Neural Reflex Control of Inflammation During Sepsis Syndromes

“Healthy organs behave as ‘biological oscillators’, which couple to one another, and this orderly coupling is maintained through a communication network, including neural, humoral, and cytokine components” (Godin & Buchman, 1996). The nervous system –acting through the autonomic nervous system (ANS)– coordinates the fine-tuning of cardiorespiratory interplay, to maintain an appropriate oxygen delivery to the tissues (Abboud & Thames, 1983; Eyzaguirre et al., 1983). Autonomic (sympatheticparasympathetic) balance is maintained by several reflex arcs, like arterial baroreflexes (Kirchheim, 1976), central chemoreflexes, peripheral arterial chemoreflexes, and pulmonary stretch reflexes (Liljestrand, 1958). These reflexes represent the major components of blood pressure and breathing regulation. Therefore, the interactions among these reflexes are of special clinical interest, since the overactivity of a single reflex, occurring pathophysiologically in several disorders, can lead to the suppression of opposite reflex responses (Schmidt et al., 2001).


Introduction
"Healthy organs behave as 'biological oscillators', which couple to one another, and this orderly coupling is maintained through a communication network, including neural, humoral, and cytokine components" (Godin & Buchman, 1996).The nervous system -acting through the autonomic nervous system (ANS)-coordinates the fine-tuning of cardiorespiratory interplay, to maintain an appropriate oxygen delivery to the tissues (Abboud & Thames, 1983;Eyzaguirre et al., 1983).Autonomic (sympatheticparasympathetic) balance is maintained by several reflex arcs, like arterial baroreflexes (Kirchheim, 1976), central chemoreflexes, peripheral arterial chemoreflexes, and pulmonary stretch reflexes (Liljestrand, 1958).These reflexes represent the major components of blood pressure and breathing regulation.Therefore, the interactions among these reflexes are of special clinical interest, since the overactivity of a single reflex, occurring pathophysiologically in several disorders, can lead to the suppression of opposite reflex responses (Schmidt et al., 2001).
Sepsis syndromes (SS), which include systemic inflammatory response syndrome (SIRS) and its consequences, severe sepsis and septic shock, involve many pathological processes like systemic inflammation, coagulopathies, hemodynamic abnormalities, and multiple organ dysfunction syndrome (MODS) (Riedemann et al., 2003).The progression of MODS associated to systemic inflammation is mainly due to an uncontrolled release of proinflammatory mediators, which damage parenchymatous organs.Additionally, sepsis activates and/or depress numerous other systems within the body, including neural, hormonal, and metabolic pathways (Carre & Singer, 2008;Singer et al., 2004).Thus, systemic inflammation would initiates disruption of communication and uncoupling, and subsequent MODS would reflects the progressive uncoupling of 'biological oscillators' that can become irremediable.
Increasing evidences here summarized shown that a particular neural reflection, the carotid body chemoreflexes, not only serves as a chemoreceptor for respiratory reflex responses, as traditionally accepted, but also as a sensor for the immune status, as modulator of autonomic balance tending to coordinate cardiorespiratory interplay, devoted to maintain oxygen homeostasis in different pathologies, and as a protective factor during sepsis and MODS.

Sepsis syndromes prevalence and current therapies
Sepsis is defined as "the systemic inflammatory response that occurs during infection" (Bone et al., 1992).It involves the evidence of infection and two or more of the following conditions: fever or hypothermia, tachycardia, tachypnea or a respiratory frequency resulting in an arterial PCO2 below 32 mm Hg, and altered white blood cells count; severe sepsis, sepsis associated with organ dysfunction, hypoperfusion or hypotension including lactic acidosis, oliguria, or acute alteration in mental state; septic shock, sepsis-induced hypotension despite adequate fluid resuscitation, and sustained perfusion abnormalities; and multiple organ dysfunction syndrome (MODS), by the presence of altered organ function in an acutely ill patient such that homeostasis cannot be maintained without intervention (Riedemann et al., 2003).
Significant demographic variation exists in the risk of developing sepsis.For example, from the standpoint of gender, the incidence of sepsis is higher in men, and the mean age at which men develop sepsis is younger.Case fatality rates also increase with age (Martin et al., 2006).The overall burden of severe sepsis is also increasing, in terms of both the number of patients who develop the syndrome and the extent and intensity of care that they require (Angus et al., 2001).Sepsis also poses a significant burden of disease in pediatric patients, where the incidence is highest in infants, mainly in children younger than one year of age (Watson & Carcillo, 2005).Maternal sepsis and neonatal sepsis are of particular concern.Maternal sepsis is responsible for at least 75,000 deaths annually, disproportionately affecting low-income countries (van Dillen et al., 2010).In the United States, studies of neonatal sepsis have documented rates as high as 170 cases per 1000 live births (Thaver & Zaidi, 2009).The average costs per case are US$22,100.Costs are higher in infants, nonsurvivors, intensive care unit patients, surgical patients, and patients with more organ failure.The incidence was projected to increase by 1.5% per annum (Angus et al., 2001).The international costs associated with sepsis and its management are reviewed in Chalupka & Talmor (2012).
Instead of many efforts and significant advances in maintaining therapies, SS and MODS, are the main cause of death between critical care patients (Martin et al., 2003).Increased morbi-mortality associated to SS is due to the absence of a really effective therapy (Riedemann et al., 2003).Thus, the knowledge of molecular mechanisms and pathophysiology of sepsis help us to improve current therapies (for a Review see Barochia et al., 2010) and to identify new pharmacological therapeutic targets.
Treatments of sepsis and septic shock involves antibiotic administration, intravenous fluids (crystalloids or colloids), vasopressors and/or inotropes (adrenergic agents), packed red blood cells (PRBC) transfusions, and corticosteroids (Barochia et al., 2010).Sepsis care bundles increase patients' survival.Numerous studies have demonstrated improved outcomes in life-threatening infections with early administration of appropriate antibiotics.Hemodynamic support with fluids and vasopressors is as important as antibiotic in reducing mortality (Natanson et al., 1990), but there are great differences among different patient populations.A considerable variation in the ranges of central venous pressure and mean arterial pressure prompted physicians to suggest that "the usage should be individualized to different patients, based on their own underlying medical conditions" (Perel, 2008).
Administration of PRBC decreases inotropes use (Nguyen et al., 2007), but the efficacy of administration in patients with sepsis is unclear.The usage of low-dose corticosteroids is variable between patient populations.However, as questions persist regarding the risk and benefits of these therapies for sepsis, they continue to undergo investigation (Misset et al., 2010).Although the use of these agents may be beneficial for some septic patients, the Surviving Sepsis Campaign guidelines (Dellinger et al., 2004) gave a weak recommendation for use these therapies, even the inclusion of some patients, until the knowledge of individual components that could modify the expected results.It is clear that the course of sepsis and therapies outcomes depend largely from host predisposition factors and response.
The serial evaluation of the SOFA score helps to predict outcome in critically ill patients.SOFA score can help assess organ dysfunction or failure over time and are useful to evaluate morbidity and mortality, by evaluating respiratory, coagulation, liver, cardiovascular, central nervous system (CNS), and renal variables (Peres et al., 2002).However, in spite of SOFA score assessment, "It is more important to know what sort of person this disease has, than what sort of disease this person has" (William Osler, 1849-1919).

Pathophysiology of sepsis and multiple organ dysfunction syndrome
As it was mentioned, the progression of MODS is due to an uncontrolled release of proinflammatory mediators, which damage parenchymatous organs.However, it is still unknown why sepsis progresses to MODS in only certain individuals or what the exact pathway is that leads to this.But, it is clear that if the inflammatory process becomes selfsustained and progressive, MOD results.In addition, because of marked hypotension and tissue hypoperfusion, oxygen delivery fails to meet tissue oxygen demands, which results in a compensatory increase in oxygen extraction.If the imbalance between oxygen delivery and consumption is not corrected, tissue 'dysoxia' progress to an anaerobic metabolism and lactate production (Nguyen et al., 2004).Persistent serum lactate elevation is an important marker of decreased tissue perfusion -even in the absence of arterial hypotension (Howell et al., 2007)-, and is strongly associated with mortality rate in critically ill patients (Meregalli et al., 2004).Thus, during sepsis an extraordinarily complex and intricate cascade of inflammatory mediators, extra-and intra-cellular signaling pathways are activated, resulting in microvascular dysregulation and/or mitochondrial dysfunction ('cytopathic hypoxia') (Crouser, 2004), which culminate in MODS and death.
To avoid tissue dysoxia, early in the course of sepsis, cardiac output (CO) rises to maintain blood pressure and organ perfusion in the face of reduced peripheral vascular resistance ('hyperdynamic sepsis').As sepsis progresses, CO is frequently reduced ('hypodynamic sepsis'), which has a poor prognosis.Cardiac dysfunction per se is apparent in up to 44% of critically ill septic patients, with the etiological agents suspected to be circulating depressant factors (Singh & Evans, 2006).Elevated cardiac biomarkers (e.g., Troponin I (ver Elst et al., 2000;Yucel et al., 2008)) in conjunction with electrocardiographic (ECG) changes are valuable in the diagnostic of sepsis and in the assessment of progression.Raised Troponin I levels in patients with sepsis result from various mechanisms, including hypoperfusion or direct extension of infection to cardiac tissue.Electrocardiographic changes in sepsis are not as well described.Some of them include loss of QRS amplitude, increase in corrected QT (QTc) interval, bundle branch blocks, and development of narrowed QRS intervals with deformed, positively deflected J waves (Martinez et al., 2009).
Contradictory evidences from animal studies suggest that such hypoperfusion does not invariably lead to heart dysfunction and death.But, our preliminary results (unpublished data) reveal many other ECG and vectorcardiographic changes in rats injected intraperitoneally (i.p.) with 15 mg/kg lipopolysaccharide (LPS), which are strongly associated with cardiac dysfunction and, almost certainly, left vetricular hypoperfusion and ischemia.Briefly, LPS administration decreases RR interval (RRI) and R amplitude.Also, sepsis increases QTc interval and ST height.Strikingly, when both carotid/sinus nerves are sectioned (bilateral carotid neurotomy (BCN) prior to LPS administration, the changes in the parameters mentioned above are greater than control condition (with intact carotid chemoand baro-sensory innervations).In addition, BCN decreases QRS duration, increases JT interval and T amplitude.On the other hand, the cardiac vector is significantly decreased (from ca.65º to ca. 15º) As it was mentioned, the major task toil of autonomic nervous system (ANS) is the finetuning of the cardiorespiratory interplay, in order to maintain an appropriate oxygen delivery to the tissues.However, the neural regulation of cardiorespiratory function and the role-played by peripheral reflexes during sepsis, in which organ communications networks are disrupted, is poorly understood.In addition to plasma or urinary levels of neurotransmitters or their metabolites, there are three methods to evaluate autonomic function: a) analysis of heart rate variability (HRV); b) baroreflex sensitivity (BRS); and c) cardiac chemoreflex sensitivity (CCRS).
The analysis of HRV gives a clear idea about the neural (autonomic) control of cardiorespiratory function and interaction.Decreased HRV is consistent with the pathogenesis of MODS, which involves the physiological uncoupling of vital organ systems.In fact, HRV decreases in response to human endotoxemia (Godin et al., 1996;Rassias et al., 2005), and is a good index of cardiac mortality (Schmidt et al., 2001).Moreover, patients with sepsis (Barnaby et al., 2002) and MODS (Korach et al., 2001;Schmidt et al., 2005) have an impaired sympatho-vagal balance.In fact, some evidences describe a sustained sympatho-excitation during sepsis, which accompanies the fall in blood pressure.Baroreceptors and chemoreceptors denervation accelerated the fall in mean blood pressure and increases sympathetic tone (Vayssettes-Courchay et al., 2005).Thus, under altered baro-and chemo-reflexes pathways, the sympathetic output from the medulla appears to play a key role in the correlation between heart rate and sympathetic nerve activity.On the other hand, decreased parasympathetic tone is a good predictor of risk of death in patients with sepsis (Chen et al., 2008).Altogether, these data suggest that reflex arcs involved in maintaining the autonomic balance are altered during sepsis.Vayssettes-Courchay et al. (2005) shown that baro-and chemo-reflexes are not inhibited during sepsis, and they give them a minor importance in the sympathetic activation and in the blood pressure modifications.Nevertheless, recently we described the first functional evidence of chemoreceptors inflammation and dysfunction during sepsis.In cats, local or systemic administration of LPS induces a significant reduction in chemoreceptor activity, ventilatory chemoreflexes, and ventilator chemosensory drive (Fernandez et al., 2008).In fact, LPS-induced tachypnea is prevented by prior bilateral carotid neurotomy.
Our results (unpublished data) shown that the i.p. administration of 15 mg/kg LPS to rats, decreases HRV and increases sympathetic tone, assessed by HRV frequency bands and low frequency/high frequency (LF/HF) quotient.Bilateral carotid neurotomy previous to LPS administration evokes a greater decrease in HRV and increase in LF/HF ratio than animals with intact carotid/sinus nerves.As it was mentioned, both decreased HRV and increased sympathetic tone are good markers of morbi-mortality.In fact, BCN prior to LPS administration increases the relative risk of death (Table 1).In addition, rats submitted to peripheral chemodenervation prior to the intravenous (i.v.) administration of high doses of LPS, show a smaller survival time (Tang et al., 1998).Baroreflex sensitivity describes ANS capacity to increase vagal activity and to decrease sympathetic activity after a sudden increase in blood pressure.Baroreflex activation counteract sympathetic activation (Somers et al., 1991).BRS is altered in rats treated with a lethal dose of LPS (Shen et al., 2004).Rougaush et al. reported an increased BRS after bacterial sub-pyrogenic dose of endotoxin.The change in sensitivity may underlie necessary adjustments to altered blood flow distribution after LPS administration (Rogausch et al., 2000).However, Schmidt et al. reported a marked decrease in BRS during MODS (Schmidt et al., 2005).Thus, there is no consensus about the role played by arterial baroreceptors during sepsis.Classically, the stimulation of peripheral chemoreceptors evokes respiratory and cardiovascular effects and a sympatho-excitatory response (Alanis et al., 1968;Montarolo et al., 1976).Cardiac chemoreflex sensitivity (CCRS) allow us to estimate the sympathetic influence upon cardiorespiratory responses (Schmidt et al., 1999).Hyperoxia decreases autonomic function i.e., decreased CCRS and increases BRS.CCRS gives an important component of the cardiorespiratory interactions in patients with MODS.Severity of illness is the more pronounced determinant of impaired CCRS (Schmidt et al., 2004).Recently, Schueller et al. described a reduced CCRS in critical ill patients (sepsis or cardiogenic shock).Moreover, there is a close negative correlation between the CCRS and the SOFA-score (Schueller et al., 2008).
In summary, there is consensus that uncoupling of the autonomic, respiratory and cardiovascular systems occurs over both short-and long-range time scales during sepsis and MODS.However, the origin from these altered reflex arcs is not well described.

Inflammatory mediators during sepsis
The development of sequential organ failure in critically ill patients with sepsis is strongly predictive of mortality.However, the mechanisms involved in the dynamic interaction between different organ systems are dictated by the intricate interplay of homodynamic, oxygen transport, and metabolic disturbances.Genetic predisposition is almost certainly relevant in upregulating the expression of inflammatory mediators [e.g., tumor necrosis factor (TNF), interleukin (IL)-1, IL-6, high mobility group box (HMGB) 1], thereby influencing adversely the anti-/pro-inflammatory balance.
Mammals are continuously exposed to different pathogens, like Gram-negative bacteria and/or its components, such as LPS (endotoxin).LPS exerts many different biological effects.While low-doses could be beneficial, by inducing immunostimulation and by increasing resistance to infection (Schletter et al., 1995), larger-doses of LPS in plasma evoke many pathophysiological reactions, like fever, leucopenia, tachycardia, tachypnea, hypotension, disseminated intravascular coagulation, MODS, and death (Patel et al., 2003;Hotchkiss & Karl, 2003;Pinsky, 2004).The systemic inflammatory response induced by LPS is due to host cells stimulation (monocytes/macrophages, endothelial, and polymorphonuclear cells) to produce and release endogenous mediators like reactive oxygen species (ROS) and proinflammatory cytokines (Schletter et al., 1995).Inflammatory mediators and ROS are believed to disrupt communication pathways between organs, which precedes organ failure.Indeed, endothelial dysfunction has been proposed as a common pathway for organ dysfunction in sepsis (Simon & Fernandez, 2009).During systemic inflammation, many physiological functions of endothelial cells are disrupted, contributing to multiple organ failure (Volk & Kox, 2000).
During the last decade, there has been a rapid progress in understanding innate immune response to pathogens or their component.The early concept supposed a nonspecific recognition.But, the discovery of Toll-like receptors (TLRs) showed that recognition by the innate immune system is specific (Akira et al., 2001).TLR-4 is identified as the long-sought receptor that respond to bacterial LPS (Akira et al., 2006).TLR4 forms a complex with MD-2 on the cell surface.Additional proteins such as the soluble plasma protein LPS-binding protein (LBP) and either soluble or membrane-anchored CD14 are also involved in LPS binding (Akashi-Takamura & Miyake, 2008).LPS transfer to the LPS-binding receptor (TLR-4/MD-2) (da Silva et al., 2001), activates nuclear factor-B (NF-B), a transcription factor involved in the synthesis and release of immune system-related cytotoxic factors, by stimulation of pro-inflammatory and immunoregulatory molecules synthesis in mononuclear cells (monocytes /macrophages and neutrophils), like IL-1, IL-6, TNF-, IL-10, and transforming growth factor (TGF)- (Medvedev et al., 2000;Sanlioglu et al., 2001).
Tumor necrosis factor- has been implicated as an important mediator of the lethal effect of endotoxin.Several publications have shown that by reducing the activity or the expression of TNF- significantly decrease the endotoxin-induced damages.The amount of TNF- in serum can be associated with the degree of tissue damage because of the stagnant blood capillary (Yang et al., 2007).TNF- is a well-known cytotoxic cytokine for certain tissue cells.In fact, plasma levels of several biophysical damage indicators are increased during sepsis, like liver alanine aminotransferase, aspartate aminotransferase, and bilirrubin; heart and other possible organ (such as muscle) lactic dehydrogenase and creatine phosphokinase; ureic nitrogen (BUN, renal function); and pancreatic alkaline phosphatase and amylase.

Reflex control of inflammation: Part I -Brain-to-immune communication
Inflammation is a localized protective response to infection or injury.It evokes many different effects upon the organisms tending to solve the inflammatory focus, like humoral factors which increase the blood flow or attract specific immune cells (Libert, 2003).As it was mentioned above, TNF-α, plays a pivotal role during systemic inflammation.Excessive inflammation and TNF-α synthesis increase morbi-mortality in SS.In consequence, highly conserved endogenous mechanisms normally regulate the magnitude of innate immune response and prevent excessive inflammation (Wang et al., 2003).
The CNS regulates systemic inflammatory responses to endotoxin through neural and humoral mechanisms.Evidence accumulated over the last 30 years suggests that norepinephrine (NE), the main neurotransmitter of the sympathetic nervous system, fulfills the criteria for neurotransmitter/neuromodulator in lymphoid organs: i) primary and secondary lymphoid organs receive extensive sympathetic/noradrenergic innervation; ii) under stimulation, NE is released from the sympathetic nerve terminals in these organs; and iii) the target immune cells, including lymphocytes and macrophages, express adrenergic receptors (AR).Adrenoceptors are G-protein coupled receptors that can be divided into two subgroups: the and -AR, which can be further subdivided into different subtypes.Neutrophils, mononuclear, and natural killer cells, also T-and B-lymphocytes express and -AR.The most important adrenoceptor -in terms of the immune system-is the 2-AR.
Activation of 2-AR results in an increase in cAMP concentrations, which can modulate cytokine expression, i.e., decreasing TNF- and increasing IL-8 (Elenkov & Chrousos, 1999).However, recently was described that 2A-AR stimulation increases TNF- gene expression in Kupffer cells and plasma TNF- during sepsis (Miksa et al., 2009).Thus, through AR stimulation, locally released NE, or circulating catecholamines, affect lymphocyte traffic, circulation, and proliferation, and modulate cytokine production and the functional activity of different lymphoid cells (Elenkov et al., 2000), just as they control heart rate and other vital functions.Serum levels of sympatoadrenergic transmitters -i.e., Neuropeptide-Y, ATP, and vanillyl mandelic acid (VMA, as an indirect measurement of catecholamine levels)-, are also increased during sepsis (Donoso et al., 2008).
A growing body of literature is aimed at studying -blockade as a treatment of sepsis.Their effects on metabolism and glucose homeostasis, cytokine expression, and myocardial function may be beneficial in the setting of sepsis.Sepsis induces an overall catabolic state, mainly due to excessive adrenergic stimulation (Bergmann et al., 1999).-Blockade has been proposed as a strategy to counteract the devastating consequences of this hyperadrenergic state.But treating a potentially hypotensive condition with a drug with antihypertensive properties may initially seem detrimental (Novotny et al., 2009).Peripheral (i.p.) 1-AR blockade prior to endotoxemia increases survival time, reduces hepatic expression of proinflammatory cytokines, decreases protein expression of cardiac dysfunction markers, and preserves arterial blood pressure and left ventricular contractility (Ackland et al., 2010).Surprisingly, few studies report overall mortality in the published -blocker trials in sepsis.Interestingly, of those investigators that do report mortality in sepsis models, one out of four show increased mortality in -blockade groups.
Vasopressor and inotropic therapies for sepsis employ adrenergic support.In fact, a recent publication about the "Efficacy and Safety of Dopamine Versus Norepinephrine in the Management of Septic Shock" showed that NE treatment decreases 28-day mortality and has a lower risk of sinus tachycardia and arrhythmias than dopamine (DA) (Patel et al., 2010).This work concludes that arrhythmias are significant predictors of sepsis morbi-mortality, and that "patients receiving DA should be monitored for the development of cardiac arrhythmias", but does not consider a potential increase of MOD indicators induced by DA infusion, since high doses of DA (> 20 μg/kg/min) has a predominant -AR effect, a potent immunostimulator (Povoa & Carneiro, 2010).Recently De Baker et al. reported that DA administration is associated with greater mortality and a higher incidence of arrhythmic events compared to NE administration (De Backer et al., 2012).
It should be noted that different cathecholamines used to treat patients with septic shock, have relative and -AR effects (depending on the dose).Thus, in addition to individual differences, it is necessary to consider the fine-tuning of both, immune system and cardiovascular effects of adrenergic drugs used for sepsis treatment.
Recent work on the anatomical basis of the cholinergic anti-inflammatory pathway indicates that the spleen is required for vagus nerve control of inflammation (Huston et al., 2006).The spleen is the major source of serum TNF- during endotoxemia (Mignini et al., 2003).In splenectomized rats injected with endotoxin, serum TNF- is reduced by 70%, and vagus nerve stimulation fails to further suppress TNF-.The celiac branches of the vagus terminate in the celiac-superior mesenteric plexus and not in the spleen (Berthoud & Powley, 1996).The spleen is innervated by the splenic nerve, which originates in celiac-superior mesenteric plexus.The splenic nerve is composed mainly by catecholaminergic fibers, which terminate in close apposition to immune cells (Felten et al., 1987).Thus, attenuation of TNF- production by spleen macrophages induced by vagus nerve stimulation is mediated by norepinephrine released from splenic nerve endings.These data confirms the importance of the adrenergic transmitters in the regulation of immune response.It must be noted that immune cells have all the essential components of a non-neuronal cholinergic system and that ACh synthesized and released from lymphocytes acts as an immunomodulator via both muscarinic (mAChR) and nicotinic ACh receptors (nAChR) (Kawashima & Fujii, 2000;Kawashima & Fujii, 2003).Most evidences points towards a crucial role for the 7 nAChR in the cholinergic regulation of macrophage activity (Wang et al., 2003).Nicotine exerts anti-inflammatory effects through 7 nAChR (Ulloa, 2005).Acetylcholine (and nicotine), also has cardiorespiratory effects (Fernandez et al., 2002;Zapata et al., 2002).Acting through the peripheral arterial chemoreceptors, ACh, nicotine, and epibatidine (a selective agonist for neuronal nAChRs) increases tidal volume and blood pressure in anesthetized cats (Zapata et al., 2003;Reyes et al., 2007), which support the idea that cholinergic nicotinic treatment can also improve cardiorespiratory performance during sepsis, and prevent tissue dysoxia, lactic acidosis and MODS.In addition, nicotine inhibit cardiac apoptosis induced by LPS in rats (Suzuki et al., 2003).
Finally, both endotoxin and cytokines, stimulates HPA anti-inflammatory responses, either by adrenal glucocorticoids (Turnbull & Rivier, 1999) or by inhibiting prolactin secretion, a potent regulator of humoral and cellular immune response during physiological and pathological states (Freeman et al., 2000).Thus, it is clear that the nervous system reflexively regulates the inflammatory response in real time, just as it controls heart rate and other vital functions.

Reflex control of inflammation: Part II -Immune-to-brain communication
Much less is known about the effect of the immune system on the CNS.Immune systemderived signals act on the CNS through four different pathways: i) by saturable transport across the blood-brain barrier (BBB) (Banks & Kastin, 1987); ii) by brain circumventricular organs (CVOs) (Stitt, 1990); iii) by cytokine binding to brain endothelial cells, which evokes paracrine mediators release (Fabry et al., 1993;Cao et al., 1998); and iv) by the activation of peripheral sensory nerves (i.e., vagus nerve) (Goehler et al., 1997).
The role of peripheral sensory nerves in immunomodulation is controversial.It is believed that chemosensory transduction begins in immune cells, which release inflammatory mediators to activate neural elements, including vagal paraganglia (Goehler et al., 1997;Goehler et al., 1999) and primary afferent neurons located in sensory ganglia, which evokes host defense reflexes.Two cell types compose vagal paraganglia: type I (glomus) cells and type II (sustentacular) cells (Berthoud et al., 1995).Vagal glomus cells (GC) are innervated by vagal afferent neurons, whose cell bodies are located in the nodose ganglion, and their central projection end primarily within the dorsal vagal complex (DVC) of the medulla oblongata.Thus, immunosensory inputs could initiate local cardiorespiratory reflexes and carry information about the state of inflammation.
The DVC consists of the NTS, the dorsal motor nucleus of the vagus (DMN), and the area postrema (AP) (Berthoud & Neuhuber, 2000).The DMN is the main site of origin of preganglionic vagus efferent fibers; while cardiovascular vagal efferences originate within the medullar nucleus ambiguus (NA).The AP, which lacks of BBB, is an important CVO and an important site for humoral immune-to-brain communication.The main portion of vagal sensory input is received by neurons in the NTS, which coordinate autonomic function and interaction with the endocrine system.Ascending projections from the NTS reach hypothalamic paraventricular nucleus (PVN), an important structure in the HPA axis activation.Synaptic contacts also exist between the neurons in the NTS and rostral ventrolateral medulla (RVM), which occupies an important role in control of cardiovascular and respiratory homeostasis.The neurons from RVM project to the locus coeruleus (LC), which innervates higher brain sites, like hypothalamus and PVN.Neuronal projections emanate from the RVM and LC to sympathetic preganglionic neurons in the spinal cord.
There are also descending pathways from the PVN to the RVM and NTS (Pavlov et al., 2003).Thus, these ascending and descending connections provide a neuronal substrate for interaction between HPA axis and the ANS as an immunomodulatory mechanism.
In response to plasma levels of TNF-α, vagal immunosensory activity increases (Emch et al., 2000) or decreases (Emch et al., 2002) vagal motor activity.Transection of abdominal vagal trunks suppresses fever and hyperalgesia caused by i.p.LPS but has little effect on the febrile response to i.v. or intramuscular LPS.To elucidate the importance of visceral afferent innervation on the response to LPS, Wan et al. studied the expression of the immediate early gene c-fos in the hypothalamus and brain stem of the rat following peripheral -either i.v. or i.p.-injection of LPS.Subdiaphragmatic vagotomy completely blocked the induction of c-Fos protein following i.p. injection of LPS; however, vagotomy had a minimal effect on c-Fos protein induction following i.v.LPS administration (Wan et al., 1994).In addition, c-Fos activation of NTS neurons induced by LPS persists after cervical bilateral vagotomy (Hermann et al., 2001).Both subdiaphragmatic and cervical bilateral vagotomy abolition of CNS c-Fos activation induced by i.p.LPS are controversial, since it could be due to the section of neurons from the abdominal region that mediate the response to LPS per se or, merely, because of the role played by the vagus efferent fibers -perhaps those within the celiac branches-in LPS transport from the peritoneal cavity to the blood.Thus, when these fibers are cut, LPS escape to systemic circulation is limited, and systemic responses to LPS would be diminished (e.g., c-Fos protein induction in the CNS) (Lenczowski et al., 1997;Romanovsky et al., 2000).
The number of neurons within the DVC that expressed c-Fos activation after peripheral administration of LPS is correlated with plasma levels of TNF-.Thus, the activation of DVC neurons did not require intact vagal pathways, suggesting that TNF- generated peripherally could acts directly on these neurons, because DVC exhibits the characteristics of CVOs (i.e., fenestrated capillary network and absence of functional BBB) (Hermann et al., 2001) or, more probably, through another neural afferent pathway.In consequence, it is possible to suggest that prominent CNS manifestations of endotoxemia are apparently caused by incoming neural signals provided by other peripheral receptors, distinct from vagal paraganglia, like the carotid arterial chemoreceptors, which function is intact after bilateral cervical vagotomy.Our results shown that LPS-induced c-Fos activation in NTS neurons and plasmatic cortisol increases in septic rats (treated i.p. with 15 mg/kg LPS) are suppressed by bilateral carotid neurotomy (Reyes et al., 2012. Adv Exp Med Biol.In press) (Table 1).
Seen from an anatomical standpoint, the carotid body (CB) is the largest paraganglia in the body (Mascorro & Yates, 1980), and like other paraganglia, it receives sensory innervation, and has specialized glomus cells with abundant synapses with the sensory nervous fibers (Verna, 1997).

The arterial chemoreceptors in neuroimmunomodulation
The CB is the main peripheral chemoreceptor responsible for the detection of blood oxygen levels.The CB consists of groups of glomus (type I) cells arranged around capillaries, ensheathed by sustentacular (type II) cells, and surrounded by connective tissue.It receives profuse sensory innervation from the carotid (sinus) nerve (CSN), a branch of the glossopharyngeal nerve, whose sensory nerve endings are in close contact with glomus cells (GC) (Hess & Zapata, 1972).CB innervation is essentially by sensory neurons residing mainly in the petrosal ganglion (Kalia & Davies, 1978;Berger, 1980).Interestingly, the first synapsis at the CNS for afferent CSN fibers occurs in the NTS (Donoghue et al., 1984;Finley & Katz, 1992).Thus, inflammation-derived sensory input originated from arterial chemoreceptors (Zapata et al., 2011) can be differentially processed in the peripheral chemoreceptor per se, in the sensory ganglion, and/or in the brainstem, and modify cardiorespiratory chemoreflexes, endocrine, and autonomic functions, like the neural control on the immune system.In rats, petrosal ganglion is a constituent of a ganglion complex, composed by nodose, petrosal and jugular ganglia, the nodose-petrosal-jugular ganglion complex (NPJgc).
Many reports allow us to propose that peripheral arterial chemoreceptors play a pivotal role in afferent signaling during sepsis.Recently, we demonstrated that i.v.administration of LPS to pentobarbitone-anesthetized cats evokes similar symptoms to those observed in patients with severe sepsis and septic shock, with tachycardia, tachypnea and hypotension, and that the increased respiratory frequency is prevented by bilateral section of the carotid and aortic nerves (Fernandez et al., 2008).In addition, LPS enhances tonic CB chemosensory activity (measured by recording the frequency of chemosensory discharges) but reduces its responsiveness to transient excitatory (hypoxia and nicotine) or depressant (pure oxygen) stimuli.Diminished ventilatory responses to moderate and severe hypoxia in cats reproduces the diminished ventilatory responses to hypoxia observed in unanesthetized newborn piglets subjected to Escherichia coli endotoxin infusion (McDeigan et al., 2003), as well as in rats, in a process that is in part mediated by an inhibitory effect of endothelial nitric oxide on the respiratory control mechanisms (Ladino et al., 2007).Apoptosis studies carried out in CB excised from endotoxemic cats discard that CB diminished chemosensory activity observed in LPS-treated animals resulted from a reduction of functional tissue (Fernandez et al., 2008), and suggest the participation of systemic soluble factors (e.g., cytokines), or locally produced by either resident monocytes/macrophages (Dvorakova et al., 2000), or parenchyma cells.
Lipopolysaccharide administration increases cytokine plasma levels in many species, including rats (Waage, 1987), bovines (Ohtsuka et al., 1997) and cats (Otto & Rawlings, 1995).Thus, by using in vitro experiments, where the carotid artery is perfused and the entire preparation (including the CB) is superfused, the frequency of carotid nerve discharges recorded under normoxic conditions was not significantly modified by TNF-α, but the enhanced CB chemosensory discharges recorded along responses to hypoxic stimulation was transiently diminished, in a dose-dependent manner (Fernandez et al., 2008).The cat CB expresses both type-1 and type-2 TNF-α receptor mRNA.Immunohistochemical studies with specific antibodies, determined that TNF-R1 protein is located mainly in the GC.In addition, a strong positive TNF- protein immunoreactivity was also found in the GC cytoplasm (Fernandez et al., 2008).These observations suggest that locally or systemically produced and secreted TNF-α, acting in an autocrine or paracrine fashion, could modify GC function.
Apart from the presence of TNF-α and TNF-R1, it is known that GC from rat CB express IL-1 receptor type I (Wang et al., 2002) and IL-6 receptor α (Wang et al., 2006), and that GC respond to IL-1β application with depolarization and a transient rise in intracellular calcium (Shu et al., 2007).On the other hand, i.p. administration of IL-1 evokes IL-1 receptor type I and tyrosine hydroxylase (TH) up-regulation in the rat CB (Zhang et al., 2007).The fact that pro-inflammatory cytokines and their receptors are functionally expressed in the CB type I cells, suggests that inflammatory mediators may have different functional roles in the activation of neurons in the NPJgc, even in the absence of sepsis syndromes -e.g., exerting a tonic control of cardiorespiratory, endocrine, autonomic, and/or immune functions-.In fact, hypoxia, the natural stimulus for peripheral arterial chemoreceptors upregulates the expression and function of proinflammatory cytokines in the rat CB (Lam et al., 2008), and the adaptation to chronic hypoxia involves immune cell invasion and increased expression of inflammatory cytokines in rat CB (Liu et al., 2009).Thus, it is possible to suggest that local or systemic pro-inflammatory cytokines, recognized by membrane receptors located in the GC, modify CB chemosensory activity and, through afferent pathways projecting to the NTS, stimulate or inhibit specific components of the systemic inflammatory response.It must be noted that, regarding the source of immune signals, neural pathways provide faster and more precise information than humoral pathways.
In view of data mentioned above, we tested whether LPS-induced systemic inflammation exerts a direct effect upon CB chemoreceptors.We determined that the rat CB and NPJgc constitutively express the mRNAs for TLR4, MyD88, TNF- and its receptors (TNF-R1 and TNF-R2).Intraperitoneal administration of 15 mg/kg LPS evokes IKB degradation, and subsequent NF-B p65 translocation into the nucleus from GC and NPJgc chemosensory neurons.LPS also evokes p38 MAPK and ERK phosphorylation.Consistently, LPS treatment increases both mRNA and protein levels of TNF-, TNF-R2, and TH.Double-labeling studies show that TLR4, TNF-α, and TNF-R1 are localized in TH-containing GC and neurons from CB and NPJgc, respectively, suggesting that the expression was confined to the chemoafferent neural pathway.TNF-R2 is also present surrounding GC clusters within the CB and in chemosensitive neurons.TNF-α, and TNF-R2 expression are increased in the carotid chemoreceptors from endotoxemic rats (Fernandez et al., 2011).Thus -in addition to systemic LPS effect-our results suggest that LPS acting directly through TLR-4 modifies TNF- and its receptors expression on chemosensory cells of the carotid chemoreceptors neural pathway.These results show a novel afferent pathway to the CNS during physiological conditions and endotoxemia, and could be relevant in understanding sepsis pathophysiology and therapy.
Thus, it is very interesting to highlight that during sepsis syndromes, LPS acting directly upon carotid chemoreceptors, modify TNF- expression.In addition systemic or local inflammatory mediators could change arterial chemoreceptors function and afferent signaling through TNF- receptors, whose expression is also modified during sepsis (our results), or through IL-1 and/or IL-6 receptors (Figure 1).Interestingly, TNF- stimulates c-Fos activation of neurons in the NTS (Hermann et al., 2001).Results here obtained would imply that arterial chemoreflexes, not only serves as a chemoreceptor for respiratory reflex responses, as traditionally accepted, but also as a sensor for the immune status, as modulator of autonomic balance tending to coordinate cardiorespiratory interplay devoted to maintain oxygen homeostasis in different pathologies, and as a protective factor during sepsis and MODS.ventrolateral medulla (RVM) and subsequent projection to the locus coeruleus (LC) innervates higher brain sites, like PVN.Also, neuronal projections emanate from the RVM and LC to sympathetic preganglionic neurons in the spinal cord, which in turn activates adrenal epinephrine (EN) secretion and norepinephrine (NE), reducing pro-inflammatory activity.Thus, these ascending and descending connections provide a neuronal substrate for interaction between HPA axis and the ANS as an immunomodulatory mechanism.PG, petrosal ganglion; NG, nodose ganglion; CSN, carotid/sinus nerve; VN, vagus nerve; GPN, glossopharyngeal nerve; NS, nervous system; ACTH, adrenocorticotrophic hormone.
The disruption of continuous detection of the 'inflammatory status' of the body exerted by carotid chemoreceptors could be responsible for modifying the activity of the ANS, thus altering the control exerted by the nervous system on the immune system, and evoking an uncontrolled cytokine production.This excessive and uncontrolled systemic inflammatory response and dysautonomy could be responsible for subsequent neural uncoupling of the vital organs and MODS.

Conclusion
Sepsis syndromes are the main cause of death between critical care patients.They result from neural, cardiovascular, respiratory, and immune systems uncoupling.Multiple organ dysfunction syndrome (MODS) is due to an uncontrolled release of pro-inflammatory mediators, which damage parenchymatous organs.However, it is still unknown why sepsis progresses to MODS in only certain individuals.
The effects of sepsis therapies are controversial and strongly dependent of individual components, like individual response and genetic predisposition.Thus, the course of sepsis and therapies outcomes depends largely from host factors.
Increasing evidences shown that peripheral carotid chemoreceptors act as sensor for the immune status, as modulator of autonomic balance tending to coordinate cardiorespiratory interplay devoted to maintain oxygen homeostasis in different pathologies, and as a protective factor during sepsis and MODS.
As result of the autonomic and immune imbalance originated from carotid chemoreceptors, neural and cytokine communication networks between healthy organs are disrupted.So, the impaired autonomic function would decrease cardiorespiratory function, oxygen delivery to the tissues, and the reflex control of inflammation.The heterostasis induced by systemic inflammation worsens the uncoupling of biological oscillators, what would lead to MODS and death.

Figure 1 .
Figure 1.Proposed model for neural reflex control of inflammation during sepsis syndromes.Lypopolysaccharide (LPS) acting through macrophages or other cytokine-producing cell, increases plasma levels of pro-inflammatory cytokines (e.g., TNF-) which in activates immunosensory inputs from vagal paraganglia or carotid body (CB) chemoreceptors.Immunosensory signals reach the nucleus tractus solitarii (NTS) neurons.The dorsal motor nucleus (DMN) is the main site of origin of preganglionic vagus efferent fibers, activating the cholinergic anti-inflammatory reflex, by secreting acetylcholine (ACh), which decreased immune response.The main portion of vagal sensory inputs received by NTS neurons coordinates autonomic function and interaction with the endocrine system.Ascending projections from the NTS reach hypothalamic paraventricular nucleus (PVN), activating the hypothalamic-pituitary-adrenal (HPA) axis for glucocorticoids production and immunosupression.Synaptic contacts with the rostral

Table 1 .
Summary of observations in rats submitted to bilateral carotid neurotomy (BCN) or simulated surgery (SHAM) prior to the i.
p. administration of 15 mg/kg LPS (LPS) or vehicle (saline).The data were assessed 90-min after LPS or vehicle administration.Table prepared from part of the data presented inReyes et al., 2012 (In press.Adv Exp Med Biol)