improvement in sympathovagal balance (HRV analysis) was observed after the first TAVNS session. Only patients treated with TAVNS for 8 weeks had a significant decrease in office BP and HR, a further improvement in sympathovagal balance, with a shift of circulating monocytes towards an anti-inflammatory phenotype and endothelial cells to a reparative vascular profile. Conclusion These results are of interest for further study of TAVNS as treatment of MetS.

Background: In this investigation, we explored the effects of pharmacological cholinergic stimulation on cardiac function and renal inflammation following acute myocardial infarction (AMI) in spontaneously hypertensive rats (SHRs). Methods: Adult male SHRs were randomized into three experimental groups: sham-operated; AMI + Veh (infarcted, treated with vehicle); and AMI + PY (infarcted, treated with the cholinesterase inhibitor, pyridostigmine bromide (PY)—40 mg/kg, once daily for seven days). Rats were euthanized 7 or 30 days post-surgery. The clinical parameters were assessed on the day before euthanasia. Subsequent to euthanasia, blood samples were collected and renal tissues were harvested for histological and gene expression analyses aimed to evaluate inflammation and injury. Results: Seven days post-surgery, the AMI + PY group demonstrated improvements in left ventricular diastolic function and autonomic regulation, and a reduction in renal macrophage infiltration compared to the AMI + Veh group. Furthermore, there was a notable downregulation in pro-inflammatory gene expression and an upregulation in anti-inflammatory gene expression. Analysis 30 days post-surgery showed that PY treatment had a sustained positive effect on renal gene expression, correlated with a decrease in biomarkers, indicative of subclinical kidney injury. Conclusions: Short-term cholinergic stimulation with PY provides both cardiac and renal protection by mitigating the inflammatory response after AMI.

Abstract Background Inflammation, the physiological response to infection and injury, is coordinated by the immune and nervous systems. Interleukin-1β (IL-1β) and other cytokines produced during inflammatory responses activate sensory neurons (nociceptors) to mediate the onset of pain, sickness behavior, and metabolic responses. Although nociceptors expressing Transient Receptor Potential Ankyrin-1 (TRPA1) can initiate inflammation, comparatively little is known about the role of TRPA1 nociceptors in the physiological responses to specific cytokines. Methods To monitor body temperature in conscious and unrestrained mice, telemetry probes were implanted into peritoneal cavity of mice. Using transgenic and tissue specific knockouts and chemogenetic techniques, we recorded temperature responses to the potent pro-inflammatory cytokine IL-1β. Using calcium imaging, whole cell patch clamping and whole nerve recordings, we investigated the role of TRPA1 during IL-1β-mediated neuronal activation. Mouse models of acute endotoxemia and sepsis were used to elucidate how specific activation, with optogenetics and chemogenetics, or ablation of TRPA1 neurons can affect the outcomes of inflammatory insults. All statistical tests were performed with GraphPad Prism 9 software and for all analyses, P ≤ 0.05 was considered statistically significant. Results Here, we describe a previously unrecognized mechanism by which IL-1β activates afferent vagus nerve fibers to trigger hypothermia, a response which is abolished by selective silencing of neuronal TRPA1. Afferent vagus nerve TRPA1 signaling also inhibits endotoxin-stimulated cytokine storm and significantly reduces the lethality of bacterial sepsis. Conclusion Thus, IL-1β activates TRPA1 vagus nerve signaling in the afferent arm of a reflex anti-inflammatory response which inhibits cytokine release, induces hypothermia, and reduces the mortality of infection. This discovery establishes that TRPA1, an ion channel known previously as a pro-inflammatory detector of cold, pain, itch, and a wide variety of noxious molecules, also plays a specific anti-inflammatory role via activating reflex anti-inflammatory activity.

Abstract Background Autoinflammatory diseases, a diverse group of inherited conditions characterized by excessive innate immune activation, have limited therapeutic options. Neuroimmune circuits of the inflammatory reflex control innate immune overactivation and can be stimulated to treat disease using the acetylcholinesterase inhibitor galantamine. Methods We tested the efficacy of galantamine in a rodent model of the prototypical autoinflammatory disease familial Mediterranean fever (FMF). Multiple chronic disease markers were evaluated in animals that received long-term galantamine treatment compared to vehicle. Results Long-term treatment with galantamine attenuated the associated splenomegaly and anemia which are characteristic features of this disease. Further, treatment reduced inflammatory cell infiltration into affected organs and a subcutaneous air pouch. Conclusions These findings suggest that galantamine attenuates chronic inflammation in this mouse model of FMF. Further research is warranted to explore the therapeutic potential of galantamine in FMF and other autoinflammatory diseases.

Abstract Background Brain metabolic alterations and neuroinflammation have been reported in several peripheral inflammatory conditions and present significant potential for targeting with new diagnostic approaches and treatments. However, non-invasive evaluation of these alterations remains a challenge. Methods Here, we studied the utility of a micro positron emission tomography (microPET) dual tracer ([11C]PBR28 – for microglial activation and [18F]FDG for energy metabolism) approach to assess brain dysfunction, including neuroinflammation in murine endotoxemia. MicroPET imaging data were subjected to advanced conjunction and individual analyses, followed by post-hoc analysis. Results There were significant increases in [11C]PBR28 and [18F]FDG uptake in the hippocampus of C57BL/6 J mice 6 h following LPS (2 mg/kg) intraperitoneal (i.p.) administration compared with saline administration. These results confirmed previous postmortem observations. In addition, patterns of significant simultaneous activation were demonstrated in the hippocampus, the thalamus, and the hypothalamus in parallel with other tracer-specific and region-specific alterations. These changes were observed in the presence of robust systemic inflammatory responses manifested by significantly increased serum cytokine levels. Conclusions Together, these findings demonstrate the applicability of [11C]PBR28 - [18F]FDG dual tracer microPET imaging for assessing neuroinflammation and brain metabolic alterations in conditions “classically” characterized by peripheral inflammatory and metabolic pathogenesis.

IntroductionInflammation is an inherently self-amplifying process, resulting in progressive tissue damage when unresolved. A brake on this positive feedback system is provided by the nervous system which has evolved to detect inflammatory signals and respond by activating anti-inflammatory processes, including the cholinergic anti-inflammatory pathway mediated by the vagus nerve. Acute pancreatitis, a common and serious condition without effective therapy, develops when acinar cell injury activates intrapancreatic inflammation. Prior study has shown that electrical stimulation of the carotid sheath, which contains the vagus nerve, boosts the endogenous anti-inflammatory response and ameliorates acute pancreatitis, but it remains unknown whether these anti-inflammatory signals originate in the brain.MethodsHere, we used optogenetics to selectively activate efferent vagus nerve fibers originating in the brainstem dorsal motor nucleus of the vagus (DMN) and evaluated the effects on caerulein-induced pancreatitis.ResultsStimulation of the cholinergic neurons in the DMN significantly attenuates the severity of pancreatitis as indicated by reduced serum amylase, pancreatic cytokines, tissue damage, and edema. Either vagotomy or silencing cholinergic nicotinic receptor signaling by pre-administration of the antagonist mecamylamine abolishes the beneficial effects.DiscussionThese results provide the first evidence that efferent vagus cholinergic neurons residing in the brainstem DMN can inhibit pancreatic inflammation and implicate the cholinergic anti-inflammatory pathway as a potential therapeutic target for acute pancreatitis.

Abstract Background Severe COVID-19 is characterized by pro-inflammatory cytokine release syndrome (cytokine storm) which causes high morbidity and mortality. Recent observational and clinical studies suggest famotidine, a histamine 2 receptor (H2R) antagonist widely used to treat gastroesophageal reflux disease, attenuates the clinical course of COVID-19. Because evidence is lacking for a direct antiviral activity of famotidine, a proposed mechanism of action is blocking the effects of histamine released by mast cells. Here we hypothesized that famotidine activates the inflammatory reflex, a brain-integrated vagus nerve mechanism which inhibits inflammation via alpha 7 nicotinic acetylcholine receptor (α7nAChR) signal transduction, to prevent cytokine storm. Methods The potential anti-inflammatory effects of famotidine and other H2R antagonists were assessed in mice exposed to lipopolysaccharide (LPS)-induced cytokine storm. As the inflammatory reflex is integrated and can be stimulated in the brain, and H2R antagonists penetrate the blood brain barrier poorly, famotidine was administered by intracerebroventricular (ICV) or intraperitoneal (IP) routes. Results Famotidine administered IP significantly reduced serum and splenic LPS-stimulated tumor necrosis factor (TNF) and IL-6 concentrations, significantly improving survival. The effects of ICV famotidine were significantly more potent as compared to the peripheral route. Mice lacking mast cells by genetic deletion also responded to famotidine, indicating the anti-inflammatory effects are not mast cell-dependent. Either bilateral sub-diaphragmatic vagotomy or genetic knock-out of α7nAChR abolished the anti-inflammatory effects of famotidine, indicating the inflammatory reflex as famotidine’s mechanism of action. While the structurally similar H2R antagonist tiotidine displayed equivalent anti-inflammatory activity, the H2R antagonists cimetidine or ranitidine were ineffective even at very high dosages. Conclusions These observations reveal a previously unidentified vagus nerve-dependent anti-inflammatory effect of famotidine in the setting of cytokine storm which is not replicated by high dosages of other H2R antagonists in clinical use. Because famotidine is more potent when administered intrathecally, these findings are also consistent with a primarily central nervous system mechanism of action.

Abstract There is a broad and growing interest in Bioelectronic Medicine, a dynamic field that continues to generate new approaches in disease treatment. The fourth bioelectronic medicine summit “Technology targeting molecular mechanisms” took place on September 23 and 24, 2020. This virtual meeting was hosted by the Feinstein Institutes for Medical Research, Northwell Health. The summit called international attention to Bioelectronic Medicine as a platform for new developments in science, technology, and healthcare. The meeting was an arena for exchanging new ideas and seeding potential collaborations involving teams in academia and industry. The summit provided a forum for leaders in the field to discuss current progress, challenges, and future developments in Bioelectronic Medicine. The main topics discussed at the summit are outlined here.

Abstract The mechanisms regulating immune cells recruitment into the heart during healing after an acute myocardial infarction (AMI) have major clinical implications. We investigated whether cholinergic stimulation with pyridostigmine, a cholinesterase inhibitor, modulates heart and spleen immune responses and cardiac remodeling after AMI in spontaneous hypertensive rats (SHRs). Male adult SHRs underwent sham surgery or ligation of the left coronary artery and were randomly allocated to remain untreated or to pyridostigmine treatment (40 mg/kg once a day by gavage). Blood pressure and heart rate variability were determined, and echocardiography was performed at day six after MI. The heart and spleen were processed for immunohistochemistry cellular analyses (CD3+ and CD4+ lymphocytes, and CD68+ and CD206+ macrophages), and TNF levels were determined at day seven after MI. Pyridostigmine treatment increased the parasympathetic tone and T CD4+ lymphocytes in the myocardium, but lowered M1/M2 macrophage ratio towards an anti-inflammatory profile that was associated with decreased TNF levels in the heart and spleen. Treatment with this cholinergic agent improved heart remodeling manifested by lower ventricular diameters and better functional parameters. In summary, cholinergic stimulation by pyridostigmine enhances the parasympathetic tone and induces anti-inflammatory responses in the heart and spleen fostering cardiac recovery after AMI in SHRs.

Abstract Background Oxygen therapy, using supraphysiological concentrations of oxygen (hyperoxia), is routinely administered to patients who require respiratory support including mechanical ventilation (MV). However, prolonged exposure to hyperoxia results in acute lung injury (ALI) and accumulation of high mobility group box 1 (HMGB1) in the airways. We previously showed that airway HMGB1 mediates hyperoxia-induced lung injury in a mouse model of ALI. Cholinergic signaling through the α7 nicotinic acetylcholine receptor (α7nAChR) attenuates several inflammatory conditions. The aim of this study was to determine whether 3–(2,4 dimethoxy-benzylidene)-anabaseine dihydrochloride, GTS-21, an α7nAChR partial agonist, inhibits hyperoxia-induced HMGB1 accumulation in the airways and circulation, and consequently attenuates inflammatory lung injury. Methods Mice were exposed to hyperoxia (≥99% O2) for 3 days and treated concurrently with GTS-21 (0.04, 0.4 and 4 mg/kg, i.p.) or the control vehicle, saline. Results The systemic administration of GTS-21 (4 mg/kg) significantly decreased levels of HMGB1 in the airways and the serum. Moreover, GTS-21 (4 mg/kg) significantly reduced hyperoxia-induced acute inflammatory lung injury, as indicated by the decreased total protein content in the airways, reduced infiltration of inflammatory monocytes/macrophages and neutrophils into the lung tissue and airways, and improved lung injury histopathology. Conclusions Our results indicate that GTS-21 can attenuate hyperoxia-induced ALI by inhibiting extracellular HMGB1-mediated inflammatory responses. This suggests that the α7nAChR represents a potential pharmacological target for the treatment regimen of oxidative inflammatory lung injury in patients receiving oxygen therapy.

Abstract The recent opioid crisis is one of the rising challenges in the history of modern health care. New and effective treatment modalities with less adverse effects to alleviate and manage this modern epidemic are critically needed. The FDA has recently approved two non-invasive electrical nerve stimulators for the adjunct treatment of symptoms of acute opioid withdrawal. These devices, placed behind the ear, stimulate certain cranial nerves with auricular projections. This neural stimulation reportedly generates a prompt effect in terms of alleviation of withdrawal symptoms resulting from acute discontinuation of opioid use. Current experimental evidence indicates that this type of non-invasive neural stimulation has excellent potential to supplement medication assisted treatment in opioid detoxification with lower side effects and increased adherence to treatment. Here, we review current findings supporting the use of non-invasive neural stimulation in detoxification from opioid use. We briefly outline the neurophysiology underlying this approach of auricular electrical neural stimulation and its role in enhancing medication assisted treatment in treating symptoms of opioid withdrawal. Considering the growing deleterious impact of addictive disorders on our society, further studies on this emerging treatment modality are warranted.

Abstract Background The sequelae of sepsis were once thought to be independent of sepsis itself and assumed to be either comorbid to sick patients or complications of critical illness. Recent studies have reported consistent patterns of functional disabilities in sepsis survivors that can last from months to years after symptoms of active sepsis had resolved. Body Post-sepsis syndrome is an emerging pathological entity that has garnered significant interest amongst clinicians and researchers over the last two decades. It is marked by a significantly increased risk of death and a poor health-related quality of life associated with a constellation of long-term effects that persist following the patient’s bout with sepsis. These include neurocognitive impairment, functional disability, psychological deficits, and worsening medical conditions. Conclusion This “post-sepsis syndrome” has been the subject of active preclinical and clinical research providing new mechanistic insights and approaches linked to survivor well-being. Here we review important aspects of these research efforts and goals of care for patients who survive sepsis.

Abstract Background Animal models of diet‐induced obesity (DIO) are commonly used in medical research for mimicking human diseases. There is no universal animal model, and careful evaluation of variety of factors needs to be considered when designing new experiments. Here, we investigated the effect of 9 weeks high‐fat diet (HFD) intervention, providing 60% energy from fat, on parameters of inflammation and insulin resistance in male C57BL/6J mice. Methods Six weeks old mice were initiated on regular diet (RD) or HFD providing 60 kcal energy from fat for 9 weeks. Fasting blood glucose levels were measured by glucometer, and fasting plasma levels of insulin and proinflammatory cytokines by Luminex assay. Insulin sensitivity was evaluated by using QUICKI and HOMA2 indexes. Results HFD mice showed ~ 40% higher body weight and ~ 20% larger abdominal circumference, due to an increase in the white adipose tissue mass. Liver examination revealed increased size and higher hepatic lipid accumulation in livers from HFD mice compared to their RD counterparts. Animals from the HFD group were characterized with significantly higher presence of crown‐like structures (CLS) in WAT and higher plasma levels of proinflammatory cytokines (TNF‐α, IL‐6, leptin, MCP‐1, PAI‐1, and resistin). HFD‐fed mice also demonstrated impaired insulin sensitivity (lower QUICKI, higher HOMA‐insulin resistance (HOMA‐IR), and lower HOMA‐percent sensitivity (HOMA‐%S)) index values. Conclusion Male C57BL/6J mice on 9 weeks HFD providing 60 kcal energy from fat display impaired insulin sensitivity and chronic inflammation, thus making this DIO mouse model appropriate for studies of early stages of obesity‐related pathology.

Abstract Studies on the role of the vagus nerve in the regulation of immunity and inflammation have contributed to current preclinical and clinical efforts in bioelectronic medicine. In parallel, this research has generated new insights into the cellular and molecular mechanisms underlying the immunoregulatory functions of the vagus nerve within the inflammatory reflex. The vagus nerve and other cellular components of the inflammatory reflex are implicated in the regulation of bleeding, cancer, obesity, blood pressure, viral infections and other conditions. This collateral benefit broadens scientific horizons and provides new rationale for technological advances and therapeutic implications.

Abstract Background Rheumatoid arthritis (RA) is a chronic and debilitating inflammatory disease characterized by extensive joint tissue inflammation. Implantable bioelectronic devices targeting the inflammatory reflex reduce TNF production and inflammation in preclinical models of inflammatory disease, and in patients with RA and Crohn’s disease. Here, we assessed the effect of applying a vibrotactile device to the cymba concha of the external ear on inflammatory responses in healthy subjects, as well as its effect on disease activity in RA patients. Methods Six healthy subjects received vibrotactile treatment at the cymba concha, and TNF production was analyzed at different time points post-stimulation. In a separate study, nineteen healthy subjects were enrolled in a randomized cross-over study, and effects of vibrotactile treatment at either the cymba concha or gastrocnemius on cytokine levels were assessed. In addition, the clinical efficacy of vibrotactile treatment on disease activity in RA was assessed in nine patients with RA in a prospective interventional study. Results Vibrotactile treatment at the cymba concha reduced TNF levels, and the suppressive effect persisted up to 24 h. In the cross-over study with 19 healthy subjects, vibrotactile treatment at the cymba concha but not at the gastrocnemius significantly reduced TNF, IL-1β, and IL-6 levels compared to pre-treatment baseline (TNF p

Acute respiratory distress syndrome (ARDS) is a critical illness complication that is associated with high mortality. ARDS is documented in severe cases of COVID-19. No effective pharmacological treatments for ARDS are currently available. Dysfunctional immune responses and pulmonary and systemic inflammation are characteristic features of ARDS pathogenesis. Recent advances in our understanding of the regulation of inflammation point to an important role of the vagus-nerve-mediated inflammatory reflex and neural cholinergic signaling. We examined whether pharmacological cholinergic activation using a clinically approved (for myasthenia gravis) cholinergic drug, the acetylcholinesterase inhibitor pyridostigmine alters pulmonary and systemic inflammation in mice with lipopolysaccharide (LPS)-induced ARDS. Male C57Bl/6 mice received one intratracheal instillation of LPS or were sham manipulated (control). Both groups were treated with either vehicle or pyridostigmine (1.5 mg/kg twice daily, 3 mg/day) administered by oral gavage starting at 1 h post-LPS and euthanized 24 h after LPS administration. Other groups were either sham manipulated or received LPS for 3 days and were treated with vehicle or pyridostigmine and euthanized at 72 h. Pyridostigmine treatment reduced the increased total number of cells and neutrophils in the bronchoalveolar lavage fluid (BALF) in mice with ARDS at 24 and 72 h. Pyridostigmine also reduced the number of macrophages and lymphocytes at 72 h. In addition, pyridostigmine suppressed the levels of TNF, IL-1β, IL-6, and IFN-γ in BALF and plasma at 24 and 72 h. However, this cholinergic agent did not significantly altered BALF and plasma levels of the anti-inflammatory cytokine IL-10. Neither LPS nor pyridostigmine affected BALF IFN-γ and IL-10 levels at 24 h post-LPS. In conclusion, treatments with the cholinergic agent pyridostigmine ameliorate pulmonary and systemic inflammatory responses in mice with endotoxin-induced ARDS. Considering that pyridostigmine is a clinically approved drug, these findings are of substantial interest for implementing pyridostigmine in therapeutic strategies for ARDS.

Background: The metabolic syndrome (MetS) is an obesity-associated disorder of pandemic proportions and limited treatment options. Oxidative stress, low-grade inflammation and altered neural autonomic regulation, are important components and drivers of pathogenesis. Galantamine, an acetylcholinesterase inhibitor and a cholinergic drug that is clinically-approved (for Alzheimer's disease) has been implicated in neural cholinergic regulation of inflammation in several conditions characterized with immune and metabolic derangements. Here we examined the effects of galantamine on oxidative stress in parallel with inflammatory and cardio-metabolic parameters in subjects with MetS.Trial Design and Methods: The effects of galantamine treatment, 8 mg daily for 4 weeks or placebo, followed by 16 mg daily for 8 weeks or placebo were studied in randomly assigned subjects with MetS (n = 22 per group) of both genders. Oxidative stress, including superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase activities, lipid and protein peroxidation, and nitrite levels were analyzed before and at the end of the treatment. In addition, plasma cytokine and adipokine levels, insulin resistance (HOMA-IR) and other relevant cardio-metabolic indices were analyzed. Autonomic regulation was also examined by heart rate variability (HRV) before treatment, and at every 4 weeks of treatment.Results: Galantamine treatment significantly increased antioxidant enzyme activities, including SOD [+1.65 USOD/mg protein, [95% CI 0.39–2.92], P = 0.004] and CAT [+0.93 nmol/mg, [95% CI 0.34–1.51], P = 0.01], decreased lipid peroxidation [thiobarbituric acid reactive substances [log scale 0.72 pmol/mg, [95% CI 0.46–1.07], P = 0.05], and systemic nitrite levels [log scale 0.83 μmol/mg protein, [95% CI 0.57–1.20], P = 0.04] compared with placebo. In addition, galantamine significantly alleviated the inflammatory state and insulin resistance, and decreased the low frequency/high frequency ratio of HRV, following 8 and 12 weeks of drug treatment.Conclusion: Low-dose galantamine alleviates oxidative stress, alongside beneficial anti-inflammatory, and metabolic effects, and modulates neural autonomic regulation in subjects with MetS. These findings are of considerable interest for further studies with the cholinergic drug galantamine to ameliorate MetS.

Abstract Reperfusion injury following acute myocardial infarction is associated with significant morbidity. Activation of neuronal or non-neuronal cholinergic pathways in the heart has been shown to reduce ischemic injury, and this effect has been attributed primarily to muscarinic acetylcholine receptors. In contrast, the role of nicotinic receptors, specifically α-7 subtype (α7nAChR), in the myocardium remains unknown, which offers an opportunity to potentially repurpose several agonists/modulators that are currently under development for neurologic indications. Treatment of ex vivo and in vivo rat models of cardiac ischemia/reperfusion (I/R) with a selective α7nAChR agonist (GTS21) showed significant increases in left ventricular developing pressure and rates of pressure development, without effects on heart rate. These positive functional effects were blocked by co-administration with methyllycaconitine (MLA), a selective antagonist of α7nAChRs. In vivo, delivery of GTS21 at the initiation of reperfusion reduced infarct size by 42% (p < 0.01) and decreased tissue reactive oxygen species (ROS) by 62% (p < 0.01). Flow cytometry of MitoTracker Red-stained mitochondria showed that mitochondrial membrane potential was normalized in mitochondria isolated from GTS21-treated compared with untreated I/R hearts. Intracellular adenosine triphosphate (ATP) concentration in cultured cardiomyocytes exposed to hypoxia/reoxygenation was reduced (p < 0.001), but significantly increased to normoxic levels with GTS21 treatment, which was abrogated by MLA pretreatment. Activation of stress-activated kinases JNK and p38MAPK was significantly reduced by GTS21 in I/R. We conclude that targeting myocardial α7nAChRs in I/R may provide therapeutic benefit by improving cardiac contractile function through a mechanism that preserves mitochondrial membrane potential, maintains intracellular ATP and reduces ROS generation, thus limiting infarct size.

Neural reflexes regulate inflammation and electrical activation of the vagus nerve reduces inflammation in models of inflammatory disease. These discoveries have generated an increasing interest in targeted neurostimulation as treatment for chronic inflammatory diseases. Data from the first clinical trials that use vagus nerve stimulation (VNS) in treatment of rheumatoid arthritis and Crohn’s disease suggest that there is a therapeutic potential of electrical VNS in diseases characterized by excessive inflammation. Accordingly, there is an interest to further explore the molecular mechanisms and therapeutic potential of electrical VNS in a range of experimental settings and available genetic mouse models of disease. Here, we describe a method for electrical VNS in experimental inflammation in mice.

The brain regulates physiological functions integral to survival. However, the insight into brain neuronal regulation of peripheral immune function and the neuromediator systems and pathways involved remains limited. Here, utilizing selective genetic and pharmacological approaches, we studied the role of forebrain cholinergic signaling in the regulation of peripheral immune function and inflammation. Forebrain-selective genetic ablation of acetylcholine release and vagotomy abolished the suppression of serum TNF by the centrally-acting cholinergic drug galantamine in murine endotoxemia. Selective stimulation of acetylcholine action on the M1 muscarinic acetylcholine receptor (M1 mAChR) by central administration of the positive allosteric modulator benzyl quinolone carboxylic acid (BQCA) suppressed serum TNF (TNFα) levels in murine endotoxemia. This effect was recapitulated by peripheral administration of the compound. BQCA also improved survival in murine endotoxemia and these effects were abolished in M1 mAChR knockout (KO) mice. Selective optogenetic stimulation of basal forebrain cholinergic neurons innervating brain regions with abundant M1 mAChR localization reduced serum TNF in endotoxemic mice. These findings reveal that forebrain cholinergic neurons regulate innate immune responses and inflammation, suggesting the possibility that in diseases associated with cholinergic dysfunction, including Alzheimer's disease this anti-inflammatory regulation can be impaired. These results also suggest novel anti-inflammatory approaches based on targeting forebrain cholinergic signaling in sepsis and other disorders characterized by immune dysregulation.

Obesity and obesity-associated disorders have become world-wide epidemics, substantially increasing the risk of debilitating morbidity and mortality. A characteristic feature of these disorders, which include the metabolic syndrome (MetS) and type 2 diabetes, is chronic low-grade inflammation stemming from metabolic and immune dysregulation. Inflammation in the CNS (neuroinflammation) and cognitive impairment have also been associated with obesity-driven disorders. The nervous system has a documented role in the regulation of metabolic homeostasis and immune function, and recent studies have indicated the important role of vagus nerve and brain cholinergic signaling in this context. In this review, we outline relevant aspects of this regulation with a specific focus on obesity-associated conditions. We outline accumulating preclinical evidence for the therapeutic efficacy of cholinergic stimulation in alleviating obesity-associated inflammation, neuroinflammation, and metabolic derangements. Recently demonstrated beneficial effects of galantamine, a centrally acting cholinergic drug and cognitive enhancer, in patients with MetS are also summarized. These studies provide a rationale for further therapeutic developments using pharmacological and bioelectronic cholinergic modulation for clinical benefit in obesity-associated disorders.

Macrophage cytokine production is regulated by neural signals, for example in the inflammatory reflex. Signals in the vagus and splenic nerves are relayed by choline acetyltransferase+ T cells that release acetylcholine, the cognate ligand for alpha7 nicotinic acetylcholine subunit-containing receptors (α7nAChR), and suppress TNF release in macrophages. Here, we observed that electrical vagus nerve stimulation with a duration of 0.1–60 s significantly reduced systemic TNF release in experimental endotoxemia. This suppression of TNF was sustained for more than 24 h, but abolished in mice deficient in the α7nAChR subunit. Exposure of primary human macrophages and murine RAW 264.7 macrophage-like cells to selective ligands for α7nAChR for 1 h in vitro attenuated TNF production for up to 24 h in response to endotoxin. Pharmacological inhibition of adenylyl cyclase (AC) and knockdown of adenylyl cyclase 6 (AC6) or c-FOS abolished cholinergic suppression of endotoxin-induced TNF release. These findings indicate that action potentials in the inflammatory reflex trigger a change in macrophage behavior that requires AC and phosphorylation of the cAMP response element binding protein (CREB). These observations further our mechanistic understanding of neural regulation of inflammation and may have implications for development of bioelectronic medicine treatment of inflammatory diseases.

Sepsis, a complex disorder characterized by immune, metabolic, and neurological dysregulation, is the number one killer in the intensive care unit. Mortality remains alarmingly high even in among sepsis survivors discharged from the hospital. There is no clear strategy for managing this lethal chronic sepsis illness, which is associated with severe functional disabilities and cognitive deterioration. Providing insight into the underlying pathophysiology is desperately needed to direct new therapeutic approaches. Previous studies have shown that brain cholinergic signaling importantly regulates cognition and inflammation. Here, we studied the relationship between peripheral immunometabolic alterations and brain cholinergic and inflammatory states in mouse survivors of cecal ligation and puncture (CLP)-induced sepsis. Within 6 days, CLP resulted in 50% mortality vs. 100% survival in sham-operated controls. As compared to sham controls, sepsis survivors had significantly lower body weight, higher serum TNF, interleukin (IL)-1β, IL-6, CXCL1, IL-10, and HMGB1 levels, a lower TNF response to LPS challenge, and lower serum insulin, leptin, and plasminogen activator inhibitor-1 levels on day 14. In the basal forebrain of mouse sepsis survivors, the number of cholinergic [choline acetyltransferase (ChAT)-positive] neurons was significantly reduced. In the hippocampus and the cortex of mouse sepsis survivors, the activity of acetylcholinesterase (AChE), the enzyme that degrades acetylcholine, as well as the expression of its encoding gene were significantly increased. In addition, the expression of the gene encoding the M1 muscarinic acetylcholine receptor was decreased in the hippocampus. In parallel with these forebrain cholinergic alterations, microglial activation (in the cortex) and increased Il1b and Il6 gene expression (in the cortex), and Il1b gene expression (in the hippocampus) were observed in mouse sepsis survivors. Furthermore, microglial activation was linked to decreased cortical ChAT protein expression and increased AChE activity. These results reinforce the notion of persistent inflammation-immunosuppression and catabolic syndrome in sepsis survivors and characterize a previously unrecognized relationship between forebrain cholinergic dysfunction and neuroinflammation in sepsis survivors. This insight is of interest for new therapeutic approaches that focus on brain cholinergic signaling for patients with chronic sepsis illness, a problem with no specific treatment.