The Vagus Nerve and α-7 nicotinic acetylcholine receptors (α7nAChR)

1. PART 2 – The Vagus Nerve and α-7 nicotinic acetylcholine receptors (α7nAChR) (see ‘FUNVAX’ presentation)

2. 1Inhibition of prostaglandin biosynthesis has been proposed as the mechanism of action of aspirin-like drugs (Vane, 1971, 1972a, b). Whereas the anti-inflammatory and anti-pyretic aspects of their activity could be adequately explained by this mechanism, the analgesic action was more difficult to account for, because the available data concerning the 'algesic' activity of prostaglandins were, until very recently, contradictory. The release of prostaglandin-like material by intra-splenic injections of bradykininwas demonstrated in 15 dogs. Bradykininreleased prostaglandin-like material in amounts up to a peak concentration of 10 ng/ml (assayed as prostaglandin E2). The relative contractions of the assay tissues showed that the prostaglandin-like activity was mostly E type. The amounts released varied from experiment to experiment, but the higher dose (10 Ug)of bradykinin always released greater amounts of prostaglandin-like material than the lower dose (5 ug). Adrenaline (1-5 /ug) also released prostaglandin-like material (2-10 ng/ml) into the splenic venous outflow. Bradykinininjected intra-arterially into the spleen produced a reflex rise in blood pressure which was proportional to the dose. A rise in blood pressure of more than 5 mmHg was considered to be a response. This was seen in 7 out of 13 dogs with 01 ugbradykininand in all dogswith 0-5 ugbradykinin. 11973, Prostaglandinsand the mechanism of analgesiaproduced by aspirin-like drugs

3. The discovery thataspirin-like drugs inhibit the biosynthesis of prostaglandins(Vane, 1971,; Smith & Willis, 1971, Ferreira, Moncada & Vane, 1971) suggested that prostaglandins were involved in the mediation of pain. This possibility was further strengthened by the work of Collier & Schneider (1972) who found that‘ prostaglandins were the most powerful substances known for induction of the writhing reflexin mice. This effect, in contrast to that produced by other noxious stimuli, was not antagonized by aspirin-like drugs. Recently, on the basis of the effects of prostaglandin infusions into man, Ferreira (1972) suggested that their main contribution to the pain of inflammation was their ability to sensitize the sensory nerve endings to mechanical or chemical stimulation. Our present results reinforce and extend this conclusion by using the same system by which Lim, Guzman, Rodgers, Goto, Braun, Dickerson & Engle (1964) demonstrated that aspirin-like drugs exert their analgesic action peripherally, nearto or at the site of generation of pain. Bradykininin doses (1-10 ,ug) which produced pain when injected intra-arterially into the spleenof lightly anaesthetized dogs (Guzman et al., 1964) caused a release of prostaglandin-like material from the isolated prefused spleenof the dog (Moncada et al., 1972). We have now shown that there is a basal output of prostaglandin-like material from the spleenof the dog in vivo and this output is increased by injections of bradykinin.

4. Bradykininalso causes release of prostaglandin E2 from dog kidney (McGiff, Terragno, Malik & Lonigro, 1972). It seems unlikely that prostaglandinsdirectly mediate the pain-producing activity of bradykinin, for adrenalinealso releases prostaglandinsbut does not cause pain (Guzman et a!., 1964). These results further support the idea that prostaglandin release does not mediate the pain-producing action of bradykinin, but in some way sensitizes thepain receptors. The analgesic action of aspirin-like drugs can thus be explained by the abolition of the generation of prostaglandins which normally sensitize the pain receptors to the action of bradykinin. In other tissues such as the skin, no basal prostaglandin release has been shown. However, prostaglandins are found in inflammation(Willis, 1969 Our experiments do not rule out the possibility that part of the analgesic action of aspirin-like drugs is mediated by a central effect. If it is, this could also involve the prostaglandin system, in the spinal cord and in the sensory cortex. Some prostaglandinsalso can facilitate polysynaptic reflexes (Siggins, Hoffer & Bloom, 1971). There is indeed evidence that there is a central component to the action of paracetamol. This substance crosses the blood brain barrier more easily than aspirin(Davison, Guy, Levitt & Smith, 1961) and, unlike aspirin, increases the pain threshold in both inflamed and normal paws (Randall & Selitto, 1958). Thus, it could act by removing the facilitation produced by centrally-released prostaglandins.

5. 2Current dogma holds that nonsteroidal anti-inflammatory drugs (NSAIIDs) act by inhibition of thesynthesis and release of prostaglandins. However, NSAIDs also inhibit the activation of neutrophils, which provoke inflammationby releasing products other than prostaglandins. We now report that NSAIDs(e.g., 1indomethacin, piroxicam) inhibitactivation of neutrophils by inflammatory stimuli, such as C5-derived peptides and leukotriene B4,even when cyclooxygenase products generatedin suspensions of stimulated neutrophilsprostaglandin E and thromboxanesare present. It is generally accepted that aspirin-like drugs [nonsteroidal anti-inflammatory agents (NSAIDs)] exert their anti-inflammatory effects by inhibiting the 1-cyclooxygenases (COX) of various cells, thereby blockingthe extracellular release of stable prostaglandins First, stable prostaglandins and their analogues are anti-inflammatory; they not only inhibit activation of inflammatory cells in vitro (2-4) but also ameliorate experimental adjuvant arthritis (5) and immune-complex nephritis (6) in vivo. Our data indicate that, at highconcentrations of NSAIDs, their inhibitionof cyclooxygenase is neither necessary nor sufficient to account for inhibition of neutrophil function. 21985, Modes of action of aspirin-like drugs

6. How then do NSAIDsexert an inhibitory effect on neutrophilfunction? It is therefore of interest that calcium translocations, early signals in the activation sequence, are inhibited by NSAIDs. Calcium uptake, required for optimal cell responses(30), was inhibited by each NSAID tested It is known that agents that increase intracellular levels of cAMP, such as stableprostaglandins, inhibit neutrophil function (40). This has led to speculation that there is a "threshold" concentration of cAMPthat serves as a negative feedback toneutrophil Activation(40). In summary, our studies indicate that inhibition of neutrophil activation by NSAIDs occurs independently of effects on prostaglandin synthesis. While the precise mechanism remains to be elucidated, the data suggest that inhibition is achieved through effects on early signalling events essential to the activation sequence.

7. Laughing Stimulates Your Vagus Nerve!

8. As we are connecting the VagusNerve toall of these ailments, let us continue. 1Recent advances (pre-1971-1985 up to 2016) at the intersection of immunologyand neuroscience reveal reflex neural circuit mechanisms regulating innate and adaptive immunity (7, 8). One well-characterized reflex circuit, termed the “inflammatory reflex,” is defined by signals that travel in the vagus nerve to inhibit monocyte and macrophage (M1, M2) production of tumor necrosis factor (TNF) and other cytokines Electrical stimulation of thevagus nerve in animals (e.g., mouse, rat, and dog) stimulates choline acetyltransferase-positive T cells to secrete acetylcholine in spleenand other tissues. Acetylcholine is the cognate ligand for α-7 nicotinic acetylcholine receptors (α7nAChR) expressed on cytokine-producing monocytes, macrophages, and stromal cells Ligand binding inhibits thenuclear translocation of NF-κBand inhibits inflammasome activation in macrophages activated by exposure to lipopolysaccharide (LPS or what is termed ‘endotoxins’), other Toll-like receptor(TLR) ligands, and other pro-inflammatory stimulating factors 12016, Vagus nerve stimulation inhibits cytokine production and attenuatesdisease severity in rheumatoid arthritis,

10. 1Inflammatory reflex signalling, which is enhanced by electrically stimulating the vagus nerve, significantly reducescytokine productionand attenuates disease severity in experimental models of endotoxemia, sepsis, colitis, and other preclinical animal models of inflammatory syndromes In experimental collagen induced arthritis, vagotomyor selective disruption of α7nAChRworsened disease severity, and administration of nicotine or other selective α7nAChR agonists, ameliorated disease severity 12016, Vagus nerve stimulation inhibits cytokine production and attenuates disease severity in rheumatoid arthritis,

12. 1Vagus nerve-stimulating devices have been used for decades in patients with refractory epilepsyand have been used more recently in patients with depression Note: You, like me, may have already noticed a common denominatorhere. RA (Rheumatoid Arthritis), and now epilepsyand depression. Amongst the many drugs trialled to alleviate CVS (Cyclic Vomiting Syndrome) were also epilepsy drugs so I find it worthy of investigation into that specific pathwayand what the targetor receptors ofepilepsy drugs are. 1We implanted a cohort of epilepsy patients with a vagus nerve-stimulating device (left cervical vagus nerve).and observed that transient delivery of electrical current during general anaesthesia significantly inhibitedTNF production in peripheral blood monocytes. A subsequent study of 17 RA patients in an 84-d open-label trialalso revealed significantly decreasedTNF production and significantly improved clinical signs and symptoms of disease. The inhibition of TNF release following vagus nerve stimulation during general anesthesia cannot be attributed to a placebo effect, because the subjects were unconscious and were not aware of the nerve stimulation. Whole-blood production of interleukin IL-6 and IL-1βwas also inhibited significantly by vagus nerve stimulation 12016, Vagus nerve stimulation inhibits cytokine production and attenuates disease severity in rheumatoid arthritis,

14. This is important because it proves the placebo effect to be null due to patients being unaware they are being stimulated and that there was an observable and direct action endogenously affected by the stimulation, mainly diminished inflammatory markers IL-6 and IL-1β, as well as TNF, what could be usefully termed ‘the terrible trio’. 1To our knowledge (in 2016, now 6 years ago as I proof-read this), this is the first reportthat the delivery of electric current applied directly on the cervical vagus nerve to stimulate the inflammatory reflex inhibitstheendotoxin-induced release of TNF, IL-1β, and IL-6 in humans Well, not quite… this was done some decades ago…back in 11982, 38 years ago, with a ferret model 1The effect of electrical stimulation of the vagus nerve with different patterns of impulses (the total number of stimuli remaining constant) on gastric acid secretion and gastric motility were investigated in the anaesthetized ferret. Since the vagus nerve is considered only to influence the amplitude of gastric contractions, the basic electric rhythm being responsible for the frequency and wave form of the contractions (Szurszewski, 1977), the functional significance of the modulatedefferentdischarge is unknown. 11982, EFFECT OF STIMULATION OF THE VAGUS NERVE IN BURSTS ON GASTRIC ACID SECRETION AND MOTILITY IN THE ANAESTHETIZED FERRET

15. NE (Nicotonic Receptor) (Toll-like Receptor 4) (Endotoxin) NE Inflammation

16. Vagus Nerve Stimulation – The Spleen

17. 1When the same number of stimuli were delivered in bursts of 1 sec every 10 sec a maximum response occurred with bursts at 30 impulses/sec. Bursts at higher frequencies failed to produce a further increase in acid production. it can be seen that stimulating in bursts of 60 and 120 impulses/sec significantly reduced the output of gastric acid while bursts of 10 impulses/sec gave a significant increase in acid output The gastric contractions could be driven at frequencies up to 12/min (1 sec stimulation/4 sec off), but at intervals less than this the contractions summated, giving rise to a change in tonus similar to that seen during continuous stimulation. The period between bursts was therefore important in allowing individual contractions to occur in the absence of a change in tonus. Another possibility, however, is that continuous stimulation results in saturation of either the ganglion or target cell with transmitter, which results in a type of depolarization block which may be overcome by stimulating in bursts. Note: This1982study was also considering the importance ofthe choline pathway (“activating both the excitatory cholinergic pathwayand the non-adrenergic, non-cholinergic inhibitory pathway to the gastric smooth muscle” and “This is probably due to temporal summation within the enteric plexiwhereby successivepost-synaptic events are superimposed, resulting in an increase in acetylcholine output during stimulation in bursts, as has been described in cat superior cervical ganglion) but no data on inflammation biomarkers such as IL-6, TNF or IL-1b are mentioned as, it was not that kind of a study. 11982, EFFECT OF STIMULATION OF THE VAGUS NERVE IN BURSTS ON GASTRIC ACID SECRETION AND MOTILITY IN THE ANAESTHETIZED FERRET

18. That last statement is a powerful one, and will become more relevant as this thesis progresses. There are also other processes and pathways being activated, such as the a7NChR nicotine receptor and the involvement of the acetylcholine receptor, the FAAHand STAT3/JAK pathway, as well as endocannabinoid AG-2 and CN1R receptorsin down-regulating COX-2, prostaglandin E2 (seen in allergiesas IgE), and the balancing protacyclin PGI2, and the subunitsregulating and triggering NF-kB production, another harmful inflammatory process. In my studies with vitamin C (Ascorbic Acid) when it was being tested for concomitant use in conjunction with cortisone or hydrocotisone (more on this later) the studies went back into the late 1940s and 1950s and the same can be said for Rheumatoid Arthritis (RA) drug research, such as Methotrexate..1RA patients in cohort I are in early stages of disease not responding to therapy with methotrexate. These patients are frequently candidates for subsequent therapy with a biological agent that inhibits TNF, which is Methotrexate 1The RA patients with active disease were studied in two cohorts. Cohort I (n = 7) included patients with active disease despite therapy with methotrexate. They had never received a biological TNF antagonist or had previously failed treatment with TNF antagonists because of drug toxicity.

19. With most pharmaceutical drugs, the toxicity and tolerance of them and their metabolites are also the causes of tertiary problems on top of the source disease, therebycompounding the problem, complicating treatment, and pouring more financial burden via hospitalisation, admissions, the need for moreknowledgeable professionals, and/or specialists, due to severe adverse reactions, other health consequences such as depression and overall lower quality of life, brought on by the pharmaceutical drug used. The Liver and Kidneys take particular punishment Paracetamol Aspirin 12016, Vagus nerve stimulation inhibits cytokine production and attenuates disease severity in rheumatoid arthritis,

20. The vagus nerve was stimulated during surgery (day −14) to measure electrode impedance and to verify device function. During the 14-d postoperative recovery period (day −14 to day 0), the device was turned off, and no current was delivered to the vagus nerve. We observed that TNF production in cultured peripheral blood obtained from the combined RA study cohort on day 42 was significantly reduced from baseline day −21 (TNF = 2,900 ± 566pg/mL on day −21 vs. 1,776 ± 342pg/mL on day 42, On day 42 the vagus nerve stimulator was turned off. After a 14-d hiatus, it was restarted on day 56, and patients were followed through day 84. After the vagus nerve stimulator was turned off, TNF production increased significantly by day 56; when the stimulator was turned on again, TNF production again decreased significantly by day 84(1,776± 342pg/mL on day 42 vs. 2,617 ± 342pg/mL on day 56 and 1,975± 407pg/mL on day 84,. This finding indicates that active electrical stimulation of the vagus nerve inhibitsTNF production in patients with RA (Rheumatoid Arthritis). No ‘could’, or ‘may’, ‘possibly’, ‘hope’ or ‘might’.. It unequivocally does. Improvement was observed in all constituent components of the composite end points (tender joint count, swollen joint count, patient’s assessment of pain, patient’s global assessment, physician’s global assessment, and CRP). Together, these data indicate that vagus nerve stimulation inhibits TNF and significantly attenuatesRA disease severity. 12016, Vagus nerve stimulation inhibits cytokine production and attenuates disease severity in rheumatoid arthritis,

21. 1Serum IL-6 levels in subjects who improved by EULAR criteria were significantly decreased compared with subjects who failed to improve: IL-6 levels were 15.4 ± 2.4 pg/mL in non-responders vs. 5.0 ± 1.4pg/mLin responders. Decreased IL-6 levelsin the patients who responded to therapy correlated with improvement in disease severity between day −21 and day 42 IL-6 Down-regulated/inhibited 12016, Vagus nerve stimulation inhibits cytokine production and attenuates disease severity in rheumatoid arthritis,

22. 1The discovery of the inflammatory reflex affords a unique opportunity for developing aneuromodulating device to regulate immune cell function by targeting a neural pathway that regulatescytokine production, a surrogate marker of molecular mechanism After electrical stimulation of the vagus nerve theDAS28-CRPimproved significantly in both cohorts, and withdrawal of treatment significantly worsened the severity of disease. Reactivating the device on day 56 restored significant clinical improvement.. The clinical responses were accompanied by significant reductions in TNF release during periods of disease remission and significant increases in TNF release during disease exacerbation. A large body of preclinical evidence has delineated the molecular and physiological mechanisms of the inflammatory reflex modulating TNF, IL-6, HMGB1, and other cytokines (7–9, 11–20). The molecular mechanisms ofcytokine inhibition implicate acetylcholine derived from TChAtcells, the subset of choline acetyltransferase-positive T cellsthat we identifiedin the inflammatory reflex (9). In future clinical trials it should be interesting to study whether TChAt cells participate in mediating anti-inflammatory reflex mechanisms. 12016, Vagus nerve stimulation inhibits cytokine production and attenuates disease severity in rheumatoid arthritis,

24. The adverse events reported here were mild to moderate in severity and were comparable in type and frequency to those seen in prior studies of vagus nerve stimulation therapyinepilepsy patients. These adverse events included transient hoarseness, post-operative hoarseness from neuropraxis, and transient intraoperative bradycardiaduringsurgery. None of the patients developed infection. That last paragraph is quite seminal. InhibitingTNF-aas a key marker for infection, as is macrophage signalling via TLR2 as a pattern recogniser in pathogens. More on this later, Preclinical studies have established that stimulation of the inflammatory reflex for as little as 60 sconfers significant inhibition of cytokine production for up to 24 h.

27. 1The vagus nerve (VN), the principal component of the parasympathetic nervous system, is a mixed nerve composed of 80% afferent and 20% efferent fibers. The VN, because of its role in interoceptiveawareness, is able to sense the microbiota metabolites through its afferents, to transfer this gut information to the central nervous system where it is integrated in the central autonomic network, and then to generate an adapted or inappropriate response. A cholinergic anti-inflammatory pathway (CAP) has been described through VN’s fibers, which is able to dampen peripheral inflammation and to decrease intestinal permeability, thus very probably modulating microbiota composition. Stress inhibits the VNand has deleterious effects on the gastrointestinal tract and on the microbiota, and is involved in the pathophysiology of gastrointestinal disorders such as irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD) which are both characterized by a dysbiosis. Targeting the VN, for example through VN stimulation which has anti-inflammatory properties, would be of interest to restore homeostasis in the microbiota-gut-brain axis. The microbiota, the gut, and the brain communicate through the microbiota-gut-brain axis and a perturbation of this axisis involved in the pathophysiology of neurodegenerative disorders. The brain and the gut communicate in a bidirectional way, through the autonomic nervous system(ANS) and the circumventricularorgans 12018The Vagus Nerve at the Interface of the Mircobiota-Gut-Brain Axis - LactibacillusRhanmosa

28. 80% afferent fibers 20% efferent fibers

30. 1The vagus nerve (VN), the principal component of the parasympathetic nervous system, is considered as the sixth sensebecause of its role in interoceptive awareness Stress(cortisol) stimulates the sympathetic nervous system while inhibiting the VN. The VN, a mixed nerve with anti-inflammatory properties both through its afferent and efferent fibers, is at the interface of the brain-gut axis. Targeting the VN could restore homeostasis in such diseases. as IBS and IBD. In particular, VN stimulation (VNS), approved for the treatment of depression and epilepsyand for its anti-inflammatory properties The communication between the brain and the microbiota is bidirectional, through multiplepathways: neural through the VN and/or spinal cord, endocrine(through the hypothalamic pituitary adrenal, HPA, axis), immune(cytokines), and metabolic[short chain fatty acids, (SCFAs), and tryptophan Neuroactive compounds are released by bacteria such as g-aminobutyricacid (GABA), serotonin, dopamine, acetylcholine(ACh), and essentially act locally on the enteric nervous system i.e., the gut-brain 12018The Vagus Nerve at the Interface of the Mircobiota-Gut-Brain Axis - LactibacillusRhanmosa

32. COX-2

33. 1Enteroendocrine cells (EECs), 1% of intestinal epithelial cells, interact with vagal afferents either directly through the release of serotonin (5-hydroxytryptamine, 5-HT) activating 5-HT3 receptors located on vagal afferent fibers. EECs detect signals from microbiota throughtoll-like receptors (TLR) which recognize bacterial products such as lipopolysaccharides (LPS/Endotoxins) and others. Thus, EECs are key players in the detection of luminal bacterial content and bacterial products that can regulate GI motility, secretion, food intake, through their indirect effect on vagal afferent fibers The TLRs are an important contributor to pathogen signalling responsesand shall be discussed later in the powerpoint. But, as you can see, there are definitive pathways to the brain and serotonin, dopamine(Parkinson’s Disease) and Acetylcholine Receptors (a7nAChR) Victims of CVSare generally administered 5-HT3 antagonist drugs (Serotonin Type 3/L-tryptophan) including for depression and fibromyalgia and triptanssuch as Sumatripan, Trazadone (a 5-HT2A antagonist) and Ondansetronto abort the vomiting stage which cross the blood-brain barrier (BBB) In addition, TLR4 are expressed on vagal afferent fibers and these fibers could sense bacterial products such as LPSto activate the brain. 12018The Vagus Nerve at the Interface of the Mircobiota-Gut-Brain Axis - LactibacillusRhanmosa

36. Healthy mice chronically treated with Lactobacillus rhamnosuspresented GABA brain expression modificationswhich increased in the cingulate cortex and decreased in the hippocampus, amygdala, and locus coeruleus. These animals had also reduced stress-induced corticosterone and anxiety and depression-related behaviour. These effects were not observed after vagotomy. Hence, L. rhamnosus have potential therapeutic indications instress-related disorderspointing out the vagally-mediated microbiota effect on mood.

37. As electrical stimulation of vagal afferent fibers modifies brain concentration of serotonin, GABA, and glutamate and is used in the treatment of drug resistant epilepsy and depression this study suggests that probiotic-mediated VN activation could have beneficial effects in these diseases The VN is able to inhibit M1 pro-inflammatory macrophages.Central vagal stimulation with a thyrotropin-releasing hormone analog, known to activate preganglionic neurons of the dorsal motor nucleusof the VN, activates M2 macrophages which are in close connection with gastric cholinergic myenteric neurons and have anti-inflammatory properties, and deactivatespro-inflammatory M1 macrophages in the stomachwhich are involved in abdominal surgery-induced gastric inflammation and gastric ileus and this anti-inflammatory effect could modify intestinal permeability and/or the gut microbiota. Vagal afferents activate vagal efferentsin an inflammatory reflexdescribed in 2000 by the group of Tracey in a model of septic shock and called the cholinergic anti-inflammatory pathway (CAP) This CAP mechanism will be explored more as we discuss the a7nAChR Nicotine receptor and NO (Nitric Oxide) production to reduce inflammation; the ‘smoking paradox’

38. Activation of the vagus nerve leads to the release of acetylcholine (Ach) that binds to α-7 nicotinic receptors (α7 nAChR) on cytokine producing cells. This inhibits the activation of NF-κBand activates STAT3 phosphorylation. Phosphorylated STAT3 triggers an anti-inflammatory signal by increasing SOCS3 expression,which suppresses signallingfromcytokine receptors. Together, these signals prevent the releaseofTNF, HMGB1, and other cytokines implicated in the pathogenesis of inflammatory disease.

39. Indeed, ACh released at the distal end of vagal efferents inhibits the release of TNFa bymacrophagesthrough their a7nicotinicACh receptors(a7nAChR). In the same way, a vago-sympathetic pathway to the spleeninhibitingTNFa release by splenic macrophageshas been described and for some authors the splanchnic pathway is the efferent arm of the inflammatory reflex. When targeting vagal afferents to the brain, the gut microbiota could modulate this inflammatory reflexeither activating or inhibiting the VN thus being anti-or pro-inflammatory. In the same model, VNShad a protective effect, independent of the spleenbut involving a cholinergic nicotinic receptor since this effect was reproduced with nicotine. The a7nAChR protects againstburn-induced gut barrier injury by preventing the decreased expression and altered localization ofoccludinandzonula occludens-1 (ZO-1). The mechanism through which vagal efferentsprotect barrier epitheliumis not well-known. One of the mechanismscouldbe the connection of the VN with the enteric nervous system which communicates with enteric glia cells through nicotinic cholinergic signalling. However, a recent study has shown that enteric glia is not required for maintenance of the intestinal epithelium Consequently, vagal activity provides a protective function to the intestinal epithelial barrierand a low vagal activity makes intestinal epithelium more permeablethus promoting systemic inflammation and chronic disease. We can hypothesize that the VN could modulate gut microbiota compositionthat depends on these two factors

41. Stress in the GI tract, through its neuromediators, corticotrophin-releasingfactor(CRF) and urocortinare released by mastocytes of the lamina propriawhich have CRF1-2 receptors, activation of which releases cytokines and other pro-inflammatory mediators by mastocytes. CRF also increases intestinal permeabilityvia mast cell release of tumor necrosis factor (TNF)-a and proteases As a note, in my research into Asthmaand COPD (emphesyma), the connection with the release of Proteasesis also worthy of note in regards to inflammation progress TargetingCRF receptors by selective antagonists to inhibit mast cell activation is a therapeutic option for chronic inflammatory disorders exacerbated by stress. Chronic early life stress induces dysbiosisin rats via modifications of intestinal permeability, which may later sensitize adult rats to visceral hypersensitivity Classically, stress inhibitsthe VN and stimulates the sympathetic nervous system through autonomicrelated projection neurons of the PVHto the dorsal motor nucleus of the VN and sympathetic pre-ganglionic neurons of the spinal cord. Since the VN has anti-inflammatory properties through its afferent and efferent fibers, stress has thus pro-inflammatory properties. A single acute stress induces a prolonged increase of pro-inflammatory cytokines after the end of stress exposition, at the moment of the recovery period which is a critical period since it corresponds to the parasympathetic rebound. Exposition to multiple repeated stressors counteracts the parasympathetic tone recovery, favouring an *allostatic load ("the wear and tear on the body" which accumulates as an individual is exposed to repeated or chronic stress) thusblunting the anti-inflammatory regulatory action of the VN.

42. Stress could counterbalancethe overall protective effect of the VN on epithelial barrierand thus favoradysbiosisby disrupting epithelial homeostasis. aamicrobial imbalance or maladaptation on or inside the body, such as an impaired microbiota  gut flora, can become deranged, with normally dominating species underrepresented and normally outcompeted or contained species increasing to fill the void

43. In summary, I’ve shown you how the Vagus Nerve is a central controller for neurotransmitter and neural pathways controlling inflammation(leading to incoceptive pain) and the inhibition of pro-inflammatory cytokine TNF-1aagain. Using the CAP (cholinergic anti-inflammatory pathway)and the targeting of a7nAChR receptor and Nicotine is something we’ll further explore as it relates to endogenous production of CB1R receptors in the synthesis of Anandamine via 2-AG, and increasing innate immunity via conversion of Long chain Polyunsaturated Fatty Acids (PUFAs). We may touch upon Toll-like Receptors TLR2 and TLR4 at some point as they relate to pathogens/bacteriathat can disturb and disrupt gut microbiota 2Animal and human studies have shown that VNS influences the activity of norepinephrine, serotonin, and other neurotransmitters implicated in mood disorders. Like other anti-depressant therapies, VNS increases the expression of the neurotrophin brain-derived neurotrophicfactor(BDNF) and activates its receptor, and also stimulates hippocampal neurogenesis 22014, Vagus Nerve Stimulation Keywords: CAP, TLR, CB1R, TNF-a, PUFA, Vagus Nerve (VN), Vagus Nerve Stimulation (VNS), parasympathetic rebound, cytokines, a7nAChR, nicotine, macrophage, 5-HT3receptors, g-aminobutyric acid (GABA), Dopamine, Microbiota, Sympathetic Nervous System, Parasympathetic Nervous System, neurotrophin brain-derived neurotrophic factor (BDNF)

46. Some other statements of interest from other studies: 1In this study, transcutaneous electrical stimulationof the auricular branch of the right vagus nerve at the tragus suppressed AF (atrial tachycardia) and decreased inflammatory cytokines. This proof-of-concept and first-in-man study raises the possibility that non-invasiveautonomic neuromodulationmay be used to treat patients with paroxysmal AF. Notably, the average stimulation voltage used in this study was less than the average discomfort threshold, suggesting that this treatment modality may be tolerated by ambulatory patients. Note: This would reduce the need for I.V or oral doses of Ondansetronor other GERD-like drugs to stop vomiting episodes of CVS (Cylical Vomiting Sydrome), and, ergo, less toxicity and decreases in pro-inflammatorycytokinesby moving from the sympathetic mode (and remaining) into >> the parasympathetic one Moreover, the magnitude of decrease in TNF-α and CRP levelsby LLTS is consistent with the difference between controls andpre-rheumatoid arthritis patients (27) as well as for patients with active versus inactive inflammatory diseases, suggesting a significant biological effect. the Long-term effects of LLTS remain unknown, these results should be interpreted cautiously.. 12015, Low-level transcutaneous electrical vagus nerve stimulation suppresses atrial fibrillation

47. Stimulation of the cholinergic anti-inflammatory pathway (CAP), which is comprised of efferent vagus nerve signals, leads to acetylcholine-dependent activation of the alpha7 nicotinic acetylcholine receptor subunit on monocytes and macrophages, resulting in reduced production of the inflammatory cytokines TNF-α, IL-1β, and IL-6,but not IL-10 IL-17, IL-22 12015, Low-level transcutaneous electrical vagus nerve stimulation suppresses atrial fibrillation

49. 1The cholinergic anti-inflammatory pathway (CAP) involves efferent vagus nerve fibers, travelling to the spleen through the celiac ganglion and the splenic nerve to inhibit inflammatory responses. Importantly, we have provided evidencethatthe cholinergic anti-inflammatory pathway (CAP) can be activatedwithoutheart rate slowing, consistent with a previous experimental study, which demonstrated that transcutaneous vagus nerve stimulation (TENS) inhibitedinflammatory cytokine production in mice with endotoxemia. Intermittent stimulation may be more efficacious than continuous, based on recent experimental evidence 2The relationship between depression, inflammation, metabolic syndrome, and heart disease mightbe mediated by the vagus nerve. VNS deserves further study for its potentially favourable effects on cardiovascular, cerebrovascular, metabolic, and other physiologicalbiomarkers associated with depression morbidity and mortality. The sympatheticand parasympathetic are both components of the autonomic nervous system (ANS); the 20% efferent cholinergic fibers80% afferent (sensory) fibres carrying information from the bodyback to the brain) are the main parasympathetic component of the ANS Electrical VNS studies were conducted during the 1930s and 1940s to understand the influence of the ANS on modulating brain activity. Cardio-respiratory stimulation of the vagus nerve may explain some of the positive emotional and cognitive benefits of deep breathing, yoga, or aerobic exercise activities. Experience in epilepsy populations has shown that VNS is effective, safe, and well tolerated in pediatric patients. 22014, Vagus Nerve Stimulation