Background: The vagus nerve plays an important role in neuroimmune interactions and in the regulation of inflammation. A major source of efferent vagus nerve fibers that contribute to the regulation of inflammation is the brainstem dorsal motor nucleus of the vagus (DMN) as recently shown using optogenetics. In contrast to optogenetics, electrical neuromodulation has broad therapeutic implications, but the antiinflammatory efficacy of electrical DMN stimulation (eDMNS) was not previously investigated. Here, we examined the effects of eDMNS on heart rate (HR) and cytokine levels in murine endotoxemia as well as the cecal ligation and puncture (CLP) model of sepsis. Methods: Anesthetized male C57BL/6 mice on a stereotaxic frame were subjected to eDMNS using a concentric bipolar electrode inserted into the left or right DMN or sham stimulation. eDMNS (50, 250 or 500 microA and 30 Hz, for 1 min) was performed and HR recorded. In endotoxemia experiments, sham or eDMNS utilizing 250 microA or 50 microA was performed for 5 mins and was followed by LPS (0.5 mg/kg) i.p. administration. eDMNS was also applied in mice with cervical unilateral vagotomy or sham operation. In CLP experiments sham or left eDMNS was performed immediately post CLP. Cytokines and corticosterone were analyzed 90 mins after LPS administration or 24h after CLP. CLP survival was monitored for 14 days. Results: Either left or right eDMNS at 250 microA and 500 microA decreased HR, compared with pre- and post-stimulation. This effect was not observed at 50 microA. Left side eDMNS at 50 microA, compared with sham stimulation, significantly decreased serum and splenic levels of the proinflammatory cytokine TNF and increased serum levels of the antiinflammatory cytokine IL10 during endotoxemia. The antiinflammatory effect of eDMNS was abrogated in mice with unilateral vagotomy and were not associated with serum corticosterone alterations. Right side eDMNS suppressed serum TNF levels but had no effects on serum IL10 and on splenic cytokines. In mice with CLP, left side eDMNS suppressed serum TNF and IL6, as well as splenic IL6 and increased splenic IL10 and significantly improved the survival rate of CLP mice. Conclusions: For the first time we show that a regimen of eDMNS which does not cause bradycardia alleviates LPS-induced inflammation and these effects require an intact vagus nerve and are not associated with corticosteroid alterations. eDMNS also decreases inflammation and improves survival in a model of polymicrobial sepsis. These findings are of interest for further studies exploring bioelectronic antiinflammatory approaches targeting the brainstem DMN.

Acute respiratory distress syndrome (ARDS) is a life‐threatening form of acute lung injury and respiratory failure. The pandemic of SARS‐CoV‐2 has resulted in over 5 million deaths worldwide and many of those deaths have been attributed to complications associated with ARDS. No efficient pharmacological treatments for ARDS are currently available. In addition to lung injury and pulmonary inflammation, patients with ARDS have increased systemic inflammation. Previous insights have highlighted the role of cholinergic signalling in the vagus nerve‐based inflammatory reflex in controlling inflammation (Annu Rev Immunol, 2018, 36:783). Cholinergic anti‐inflammatory signalling can be activated by galantamine ‐ a centrally acting acetylcholinesterase inhibitor that is also clinically approved (for the treatment of Alzheimer’s disease). Here we examined the efficacy of galantamine in attenuating ARDS severity and inflammation in mice. We used a recently developed model of direct acute lung injury and ARDS, in which intratracheal (i.t.) hydrochloric acid (0.1N HCl, 2ml/kg) instillation is combined with LPS (10 mg/kg, i.t.) instillation 24h later in anesthetized mice. Male, 12‐week‐old C57BL/6 mice (n=7‐9), were subjected to this two hit ARDS model and treated 30 mins prior to each insult with galantamine (4 mg/kg, i.p.) or vehicle. Mice were euthanized 6h after LPS instillation and blood and bronchoalveolar lavage (BAL) fluid were collected. Galantamine significantly lowered the levels of BAL myeloperoxidase – an indicator of the accumulation of activated neutrophils in the airspaces of the lung (p = 0.0021) and the BAL total protein ‐ an indicator of alveolar permeability (p = 0.0076), compared with vehicle‐treated mice. BAL pro‐inflammatory cytokine levels were also reduced by galantamine, compared with vehicle‐treated mice – TNF (p = 0.0274), IL‐6 (p = 0.0009), and IL‐1β (p = 0.0380). In addition, treatment with galantamine significantly lowered the levels of serum pro‐inflammatory cytokines ‐ TNF (p = 0.0015), IL‐6 (p = 0.0089), and IL‐1β (p = 0.0149). In conclusion, galantamine reduced the severity of ARDS in mice and alleviated the local and systemic inflammatory responses. These results are of interest for further preclinical and clinical development of galantamine in the treatment of ARDS.

The inflammatory reflex is a vagus nerve‐mediated and brainstem‐integrated physiological mechanism that regulates immunity and inflammation (Annu Rev Immunol, 2018, 36:783). The efferent vagus nerve originates in brainstem nuclei, including the dorsal motor nucleus of the vagus (DMN). Recently, it was shown that optogenetic stimulation of cholinergic DMN neurons inhibits serum TNF levels during endotoxemia (Proc Natl Acad Sci USA, 2020, 117(47):29803). However, the effects of electrical DMN stimulation on cytokine levels in inflammatory conditions have not been previously evaluated. To provide insight, we examined the effect of electrical DMN stimulation in mice with endotoxemia and cecal ligation and puncture (CLP)‐induced polymicrobial sepsis. In the endotoxemia model, 10–12‐week‐old male C57BL/6 mice (n=14‐20 per group) were anesthetized using isoflurane and the head was positioned on a stereotaxic frame. A surgical intervention and a stereotaxic‐guided approach were used to insert a concentric bipolar electrode into the left DMN. Electrical stimulation (50 μA, 30 Hz, 260 μs) or sham stimulation was performed for five mins, followed by lipopolysaccharide (LPS, 0.5 mg/kg) i.p. administration. The mice were euthanized 90 mins after LPS and blood (via cardiac puncture) was collected and processed for cytokine analysis. In the CLP model, DMN stimulation or sham stimulation was performed as described above immediately after subjecting mice (n=14‐20 per group) to CLP, i.e., following a midline laparotomy in anesthetised mice, the cecum was isolated and the distal end was ligated and punctured. 24h later, the mice were euthanised and blood was collected and processed for analysis. In mice with endotoxemia, electrical DMN stimulation significantly decreased serum TNF (p = 0.0069) and IL‐6 (p = 0.0427) levels compared with sham stimulation. In addition, electrical DMN stimulation increased the serum levels of the anti‐inflammatory cytokine IL‐10 (p = 0.0284). In mice with CLP, electrical DMN stimulation resulted in significantly lower serum TNF levels compared with sham stimulation (p = 0.0291). These results indicate for the first time that electrical DMN stimulation alters circulating pro‐inflammatory and anti‐inflammatory levels in murine endotoxemia and sepsis. These findings are of interest for further development of bioelectronic approaches targeting DMN for therapeutic benefit in conditions, characterised by immune dysregulation and inflammation.

In sepsis and other disorders characterized by peripheral immune and metabolic dysregulation, the brain also is affected and brain dysfunction, including neuroinflammation, metabolic changes and cognitive impairment have been reported (Annu Rev Immunol, 2018, 36:783‐812). Non‐invasive evaluation of neuroinflammation is important for strategizing new diagnostic and therapeutic approaches targeting the brain. Here we utilized Micro Positron Emission Tomography (microPET) with [18F]Fluoro‐2‐deoxy‐2‐D‐glucose (18FDG) and 11C‐Peripheral Benzodiazepine Receptor ([11C]PBR) to evaluate brain metabolic alterations and microglia activation (indicating neuroinflammation) during murine endotoxemia. Lipopolysaccharide (LPS, endotoxin) (2 mg/kg) or saline was injected (i.p.) in male C57Bl/6 mice 6 hrs prior to microPET imaging. [11C] PBR28 (~0.5 mCi) was injected via the tail vein followed by 60 mins dynamic imaging. Subsequently, 18FDG (0.5 mCi) was injected i.p. with a 10 mins static scan acquired following 40 mins uptake. Brain images were analyzed in a standard anatomical space using Statistical Parametric Mapping. Significantly (P<0.01) increased glucose metabolism in endotoxemic mice (vs controls) was observed in the hippocampal CA2/CA3 region and other brain areas. Increased [11C]PBR28 binding (P<0.05) was also determined in the hippocampal CA2/CA3 region. These brain alterations were associated with significantly increased serum cytokine levels (TNF, IL‐6, IL‐1β, and IL‐10) at 6 hrs following administration of the same (2mg/kg, i.p.) endotoxin dose. These results indicate brain hypermetabolic activity and neuroinflammation during endotoxemia specifically affecting the hippocampus – an area with a primary role in memory and cognition. These findings support further development of PET‐based evaluation of neuroinflammation in preclinical and clinical settings of inflammatory and metabolic disorders.