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Neuronal activation in the nucleus of the solitary tract following jejunal lipopolysaccharide in the rat
Autonomic Neuroscience: Basic and Clinical 148 (2009) 63–68
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Autonomic Neuroscience: Basic and Clinical j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a u t n e u
Neuronal activation in the nucleus of the solitary tract following jejunal lipopolysaccharide in the rat☆ G. Gakis a,b, M.H. Mueller a, J. Hahn c, J. Glatzle c, D. Grundy d, M.E. Kreis a,⁎ a
Ludwig-Maximilian's University, Department of Surgery, Grosshadern, Munich, Germany Eberhard-Karl's University, Department of Urology, Tuebingen, Germany Eberhard-Karl's University, Department of General Surgery, Tuebingen, Germany d University of Sheffield, Department of Biomedical Sciences, Sheffield, United Kingdom b c
a r t i c l e
i n f o
Article history: Received 1 December 2008 Received in revised form 21 February 2009 Accepted 12 March 2009 Keywords: Autonomic innervation Brain stem Endotoxin Sepsis Vagus nerve
a b s t r a c t Introduction: Inflammation during systemic lipopolysaccharide (LPS) seems to be modulated by the CNS via afferent and efferent vagal pathways. We hypothesized that similar to systemic inflammation, local LPS in the gut lumen may also activate central neurons and aimed to identify potential molecular mechanisms. Methods: Male Wistar rats were equipped with an exteriorized canula in the proximal jejunum. LPS or vehicle were administered into the jejunum (10 mg ml− 1). For further study of molecular mechanisms, LPS or vehicle were administered systemically (1 mg kg− 1). Brain stem activation was quantified by Fosimmunohistochemistry in the vagal nucleus of the solitary tract (NTS) and the Area postrema which is exposed to systemic circulation. Serum LPS concentrations were also determined. Results: Jejunal LPS exposure entailed 91 ± 12 (n = 7) Fos-positive neurons in the NTS compared to 39 ± 9 in controls (n = 6; p b 0.01), while serum LPS concentrations and Fos-positive neurons in the Area postrema were not different. Systemic LPS triggered 150 ± 25 (n = 6) and vehicle 52 ± 6 Fos-positive neurons (n = 7; p b 0.01). The Fos count after systemic LPS was reduced to 99 ± 30 following pretreatment with the cyclooxygenase inhibitor Naproxen (10 mg kg− 1; p N 0.05 versus vehicle controls) and increased to 242 ± 66 following the iNOS-inhibitor Aminoguanidine (15 mg kg− 1; p b 0.01). In the Area postrema, 97 ± 17 (n = 6) neurons were counted in animals pretreated with systemic LPS compared to 14 ± 4 in controls (n = 7, p b 0.001). Conclusions: Central neuronal activation following inflammation after systemic LPS is modulated by cyclooxygenase and NO pathways. Local exposure to bacterial LPS in the gut lumen activates the NTS which may set the stage for efferent vagal modulation of intestinal inflammation. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The traditional concept that the immune system independently orchestrates a systemic inflammatory response to intruding pathogens during sepsis was challenged in recent years. Lipopolysaccharide (LPS) which is a component of the cell wall of gram-negative bacteria simulates a septic inflammatory response as it brings on the release of an array of proinflammatory mediators from immune cells when given systemically (Fink et al., 1987, Opal, 2007). While this action of LPS is well-established, it also has the potential to sensitize neurons in the brain stem (Lin et al., 1999) which suggests a central sensory mechanism for LPS. This observation entails the obvious question,
☆ Supported by the Deutsche Forschungsgemeinschaft (DFG; grant KR 1816/3-3). ⁎ Corresponding author. Ludwig-Maximilian's University, University Hospital Grosshadern, Marchioninistrasse 15, D-81377 Munich, Germany. Tel.: +49 897095 3561; fax: +49 897095 8894. E-mail address: [email protected] (M.E. Kreis). 1566-0702/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.autneu.2009.03.004
whether the CNS subsequently exerts an efferent modulation of the systemic inflammatory response. Indeed, Borovikova et al. (2000) demonstrated that vagal efferent stimulation attenuates this inflammatory response to lipopolysaccharide (LPS) which ultimately improved survival. The mechanism is based on efferent vagal fibers that release acetylcholine which subsequently binds to α-7-subunit-containing nicotinic acetylcholine receptors that are expressed on macrophages (Wang et al., 2003). Acetylcholine binding attenuates the release of proinflammatory cytokines such as tumour necrosis factor (TNF-α), IL-1β, IL-6 and IL-18 but not the antiinflammatory cytokine IL-10 (Borovikova et al., 2000). While Tracey's group studied systemic inflammation, recent work demonstrated the efferent vagus' modulatory potential on local inflammation in the gastrointestinal (GI-tract; Bonaz, 2007; The et al., 2007). This is of particular importance as inflammation is an important defense mechanism in the GI-tract since it is continuously exposed to an abundance of pathogens, toxins and antigens entering the organism via the oral route. These bring on a continued low-grade inflammatory
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response in the intestinal wall that needs to be balanced in order to ensure a defence barrier preventing intrusion into the organism and at the same time avoiding a detrimental extent of the inflammatory response which may also be harmful (Simmons et al., 2001). This efferent regulatory function of the vagus nerve in the intestine raises the intriguing question whether there is a sensory mechanism that provides the CNS with specific information from the GI-tract necessary to trigger efferent vagal modulation of intestinal inflammation or – in other words – whether central vagal nuclei sense local inflammation in the GI-tract. Another important question is, which is the mechanism in the gut lumen that would trigger central neuronal activation e.g. during bacterial infection and subsequent inflammation in the gut. The first question was addressed in a model of intestinal inflammation by cecal infection with the bacterium Campylobacter jejuni (Gaykema et al., 2003). Subsequent neuronal activation in visceral sensory nuclei via the vagus nerve (Goehler et al., 2005) innervating the cecum was demonstrated (Cao et al., 2007). These reports strongly suggest that brainstem activation is a consequence of local vagal sensitization in the intestine following bacterial infection rather than triggered by a systemic response with local release of mediators activating vagal afferents elsewhere, e.g. in the liver (Sehic et al., 1998), heart (Hisata et al., 2006) or lungs (Kubin et al., 2006). The latter question regarding the mechanism for vagal sensitization in the gut lumen during bacterial infection and inflammation is unresolved. One likely candidate is LPS as it brings on reflex responses such as increased motility and reduced absorption of water in order to clear bacterial content from the gut lumen supporting the defense of the immune system (Spates et al., 1998, Cullen et al., 1998). Systemic LPS given intravenously has the potential to activate vagal afferents in the GI-tract (Liu et al., 2007) which was shown in animals after previous chronic subdiaphragmatic vagotomy. It is unknown, however, whether vagal sensitisation also occurs when LPS is administered locally into the gut lumen. The aim of this study, therefore, was to determine whether a local inflammatory response in the GI-tract inflicted by LPS exposure in the intestinal lumen triggers afferent activation of central vagal sensory nuclei. Furthermore, we aimed to explore potential molecular mechanisms involved. 2. Material and methods 2.1. Animals Experiments were performed with male Wistar rats weighing 300 to 400 g. Animals received regular rat chow and were held under a 12 h/12 h light/dark cycle. The institutional guidelines for the use and care of laboratory animals at the University of Tuebingen, Germany, were followed throughout the study.
aorta and the animal was perfused with normal saline and paraformaldehyde subsequently (Sigma-Aldrich, as above). Finally, the brain was removed, postfixed overnight in phosphate-buffered saline and stored in 25% sucrose for later immunohistochemistry. 2.3. Protocols Two different protocols were performed on separate animals. In the first protocol, the effect of E. coli-derived LPS administered into the jejunum was studied (10 mg ml− 1, total volume: 2 ml). LPS or vehicle (0.9% NaCl) was administered over a period of three hours and animals remained in their cages for another three hours until they were sacrificed by pentobarbitone overdose. Saline was given in vehicle controls. In the second protocol, molecular mechanisms were studied following systemic LPS given by intraperitoneal injection (i.p., 1 mg kg− 1; volume 1 mg ml− 1) or vehicle (0.9% NaCl) as a more robust central neuronal activation was needed for this purpose. In separate subgroups, the cyclooxygenase inhibitor Naproxen (10 mg kg− 1, Sigma-Aldrich) or the iNOS-inhibitor Aminoguanidine (15 mg kg− 1, Sigma-Aldrich) were injected i.p. 30 min before LPS- or vehicle administration. Then, animals remained in their cages for another 150 min and were also sacrificed by overdose of pentobarbitone i.p. three hours after primary injection of Naproxen or Aminoguanidine. 2.4. Fos-immunohistochemistry The brainstem was cut into 30 µm slices. For immunohistochemical staining they were incubated with polyclonal anti-Fos rabbit antibody in free-floating technique (Oncogene Research Products, Cambridge, UK) and biotinylated goat-anti-rabbit secondary antibody (DIANOVA GmbH, Hamburg, Germany) as described previously (Hsu et al., 1981; Sagar et al., 1988). Avidin-biotin complex reagents were obtained from Vector, CA, USA and 3, 3’ diaminobenzidine from Sigma (as above). The quality of the performed immunohistochemistry was not always sufficient for analysis which led to minor variation in n-numbers. 2.5. Serum-LPS determination Analysis of serum-LPS levels were performed by Limulus Amoebocyte Lysate (LAL) Chromogenic Endpoint Assay (HyCult Biotech, Uden, Netherlands). The assay is based on opacity and gelation caused by endotoxin after an enzymatic reaction with the lysate (Hurley et al., 1991; Lindsay et al., 1989). The kit has a minimum detection limit of 1.4 pg ml− 1 and a measurable concentration range of 1 to 1000 pg ml− 1. Whenever necessary, serum samples were diluted so that the measurable concentration range was not exceeded. 2.6. Data evaluation and statistical analysis
2.2. Surgery — canula placement Animals were withdrawn from food the night before surgery. Following deep ether anaesthesia, animals were laparotomized under sterile conditions and the small intestine exposed. After a stab incision, a polyethylene canula with an inner diameter of 0.5 mm was inserted and secured in the proximal jejunum just distal to the ligament of Treitz and secured with a purse-string suture. The canula was exteriorized through a subcutaneous tunnel at the animal's midscapular region. The laparotomy was closed with a running suture. Four days later, LPS from E. coli (Lot number 0111:B4, Sigma-Aldrich, Seelze, Germany) was administered according to one of the two protocols outlined below. Animals were killed by an overdose of pentobarbitone. Venous blood was obtained by needle aspiration from the ventricle of the right heart for determination of serum LPS concentrations. Then, a metal cannula was inserted in the ascending
The total number of Fos-positive cells was counted with the support of a computerized imaging system (Leica Quantimet Q550, Bensheim, Germany). The nucleus of the solitary tract (NTS) was evaluated on both sides of the brain stem at 13.3, 13.8 and 14.3 mm caudal from Bregma and the Area postrema (AP) at 13.8 mm according to stereotactic atlases (Paxinos and Watson, 1998). Data were converted to normal distribution by log10 calculation and compared with one-way ANOVA and posthoc Student–Newman–Keuls method. Data are mean ± standard error of the mean. p b 0.05 was considered as significant. 3. Results In the NTS 91 ± 12 (n = 7) Fos-positive cells were counted following luminal LPS perfusion compared to 39 ± 9 (n = 6) in controls
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Fig. 1. I. Representative slides of Fos-immunohistochemistry in the NTS 13.8 mm caudal from Bregma indicating activation of Fos-positive cells following intrajejunal LPSadministration (A) versus vehicle (B). Note the Fos-positive cells visible as dark spots in the NTS in (A). Their number is increased in the NTS compared to the vehicle control in (B). CC: Canalis centralis; NTS: nucleus of the solitary tract; AP: Area postrema DMV: Dorsal motor nucleus of the vagus. II: Brainstem section at the same level from Bregma demonstrating an increased number of Fos-positive cells following systemic LPS (C) compared to vehicle (D).
receiving normal saline (p b 0.01, Figs. 1 and 2a). In the Area postrema the number of Fos-positive cells was 27 ± 6 (n = 7) in animals receiving luminal LPS, which was not different from 16 ± 3 in controls (n = 6, p N 0.05, Fig. 2c). Serum LPS levels were not different between animals receiving intrajejunal LPS with 68 ± 4 pg ml− 1 (n = 7) compared to saline with 34 ± 8 pg ml− 1 (n = 6, p N 0.05, Fig. 3). When LPS was administered systemically, the number of Fospositive cells in the NTS was 150 ± 25 (n = 6) compared to 52 ± 6 in vehicle treated controls (n = 7, p b 0.01, Figs. 1 and 2b). In the Area postrema, 97 ± 17 (n = 6) cells were counted in LPS treated animals compared to 14 ± 4 in controls (n = 7, p b 0.001, Fig. 2c). Fos-positive cells were increased to 242 ± 66 (n = 6) in the NTS of animals pretreated with the iNOS-inhibitor Aminoguanidine (15 mg kg− 1; p b 0.01 vs. controls, Fig. 2b). Following previous administration of the cyclooxygenase inhibitor Naproxen (10 mg kg− 1), the number of Fospositive cells was 99 ± 30 (n = 6), which was not different compared to vehicle controls (p N 0.05, Fig. 2b). LPS-levels were increased to 493 × 103 ± 207 × 103 pg ml− 1 (n = 6) in the serum of animals that were treated with systemic LPS compared to 34 ± 9 pg ml− 1 in vehicle controls (n = 7, p b 0.003, Fig. 3). 4. Discussion The present study shows that LPS has the potential to activate neurons in the nucleus of the solitary tract (NTS) which were detected by Fos-immunohistochemistry. This activation was not only observed
following systemic LPS administration but also following local LPS exposure in the lumen of the small intestine. Neuronal activation was enhanced after pretreatment of the animals with the iNOS-inhibitor Aminoguanidine and reduced after the cyclooxygenase-inhibitor Naproxen. Bacterial infections in the intestine may trigger central neuronal activation in the brain stem (Gaykema et al., 2003; Goehler et al., 2005; Cao et al., 2007). To elucidate whether luminal LPS in the small intestine may represent the stimulus responsible for the sensitization of peripheral nerve endings with subsequent activation of vagal central nuclei such as the NTS, we conducted a series of experiments showing that luminal LPS has the capacity to trigger neuronal activation in the NTS, while a relevant systemic uptake of LPS in the circulation was ruled out by measuring LPS concentrations in the serum. Furthermore, in this experimental setup, neuronal staining in the Area postrema that is in close association with the circulation (Bickel et al., 1998) was not observed. While a remote possibility exists that luminal LPS causes local release of a diffusable neuromodulator or hormone that may pass the blood-brain barrier and sensitize neurons in the NTS selectively, the most likely mechanism of neuronal activation in the NTS is that peripheral intestinal afferents were sensitized by luminal LPS with subsequent signalling to the NTS. The subpopulation of small intestinal afferents that respond to LPS were investigated in our previous work. It was shown that LPS sensitizes vagal afferents (Liu et al., 2007) which is in keeping with the observation in this study that the vagal NTS was activated.
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2005), direct afferent vagal activation by LPS binding to TLR-4 receptors is one explanation. Alternatively, LPS may trigger release of other mediators such as 5-HT from enterochromaffin cells (Kidd et al., 2009) or interleukin-1/tumor-necrosis-factor-alpha from macrophages (Furutani, 1994) which may indirectly sensitize vagal afferents (Ek et al., 1998). While these potential mechanisms may be responsible for sensitization of vagal afferent pathways following luminal LPS exposure, additional mechanisms may underly neuronal activation in the NTS following systemic LPS. LPS is a macromolecule which has a molecular weight of approximately 50–100 kDa (Jann et al., 1975) and is, therefore, probably not capable to cross the blood-brain barrier (Singh and Jiang, 2004). In addition, LPS does not alter blood-brain barrier permeability (Bickel et al., 1998). Even if we consider that large portions of the NTS and DMV (dorsal motor nucleus of the vagus) may be outside the blood brain barrier (Maolood and Meister, 2009), a diffusion barrier nevertheless exists between these nuclei and circumventricular organs as for example the Area postrema (Wang et al., 2008). Thus, it seems unlikely that LPS reached the NTS directly via the circulation triggering a direct activation of NTS neurons. LPS also triggered neuronal activation at the level of the Area postrema which is a region in the brain that is characterized by absence of the blood-brain barrier and with close contact between the systemic circulation and neurons (Bickel et al., 1998). Neuronal activation in the Area postrema may have occurred by direct contact of neurons with LPS since serum LPS levels were naturally elevated following intraperitoneal LPS administration. Thus, one possibility to explain sensitisation of neurons in the NTS is that neurons activated in the Area postrema relayed their signals to the NTS (Koga and Fukuda, 1992; Karcsú et al., 1985). Beside this explanation, there are two other pathways, how LPS may have sensitized neurons in the NTS. First, LPS triggers the release of an array of proinflammatory cytokines such as TNF-α and IL-1 from macrophages that have the potential to orchestrate an inflammatory response that may entail the release of other mediators such as prostaglandins (Wang et al., 2005). Some evidence exists that this occurs in cells lining the vessels in the brain i.e. those cells that represent the blood-brain barrier (Rivest et al., 2000). Once these cells release prostaglandins, the adjacent neurons may be sensitized subsequently leading to c-fos expression. The second possibility is that these mediators stimulate the NTS by sensitizing vagal afferent
Fig. 2. A: Increase of Fos-positive cells in the NTS following intrajejunal LPSadministration compared to vehicle (0.9% NaCl; p b 0.01). B: Increase of Fos-positive cells in the NTS following systemic LPS-administration compared to vehicle (0.9% NaCl; p b 0.01). C: Numbers of Fos-positive cells in the Area postrema under the different experimental conditions. Note the marked increase in neuronal activation following systemic LPS in contrast to intrajejunal LPS administration (p b 0.001).
There are several possible mechanisms, how luminal LPS may excite intestinal vagal afferents. Since LPS may bind to TLR-4 receptors which are present on neurons of the rat nodose ganglion (Hosoi et al.,
Fig. 3. Serum LPS levels following systemic and intrajejunal LPS. Following intrajejunal LPS, the serum LPS concentration is not increased compared to vehicle, while systemic LPS administration gave rise to increased serum levels as expected (in pg ml− 1; p b 0.003 versus systemic vehicle).
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nerve endings in the periphery, i.e. in the liver (Sehic et al., 1998), heart (Hisata et al., 2006), lung (Kubin et al., 2006) or gastrointestinal tract (Liu et al., 2005) to name just a few. These signals would then be relayed to the NTS with subsequent central neuronal activation. This latter pathway, however, may also be stimulated by LPS directly via TLR-4 receptors as discussed previously (Hosoi et al., 2005). The molecular mechanisms of central neuronal activation following LPS were investigated after systemic LPS and not luminal LPS exposure since we anticipated that a robust response in neuronal brainstem activation would be indispensable to reveal a potential modulation with pharmacological tools. Indeed, with this experimental approach, we identified two molecular mechanisms which may play a regulatory role for central neuronal activation following LPS. First, inhibition of inducible nitric oxide synthase by Aminoguanidine led to an increase in c-fos expression in the NTS suggesting that the release of NO attenuates central activation. There are two isoforms of nitric oxide synthase. The constitutive form is mainly expressed during physiological conditions, while the inducible form is expressed during endotoxemia and inflammation (Moncada and Higgs, 1993). In the GI-tract iNOS inhibition by Aminoguanidine was shown to enhance the inflammatory response during acute colitis (Dikopoulos et al., 2001; Kolios et al., 2004). Thus, in our study, Aminoguanidine is likely to have augmented the inflammatory response to LPS with subsequently increased central neuronal activation. Second, the number of Fos-positive neurons was reduced following pretreatment with the cyclooxygenase inhibitor Naproxen, so that the count was not different from vehicle controls. This observation suggests that the synthesis and release of prostanoids plays a major role in the central activation of neurons in the NTS after systemic LPS. Prostaglandin E2receptors are present in the GI-tract (Zhang and Rivest, 1999; Haupt et al., 2000). Thus, if the NTS sensitisation is dependent on afferent nerve fibers in peripheral organs, they may be sensitized by locally released prostanoids secondary to LPS challenge which was shown for afferents in the mesentery of the small intestine by our own group (Liu et al., 2005). Alternatively, prostanoid release may have occurred in other cell types such as the epithelial lining of blood vessels with subsequent neuronal activation in the CNS. There is evidence that prostaglandin E2 und E4 play a key role in this process mediating their effects via EP2 und EP4-receptor subtypes (Rivest et al., 2000; Zhang and Rivest, 1999). In the present study, we demonstrated that LPS has the potential to dramatically sensitize central neurons in the NTS. The NTS represents the first relay station for vagal sensory input into the brain stem. It is in close association with the dorsal motor nucleus of the vagus (DMV) forming the dorsal vagal complex (DVC). The DMV is involved in efferent vagal signalling (Travagli and Rogers, 2001) which was previously shown to attenuate the inflammatory response during sepsis induced by systemic LPS (Borovikova et al., 2000) and also reduces inflammatory mediator release during hemorrhagic shock (Guarini et al., 2003). Thus, the NTS potentially forms the sensory link for efferent modulation of the inflammatory response during these conditions. The present study adds to the existing knowledge on these mechanism as it demonstrated for the GI-tract that LPS in the intestinal lumen may stimulate central neuronal activation in the NTS during bacterial infections in the gut (Gaykema et al., 2003; Goehler et al., 2005; Cao et al., 2007). Another important aspect of this study is that the NTS activation following LPS seems to be amenable to pharmacological manipulation i.e. via the NO metabolism and the cyclooxygenase pathway. This observation may set the stage for the development of future strategies to pharmacologically modulate the inflammatory hit in patients during intestinal bacterial infections and subsequent sepsis. In conclusion, the present study demonstrates that brainstem neurons in the nucleus of the solitary tract are activated following intestinal i.e. luminal or systemic LPS exposure. Neuronal activation in the vagal NTS during intestinal LPS exposure may represent a sensory
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Autonomic Neuroscience: Basic and Clinical j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a u t n e u
Neuronal activation in the nucleus of the solitary tract following jejunal lipopolysaccharide in the rat☆ G. Gakis a,b, M.H. Mueller a, J. Hahn c, J. Glatzle c, D. Grundy d, M.E. Kreis a,⁎ a
Ludwig-Maximilian's University, Department of Surgery, Grosshadern, Munich, Germany Eberhard-Karl's University, Department of Urology, Tuebingen, Germany Eberhard-Karl's University, Department of General Surgery, Tuebingen, Germany d University of Sheffield, Department of Biomedical Sciences, Sheffield, United Kingdom b c
a r t i c l e
i n f o
Article history: Received 1 December 2008 Received in revised form 21 February 2009 Accepted 12 March 2009 Keywords: Autonomic innervation Brain stem Endotoxin Sepsis Vagus nerve
a b s t r a c t Introduction: Inflammation during systemic lipopolysaccharide (LPS) seems to be modulated by the CNS via afferent and efferent vagal pathways. We hypothesized that similar to systemic inflammation, local LPS in the gut lumen may also activate central neurons and aimed to identify potential molecular mechanisms. Methods: Male Wistar rats were equipped with an exteriorized canula in the proximal jejunum. LPS or vehicle were administered into the jejunum (10 mg ml− 1). For further study of molecular mechanisms, LPS or vehicle were administered systemically (1 mg kg− 1). Brain stem activation was quantified by Fosimmunohistochemistry in the vagal nucleus of the solitary tract (NTS) and the Area postrema which is exposed to systemic circulation. Serum LPS concentrations were also determined. Results: Jejunal LPS exposure entailed 91 ± 12 (n = 7) Fos-positive neurons in the NTS compared to 39 ± 9 in controls (n = 6; p b 0.01), while serum LPS concentrations and Fos-positive neurons in the Area postrema were not different. Systemic LPS triggered 150 ± 25 (n = 6) and vehicle 52 ± 6 Fos-positive neurons (n = 7; p b 0.01). The Fos count after systemic LPS was reduced to 99 ± 30 following pretreatment with the cyclooxygenase inhibitor Naproxen (10 mg kg− 1; p N 0.05 versus vehicle controls) and increased to 242 ± 66 following the iNOS-inhibitor Aminoguanidine (15 mg kg− 1; p b 0.01). In the Area postrema, 97 ± 17 (n = 6) neurons were counted in animals pretreated with systemic LPS compared to 14 ± 4 in controls (n = 7, p b 0.001). Conclusions: Central neuronal activation following inflammation after systemic LPS is modulated by cyclooxygenase and NO pathways. Local exposure to bacterial LPS in the gut lumen activates the NTS which may set the stage for efferent vagal modulation of intestinal inflammation. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The traditional concept that the immune system independently orchestrates a systemic inflammatory response to intruding pathogens during sepsis was challenged in recent years. Lipopolysaccharide (LPS) which is a component of the cell wall of gram-negative bacteria simulates a septic inflammatory response as it brings on the release of an array of proinflammatory mediators from immune cells when given systemically (Fink et al., 1987, Opal, 2007). While this action of LPS is well-established, it also has the potential to sensitize neurons in the brain stem (Lin et al., 1999) which suggests a central sensory mechanism for LPS. This observation entails the obvious question,
☆ Supported by the Deutsche Forschungsgemeinschaft (DFG; grant KR 1816/3-3). ⁎ Corresponding author. Ludwig-Maximilian's University, University Hospital Grosshadern, Marchioninistrasse 15, D-81377 Munich, Germany. Tel.: +49 897095 3561; fax: +49 897095 8894. E-mail address: [email protected] (M.E. Kreis). 1566-0702/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.autneu.2009.03.004
whether the CNS subsequently exerts an efferent modulation of the systemic inflammatory response. Indeed, Borovikova et al. (2000) demonstrated that vagal efferent stimulation attenuates this inflammatory response to lipopolysaccharide (LPS) which ultimately improved survival. The mechanism is based on efferent vagal fibers that release acetylcholine which subsequently binds to α-7-subunit-containing nicotinic acetylcholine receptors that are expressed on macrophages (Wang et al., 2003). Acetylcholine binding attenuates the release of proinflammatory cytokines such as tumour necrosis factor (TNF-α), IL-1β, IL-6 and IL-18 but not the antiinflammatory cytokine IL-10 (Borovikova et al., 2000). While Tracey's group studied systemic inflammation, recent work demonstrated the efferent vagus' modulatory potential on local inflammation in the gastrointestinal (GI-tract; Bonaz, 2007; The et al., 2007). This is of particular importance as inflammation is an important defense mechanism in the GI-tract since it is continuously exposed to an abundance of pathogens, toxins and antigens entering the organism via the oral route. These bring on a continued low-grade inflammatory
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response in the intestinal wall that needs to be balanced in order to ensure a defence barrier preventing intrusion into the organism and at the same time avoiding a detrimental extent of the inflammatory response which may also be harmful (Simmons et al., 2001). This efferent regulatory function of the vagus nerve in the intestine raises the intriguing question whether there is a sensory mechanism that provides the CNS with specific information from the GI-tract necessary to trigger efferent vagal modulation of intestinal inflammation or – in other words – whether central vagal nuclei sense local inflammation in the GI-tract. Another important question is, which is the mechanism in the gut lumen that would trigger central neuronal activation e.g. during bacterial infection and subsequent inflammation in the gut. The first question was addressed in a model of intestinal inflammation by cecal infection with the bacterium Campylobacter jejuni (Gaykema et al., 2003). Subsequent neuronal activation in visceral sensory nuclei via the vagus nerve (Goehler et al., 2005) innervating the cecum was demonstrated (Cao et al., 2007). These reports strongly suggest that brainstem activation is a consequence of local vagal sensitization in the intestine following bacterial infection rather than triggered by a systemic response with local release of mediators activating vagal afferents elsewhere, e.g. in the liver (Sehic et al., 1998), heart (Hisata et al., 2006) or lungs (Kubin et al., 2006). The latter question regarding the mechanism for vagal sensitization in the gut lumen during bacterial infection and inflammation is unresolved. One likely candidate is LPS as it brings on reflex responses such as increased motility and reduced absorption of water in order to clear bacterial content from the gut lumen supporting the defense of the immune system (Spates et al., 1998, Cullen et al., 1998). Systemic LPS given intravenously has the potential to activate vagal afferents in the GI-tract (Liu et al., 2007) which was shown in animals after previous chronic subdiaphragmatic vagotomy. It is unknown, however, whether vagal sensitisation also occurs when LPS is administered locally into the gut lumen. The aim of this study, therefore, was to determine whether a local inflammatory response in the GI-tract inflicted by LPS exposure in the intestinal lumen triggers afferent activation of central vagal sensory nuclei. Furthermore, we aimed to explore potential molecular mechanisms involved. 2. Material and methods 2.1. Animals Experiments were performed with male Wistar rats weighing 300 to 400 g. Animals received regular rat chow and were held under a 12 h/12 h light/dark cycle. The institutional guidelines for the use and care of laboratory animals at the University of Tuebingen, Germany, were followed throughout the study.
aorta and the animal was perfused with normal saline and paraformaldehyde subsequently (Sigma-Aldrich, as above). Finally, the brain was removed, postfixed overnight in phosphate-buffered saline and stored in 25% sucrose for later immunohistochemistry. 2.3. Protocols Two different protocols were performed on separate animals. In the first protocol, the effect of E. coli-derived LPS administered into the jejunum was studied (10 mg ml− 1, total volume: 2 ml). LPS or vehicle (0.9% NaCl) was administered over a period of three hours and animals remained in their cages for another three hours until they were sacrificed by pentobarbitone overdose. Saline was given in vehicle controls. In the second protocol, molecular mechanisms were studied following systemic LPS given by intraperitoneal injection (i.p., 1 mg kg− 1; volume 1 mg ml− 1) or vehicle (0.9% NaCl) as a more robust central neuronal activation was needed for this purpose. In separate subgroups, the cyclooxygenase inhibitor Naproxen (10 mg kg− 1, Sigma-Aldrich) or the iNOS-inhibitor Aminoguanidine (15 mg kg− 1, Sigma-Aldrich) were injected i.p. 30 min before LPS- or vehicle administration. Then, animals remained in their cages for another 150 min and were also sacrificed by overdose of pentobarbitone i.p. three hours after primary injection of Naproxen or Aminoguanidine. 2.4. Fos-immunohistochemistry The brainstem was cut into 30 µm slices. For immunohistochemical staining they were incubated with polyclonal anti-Fos rabbit antibody in free-floating technique (Oncogene Research Products, Cambridge, UK) and biotinylated goat-anti-rabbit secondary antibody (DIANOVA GmbH, Hamburg, Germany) as described previously (Hsu et al., 1981; Sagar et al., 1988). Avidin-biotin complex reagents were obtained from Vector, CA, USA and 3, 3’ diaminobenzidine from Sigma (as above). The quality of the performed immunohistochemistry was not always sufficient for analysis which led to minor variation in n-numbers. 2.5. Serum-LPS determination Analysis of serum-LPS levels were performed by Limulus Amoebocyte Lysate (LAL) Chromogenic Endpoint Assay (HyCult Biotech, Uden, Netherlands). The assay is based on opacity and gelation caused by endotoxin after an enzymatic reaction with the lysate (Hurley et al., 1991; Lindsay et al., 1989). The kit has a minimum detection limit of 1.4 pg ml− 1 and a measurable concentration range of 1 to 1000 pg ml− 1. Whenever necessary, serum samples were diluted so that the measurable concentration range was not exceeded. 2.6. Data evaluation and statistical analysis
2.2. Surgery — canula placement Animals were withdrawn from food the night before surgery. Following deep ether anaesthesia, animals were laparotomized under sterile conditions and the small intestine exposed. After a stab incision, a polyethylene canula with an inner diameter of 0.5 mm was inserted and secured in the proximal jejunum just distal to the ligament of Treitz and secured with a purse-string suture. The canula was exteriorized through a subcutaneous tunnel at the animal's midscapular region. The laparotomy was closed with a running suture. Four days later, LPS from E. coli (Lot number 0111:B4, Sigma-Aldrich, Seelze, Germany) was administered according to one of the two protocols outlined below. Animals were killed by an overdose of pentobarbitone. Venous blood was obtained by needle aspiration from the ventricle of the right heart for determination of serum LPS concentrations. Then, a metal cannula was inserted in the ascending
The total number of Fos-positive cells was counted with the support of a computerized imaging system (Leica Quantimet Q550, Bensheim, Germany). The nucleus of the solitary tract (NTS) was evaluated on both sides of the brain stem at 13.3, 13.8 and 14.3 mm caudal from Bregma and the Area postrema (AP) at 13.8 mm according to stereotactic atlases (Paxinos and Watson, 1998). Data were converted to normal distribution by log10 calculation and compared with one-way ANOVA and posthoc Student–Newman–Keuls method. Data are mean ± standard error of the mean. p b 0.05 was considered as significant. 3. Results In the NTS 91 ± 12 (n = 7) Fos-positive cells were counted following luminal LPS perfusion compared to 39 ± 9 (n = 6) in controls
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Fig. 1. I. Representative slides of Fos-immunohistochemistry in the NTS 13.8 mm caudal from Bregma indicating activation of Fos-positive cells following intrajejunal LPSadministration (A) versus vehicle (B). Note the Fos-positive cells visible as dark spots in the NTS in (A). Their number is increased in the NTS compared to the vehicle control in (B). CC: Canalis centralis; NTS: nucleus of the solitary tract; AP: Area postrema DMV: Dorsal motor nucleus of the vagus. II: Brainstem section at the same level from Bregma demonstrating an increased number of Fos-positive cells following systemic LPS (C) compared to vehicle (D).
receiving normal saline (p b 0.01, Figs. 1 and 2a). In the Area postrema the number of Fos-positive cells was 27 ± 6 (n = 7) in animals receiving luminal LPS, which was not different from 16 ± 3 in controls (n = 6, p N 0.05, Fig. 2c). Serum LPS levels were not different between animals receiving intrajejunal LPS with 68 ± 4 pg ml− 1 (n = 7) compared to saline with 34 ± 8 pg ml− 1 (n = 6, p N 0.05, Fig. 3). When LPS was administered systemically, the number of Fospositive cells in the NTS was 150 ± 25 (n = 6) compared to 52 ± 6 in vehicle treated controls (n = 7, p b 0.01, Figs. 1 and 2b). In the Area postrema, 97 ± 17 (n = 6) cells were counted in LPS treated animals compared to 14 ± 4 in controls (n = 7, p b 0.001, Fig. 2c). Fos-positive cells were increased to 242 ± 66 (n = 6) in the NTS of animals pretreated with the iNOS-inhibitor Aminoguanidine (15 mg kg− 1; p b 0.01 vs. controls, Fig. 2b). Following previous administration of the cyclooxygenase inhibitor Naproxen (10 mg kg− 1), the number of Fospositive cells was 99 ± 30 (n = 6), which was not different compared to vehicle controls (p N 0.05, Fig. 2b). LPS-levels were increased to 493 × 103 ± 207 × 103 pg ml− 1 (n = 6) in the serum of animals that were treated with systemic LPS compared to 34 ± 9 pg ml− 1 in vehicle controls (n = 7, p b 0.003, Fig. 3). 4. Discussion The present study shows that LPS has the potential to activate neurons in the nucleus of the solitary tract (NTS) which were detected by Fos-immunohistochemistry. This activation was not only observed
following systemic LPS administration but also following local LPS exposure in the lumen of the small intestine. Neuronal activation was enhanced after pretreatment of the animals with the iNOS-inhibitor Aminoguanidine and reduced after the cyclooxygenase-inhibitor Naproxen. Bacterial infections in the intestine may trigger central neuronal activation in the brain stem (Gaykema et al., 2003; Goehler et al., 2005; Cao et al., 2007). To elucidate whether luminal LPS in the small intestine may represent the stimulus responsible for the sensitization of peripheral nerve endings with subsequent activation of vagal central nuclei such as the NTS, we conducted a series of experiments showing that luminal LPS has the capacity to trigger neuronal activation in the NTS, while a relevant systemic uptake of LPS in the circulation was ruled out by measuring LPS concentrations in the serum. Furthermore, in this experimental setup, neuronal staining in the Area postrema that is in close association with the circulation (Bickel et al., 1998) was not observed. While a remote possibility exists that luminal LPS causes local release of a diffusable neuromodulator or hormone that may pass the blood-brain barrier and sensitize neurons in the NTS selectively, the most likely mechanism of neuronal activation in the NTS is that peripheral intestinal afferents were sensitized by luminal LPS with subsequent signalling to the NTS. The subpopulation of small intestinal afferents that respond to LPS were investigated in our previous work. It was shown that LPS sensitizes vagal afferents (Liu et al., 2007) which is in keeping with the observation in this study that the vagal NTS was activated.
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2005), direct afferent vagal activation by LPS binding to TLR-4 receptors is one explanation. Alternatively, LPS may trigger release of other mediators such as 5-HT from enterochromaffin cells (Kidd et al., 2009) or interleukin-1/tumor-necrosis-factor-alpha from macrophages (Furutani, 1994) which may indirectly sensitize vagal afferents (Ek et al., 1998). While these potential mechanisms may be responsible for sensitization of vagal afferent pathways following luminal LPS exposure, additional mechanisms may underly neuronal activation in the NTS following systemic LPS. LPS is a macromolecule which has a molecular weight of approximately 50–100 kDa (Jann et al., 1975) and is, therefore, probably not capable to cross the blood-brain barrier (Singh and Jiang, 2004). In addition, LPS does not alter blood-brain barrier permeability (Bickel et al., 1998). Even if we consider that large portions of the NTS and DMV (dorsal motor nucleus of the vagus) may be outside the blood brain barrier (Maolood and Meister, 2009), a diffusion barrier nevertheless exists between these nuclei and circumventricular organs as for example the Area postrema (Wang et al., 2008). Thus, it seems unlikely that LPS reached the NTS directly via the circulation triggering a direct activation of NTS neurons. LPS also triggered neuronal activation at the level of the Area postrema which is a region in the brain that is characterized by absence of the blood-brain barrier and with close contact between the systemic circulation and neurons (Bickel et al., 1998). Neuronal activation in the Area postrema may have occurred by direct contact of neurons with LPS since serum LPS levels were naturally elevated following intraperitoneal LPS administration. Thus, one possibility to explain sensitisation of neurons in the NTS is that neurons activated in the Area postrema relayed their signals to the NTS (Koga and Fukuda, 1992; Karcsú et al., 1985). Beside this explanation, there are two other pathways, how LPS may have sensitized neurons in the NTS. First, LPS triggers the release of an array of proinflammatory cytokines such as TNF-α and IL-1 from macrophages that have the potential to orchestrate an inflammatory response that may entail the release of other mediators such as prostaglandins (Wang et al., 2005). Some evidence exists that this occurs in cells lining the vessels in the brain i.e. those cells that represent the blood-brain barrier (Rivest et al., 2000). Once these cells release prostaglandins, the adjacent neurons may be sensitized subsequently leading to c-fos expression. The second possibility is that these mediators stimulate the NTS by sensitizing vagal afferent
Fig. 2. A: Increase of Fos-positive cells in the NTS following intrajejunal LPSadministration compared to vehicle (0.9% NaCl; p b 0.01). B: Increase of Fos-positive cells in the NTS following systemic LPS-administration compared to vehicle (0.9% NaCl; p b 0.01). C: Numbers of Fos-positive cells in the Area postrema under the different experimental conditions. Note the marked increase in neuronal activation following systemic LPS in contrast to intrajejunal LPS administration (p b 0.001).
There are several possible mechanisms, how luminal LPS may excite intestinal vagal afferents. Since LPS may bind to TLR-4 receptors which are present on neurons of the rat nodose ganglion (Hosoi et al.,
Fig. 3. Serum LPS levels following systemic and intrajejunal LPS. Following intrajejunal LPS, the serum LPS concentration is not increased compared to vehicle, while systemic LPS administration gave rise to increased serum levels as expected (in pg ml− 1; p b 0.003 versus systemic vehicle).
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nerve endings in the periphery, i.e. in the liver (Sehic et al., 1998), heart (Hisata et al., 2006), lung (Kubin et al., 2006) or gastrointestinal tract (Liu et al., 2005) to name just a few. These signals would then be relayed to the NTS with subsequent central neuronal activation. This latter pathway, however, may also be stimulated by LPS directly via TLR-4 receptors as discussed previously (Hosoi et al., 2005). The molecular mechanisms of central neuronal activation following LPS were investigated after systemic LPS and not luminal LPS exposure since we anticipated that a robust response in neuronal brainstem activation would be indispensable to reveal a potential modulation with pharmacological tools. Indeed, with this experimental approach, we identified two molecular mechanisms which may play a regulatory role for central neuronal activation following LPS. First, inhibition of inducible nitric oxide synthase by Aminoguanidine led to an increase in c-fos expression in the NTS suggesting that the release of NO attenuates central activation. There are two isoforms of nitric oxide synthase. The constitutive form is mainly expressed during physiological conditions, while the inducible form is expressed during endotoxemia and inflammation (Moncada and Higgs, 1993). In the GI-tract iNOS inhibition by Aminoguanidine was shown to enhance the inflammatory response during acute colitis (Dikopoulos et al., 2001; Kolios et al., 2004). Thus, in our study, Aminoguanidine is likely to have augmented the inflammatory response to LPS with subsequently increased central neuronal activation. Second, the number of Fos-positive neurons was reduced following pretreatment with the cyclooxygenase inhibitor Naproxen, so that the count was not different from vehicle controls. This observation suggests that the synthesis and release of prostanoids plays a major role in the central activation of neurons in the NTS after systemic LPS. Prostaglandin E2receptors are present in the GI-tract (Zhang and Rivest, 1999; Haupt et al., 2000). Thus, if the NTS sensitisation is dependent on afferent nerve fibers in peripheral organs, they may be sensitized by locally released prostanoids secondary to LPS challenge which was shown for afferents in the mesentery of the small intestine by our own group (Liu et al., 2005). Alternatively, prostanoid release may have occurred in other cell types such as the epithelial lining of blood vessels with subsequent neuronal activation in the CNS. There is evidence that prostaglandin E2 und E4 play a key role in this process mediating their effects via EP2 und EP4-receptor subtypes (Rivest et al., 2000; Zhang and Rivest, 1999). In the present study, we demonstrated that LPS has the potential to dramatically sensitize central neurons in the NTS. The NTS represents the first relay station for vagal sensory input into the brain stem. It is in close association with the dorsal motor nucleus of the vagus (DMV) forming the dorsal vagal complex (DVC). The DMV is involved in efferent vagal signalling (Travagli and Rogers, 2001) which was previously shown to attenuate the inflammatory response during sepsis induced by systemic LPS (Borovikova et al., 2000) and also reduces inflammatory mediator release during hemorrhagic shock (Guarini et al., 2003). Thus, the NTS potentially forms the sensory link for efferent modulation of the inflammatory response during these conditions. The present study adds to the existing knowledge on these mechanism as it demonstrated for the GI-tract that LPS in the intestinal lumen may stimulate central neuronal activation in the NTS during bacterial infections in the gut (Gaykema et al., 2003; Goehler et al., 2005; Cao et al., 2007). Another important aspect of this study is that the NTS activation following LPS seems to be amenable to pharmacological manipulation i.e. via the NO metabolism and the cyclooxygenase pathway. This observation may set the stage for the development of future strategies to pharmacologically modulate the inflammatory hit in patients during intestinal bacterial infections and subsequent sepsis. In conclusion, the present study demonstrates that brainstem neurons in the nucleus of the solitary tract are activated following intestinal i.e. luminal or systemic LPS exposure. Neuronal activation in the vagal NTS during intestinal LPS exposure may represent a sensory
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